@article {pmid41162659,
year = {2025},
author = {Campbell, IW and Basta, DW and Zingl, FG and Sullivan, EJ and Doranga, S and Waldor, MK},
title = {Anoxia activates CRISPR-Cas immunity in the mouse intestine.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {41162659},
issn = {2058-5276},
support = {P30 DK034854/DK/NIDDK NIH HHS/United States ; R01 AI042347/AI/NIAID NIH HHS/United States ; Investigator program//Howard Hughes Medical Institute (HHMI)/ ; },
abstract = {The natural context in which CRISPR-Cas systems are active in Enterobacteriaceae has remained enigmatic. Here we find that the Citrobacter rodentium type I-E CRISPR-Cas system is activated by the oxygen-responsive transcriptional regulator Fnr in the anoxic environment of the mouse intestine. Since Fnr-dependent regulation is predicted in ~41% of Enterobacteriaceae cas3 orthologues, we propose that anoxic regulation of CRISPR-Cas immunity is an adaptation that protects Enterobacteriaceae against threats from foreign DNA within the intestinal microbiome.},
}

@article {pmid41162404,
year = {2025},
author = {Yan, X and Liu, B and Zhou, S and Fan, Y and Wei, S and Qiu, D and Xiang, H and Zhou, J and Mergny, JL and Monchaud, D and Ju, H and Zhou, J},
title = {A Chimeric Photo-Controllable CRISPR/Cas12a System for Universal and Fast Diagnostics.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c04782},
pmid = {41162404},
issn = {1520-6882},
abstract = {The potential of clustered regularly interspaced short palindromic repeats (CRISPR) and corresponding CRISPR-associated (Cas) protein systems (CRISPR/Cas) systems for biomedical applications is tremendous; however, precise control of their activity is essential to better harness this potential and, beyond this, to develop reliable diagnostic reagents. Herein, we report on such a strategy by controlling the CRISPR/Cas12a activity using a photo-controllable CRISPR RNA (crRNA). To this end, the 3' end of crRNA was conjugated to a G-quadruplex (G4) block through a photocleavable linker: upon photo irradiation, the G4 trigger is removed, thus allowing for the DNA target to access and hybridize with the crRNA, and thus be processed by the CRISPR/Cas12a system. The efficiency of this approach was demonstrated by the detection of human papillomavirus 16 DNA in 50 clinical samples: our one-pot strategy was found to be as efficient as the routinely implemented method (qPCR), with 95.7% sensitivity and 100% specificity, in addition to be faster (25 versus 60 min) and both simpler and less expensive (being implementable as lateral flow test strips). Collectively, this new and fully controllable CRISPR/Cas system holds great potential for next-generation clinical diagnostics.},
}

@article {pmid41162070,
year = {2025},
author = {Gautam, V and Jambagi, SR and Muthugounder, M},
title = {Genome editing of detoxification gene repertoires in insects using clustered regularly interspaced short palindromic repeats (CRISPR): A systematic review and meta-analysis.},
journal = {Pesticide biochemistry and physiology},
volume = {215},
number = {},
pages = {106687},
doi = {10.1016/j.pestbp.2025.106687},
pmid = {41162070},
issn = {1095-9939},
mesh = {Animals ; *Gene Editing ; *Insecta/genetics ; *Insecticide Resistance/genetics ; Insecticides/pharmacology ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Inactivation, Metabolic/genetics ; },
abstract = {Decoding the molecular mechanisms underlying insect resistance to insecticides and host plant adaptation is essential for effective and sustainable Insecticide Resistance Management (IRM). Reverse genetic approaches targeting "target site resistance" can help control pests without harming pollinators and beneficial biocontrol agents. Understanding the role of mutations involved in xenobiotic resistance enables the judicious use of pesticides. CRISPR-based genome editing allows precise manipulation of detoxification genes, helping to decipher their roles in resistance development. This review provides a comprehensive overview of CRISPR-mediated genome editing in insect detoxification genes and their involvement in resistance mechanisms. In addition to synthesizing overall data trajectories, we present study-level effect sizes that highlight context-specific responses to gene editing, offering insights that can inform future experimental designs and functional validation studies.},
}

Animals
*Gene Editing
*Insecta/genetics
*Insecticide Resistance/genetics
Insecticides/pharmacology
CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Inactivation, Metabolic/genetics

@article {pmid41161594,
year = {2025},
author = {Fu, H and Xu, W and Huang, M and Cong, Y},
title = {Molecular detection of Salmonella.},
journal = {Journal of food protection},
volume = {},
number = {},
pages = {100659},
doi = {10.1016/j.jfp.2025.100659},
pmid = {41161594},
issn = {1944-9097},
abstract = {The genus Salmonella consists of a group of globally significant foodborne pathogens that pose substantial public health risks. Traditional detection methods are inadequate for rapid diagnosis and effective epidemic surveillance due to limitations such as time-consuming procedures and insufficient sensitivity. In recent years, development of molecular techniques has driven innovations in Salmonella detection. Nucleic acid-based detection methods including polymerase chain reaction (PCR), real-time fluorescent quantitative PCR (qPCR), whole genome sequencing (WGS) and more emerge as crucial approaches for Salmonella detection due to their high sensitivity, specificity, and rapidity. Our review systematically summarized technological advancements in molecular detection of Salmonella, including specific genetic targets and drug resistance genes used for molecular detection, typing technologies, and emerging techniques such as CRISPR-Cas systems and microfluidic chips. This review comprehensively covers a wide array of molecular detection and characterization technologies, including conventional PCR, qPCR, multiplex PCR, digital PCR (dPCR), isothermal amplification techniques (such as loop-mediated isothermal amplification, recombinase polymerase amplification), genotyping methods (including pulsed-field gel electrophoresis, multilocus sequence typing etc.), WGS, melting curve analysis (MCA), and other emerging technologies. The review also discusses the balance between sensitivity and specificity in complex samples, challenges regarding the cost and accessibility of advanced technologies, as well as prospects for future development directions including portable point-of-care testing devices, automated detection equipment. Ongoing optimization of molecular detection technologies will provide critical support for the prevention and control of Salmonella infections.},
}

@article {pmid41161575,
year = {2025},
author = {Verma, R and Das, G and Manjunathachar, H and Muwel, N and Choudhary, R and Kumar, S and Nath, S and Gattani, A and Gupta, V and Sharma, RK and Ajith, Y},
title = {CRISPR-Cas systems: Pioneering next-generation diagnostic tools for parasitic diseases.},
journal = {Molecular and biochemical parasitology},
volume = {},
number = {},
pages = {111708},
doi = {10.1016/j.molbiopara.2025.111708},
pmid = {41161575},
issn = {1872-9428},
abstract = {Parasitic diseases pose significant threats to both human and veterinary health, causing morbidity, mortality, and economic losses. Effective diagnostics are critical, yet conventional methods such as microscopy, serology, and polymerase chain reaction (PCR) are limited by low sensitivity, cross-reactivity, or dependence on costly equipment and skilled personnel. Isothermal amplification techniques, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), have improved point-of-care (POC) applications but remain limited by nonspecific amplification and reduced sensitivity for low-copy targets. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems have emerged as transformative tools in molecular diagnostics, offering high sensitivity, specificity, rapidity, and cost-effectiveness. This review presents an overview of CRISPR-Cas systems, their historical development, classification (Class 1 and Class 2, Types I-VI), molecular mechanisms, and diagnostic potential in parasitic diseases, with illustrative examples from studies published between 2017 and May 2025. Despite significant progress, CRISPR-based diagnostics face challenges such as off-target activity, dependence on nucleic acid amplification, and complex sample preparation. Future directions focus on amplification-free detection, multiplexed assay development, and integration with nanotechnology, microfluidics, smartphone-based devices, and artificial intelligence. CRISPR-Cas technologies thus represent a promising frontier in next-generation diagnostics for parasitic disease surveillance, control, and personalized healthcare in both human and veterinary health.},
}

@article {pmid41161091,
year = {2025},
author = {Hong, SH and Kim, I and Lee, G and Kim, EH and Bae, E and Suh, JY},
title = {Structural and mechanistic investigation of the anti-CRISPR protein AcrIE5 using NMR spectroscopy and AlphaFold modeling.},
journal = {Biochemical and biophysical research communications},
volume = {789},
number = {},
pages = {152854},
doi = {10.1016/j.bbrc.2025.152854},
pmid = {41161091},
issn = {1090-2104},
abstract = {The CRISPR-Cas system employs RNA-guided endonucleases to protect bacteria and archaea from invading bacteriophages and plasmids. In response, bacteriophages have evolved anti-CRISPR proteins that inhibit diverse types of the CRISPR-Cas system. AcrIE5 was discovered from the mobile genetic elements of Pseudomonas aeruginosa, and potently inhibits the type I-E CRISPR-Cas system of P. aeruginosa. Here, we determined the solution structure of AcrIE5 using NMR spectroscopy, which adopts a novel αβ fold comprising three α-helices and two β-strands. AcrIE5 harbors a mobile loop between the β-strands that is conserved among homologs encoded by MGEs infecting Pseudomonas species, but truncated in homologs from MGEs of other bacteria. AlphaFold correctly reproduced the experimental structure of AcrIE5 and predicted its binding at a cleft formed by Cas8e, Cas7e, and Cas5e within the P. aeruginosa type I-E Cascade. The mobile loop and α-helices of AcrIE5 mediated key interactions with Cas8e at the PAM recognition site, as well as with adjacent Cas7e and Cas5e. AcrIE5 did not bind individual subunits of Cascade with high affinity, suggesting that it recognizes a composite interface of the functional Cascade assembly. Taken together, our findings suggest that AcrIE5 may compete with DNA binding to the PAM recognition site of type I-E Cascade, similar to AcrIE3 and AcrIE4, and also highlight a potential functional role of the conserved mobile loop in host-specific anti-CRISPR activity.},
}

@article {pmid41160700,
year = {2025},
author = {Elliott, SD and Ready, PJ and Wrinn, CM and Ma, Q and Edward, M and Niescier, RF and Escobar, I and Sun, J and Ganga, AK and McAtee, CK and Atiş, İS and Koleske, AJ and Bordey, A and Breslow, DK},
title = {A CRISPR activation screen reveals a cilia disassembly pathway mutated in focal cortical dysplasia.},
journal = {Science advances},
volume = {11},
number = {44},
pages = {eaeb7238},
pmid = {41160700},
issn = {2375-2548},
mesh = {*Cilia/metabolism/genetics/pathology ; Humans ; *Mutation ; *Malformations of Cortical Development/genetics/metabolism/pathology ; Cytoskeletal Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; rhoA GTP-Binding Protein/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Armadillo Domain Proteins/genetics/metabolism ; Animals ; Signal Transduction ; Focal Cortical Dysplasia ; },
abstract = {Defective assembly of primary cilia causes ciliopathies, but cilia disassembly and its role in disease remain poorly understood. From a genome-wide CRISPR activation (CRISPRa) screen for negative regulators of ciliary function, we find here that the F2R G protein-coupled receptor, sterile alpha and TIR motif-containing 1 (SARM1) hydrolase, ryanodine receptors, peri-centrosomal calcium signaling, and RhoA form a functional pathway that is necessary and sufficient for cilia disassembly. Highlighting the significance of this pathway, several components are somatically mutated in focal cortical dysplasia (FCD), a neurological disorder characterized by intractable epilepsy. Supporting the functional impact of these variants, patient-derived SARM1 and RhoA mutations potentiate cilia loss, and a RhoA variant impairs cortical development. Conversely, SARM1 inhibition restores cilia in cells with FCD-associated alterations. Together, our work identifies a pathway for cilia disassembly, implicates aberrant pathway activation as a feature of FCD-associated mutations, and illustrates the potential of CRISPRa screening to provide insight into diseases caused by somatic mutations.},
}

*Cilia/metabolism/genetics/pathology
Humans
*Mutation
*Malformations of Cortical Development/genetics/metabolism/pathology
Cytoskeletal Proteins/genetics/metabolism
*CRISPR-Cas Systems
rhoA GTP-Binding Protein/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
Armadillo Domain Proteins/genetics/metabolism
Animals
Signal Transduction
Focal Cortical Dysplasia

@article {pmid40991409,
year = {2025},
author = {Smith, SL and Iwamoto, Y and Manimaran, A and Drubin, DG},
title = {Harnessing fusion of genome-edited human stem cells to rapidly screen for novel protein functions in vivo.},
journal = {Molecular biology of the cell},
volume = {36},
number = {11},
pages = {ar141},
doi = {10.1091/mbc.E25-06-0301},
pmid = {40991409},
issn = {1939-4586},
mesh = {Humans ; *Gene Editing/methods ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Endocytosis/genetics ; Cell Fusion/methods ; Actin Cytoskeleton/metabolism ; CRISPR-Cas Systems/genetics ; Clathrin/metabolism ; Lysosomes/metabolism ; },
abstract = {Genome editing has enabled the integration of fluorescent protein coding sequences into genomes, resulting in expression of in-frame fusion proteins under the control of their natural gene regulatory sequences. While this technique overcomes the well-documented artifacts associated with gene overexpression for biological processes sensitive to altered protein stoichiometry, such as clathrin-mediated endocytosis (CME), editing genomes of metazoan cells incurs a significant time cost compared with simpler organisms, such as yeast. Editing two or more genes to express multiple fluorescent fusion proteins in a single cell line has proven to be a powerful strategy for uncovering spatial dynamic, and therefore functional, relationships among different proteins, but it can take many months to edit each gene within the same cell line. Here, by utilizing cell fusions, we quickly generated cells expressing pairwise permutations of fluorescent fusion proteins in genome-edited human cells to reveal previously undetected protein-organelle interactions. We fused human induced pluripotent stem cells (hiPSCs) that express in-frame fusions of CME and actin cytoskeleton proteins with hiPSCs that express fluorescently tagged organelle markers, uncovering novel interactions between CME proteins, branched actin filament networks, and lysosomes.},
}

Humans
*Gene Editing/methods
*Induced Pluripotent Stem Cells/metabolism/cytology
Endocytosis/genetics
Cell Fusion/methods
Actin Cytoskeleton/metabolism
CRISPR-Cas Systems/genetics
Clathrin/metabolism
Lysosomes/metabolism

@article {pmid40963020,
year = {2025},
author = {Chauhan, VP and Sharp, PA and Langer, R},
title = {Engineered prime editors with minimal genomic errors.},
journal = {Nature},
volume = {646},
number = {8087},
pages = {1254-1260},
pmid = {40963020},
issn = {1476-4687},
mesh = {*Gene Editing/methods ; *INDEL Mutation/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Deoxyribonuclease I/metabolism/genetics ; Humans ; *Genome/genetics ; DNA/genetics/metabolism/chemistry ; *Genomics ; },
abstract = {Prime editors make programmed genome modifications by writing new sequences into extensions of nicked DNA 3' ends[1]. These edited 3' new strands must displace competing 5' strands to install edits, yet a bias towards retaining the competing 5' strands hinders efficiency and can cause indel errors[2]. Here we discover that nicked end degradation, consistent with competing 5' strand destabilization, can be promoted by Cas9-nickase mutations that relax nick positioning. We exploit this mechanism to engineer efficient prime editors with strikingly low indel errors. Combining this error-suppressing strategy with the latest efficiency-boosting architecture, we design a next-generation prime editor (vPE). Compared with previous editors, vPE features comparable efficiency yet up to 60-fold lower indel errors, enabling edit:indel ratios as high as 543:1.},
}

*Gene Editing/methods
*INDEL Mutation/genetics
CRISPR-Associated Protein 9/metabolism/genetics
*CRISPR-Cas Systems/genetics
Deoxyribonuclease I/metabolism/genetics
Humans
*Genome/genetics
DNA/genetics/metabolism/chemistry
*Genomics

@article {pmid41160667,
year = {2025},
author = {Du, X and Goh, PK and Ma, C and Coughlan, E and Greatorex, S and Porter, LH and Russ, B and Cummins, KD and Sek, K and Slaney, CY and Scott, AM and Oliaro, J and Neeson, PJ and Risbridger, GP and Taylor, RA and Trapani, JA and Turner, SJ and Darcy, PK and Wiede, F and Tiganis, T},
title = {Targeting PTPN2 enhances human CAR T cell efficacy and the development of long-term memory in mouse xenograft models.},
journal = {Science translational medicine},
volume = {17},
number = {822},
pages = {eadk0627},
doi = {10.1126/scitranslmed.adk0627},
pmid = {41160667},
issn = {1946-6242},
mesh = {Animals ; Humans ; *Protein Tyrosine Phosphatase, Non-Receptor Type 2/metabolism/antagonists & inhibitors ; *Xenograft Model Antitumor Assays ; Mice ; *Immunologic Memory ; *Receptors, Chimeric Antigen/metabolism/immunology ; Immunotherapy, Adoptive ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/immunology ; },
abstract = {Chimeric antigen receptor (CAR) T cells have been ineffective against solid tumors, where the hostile tumor microenvironment limits CAR T cell function and persistence. Protein tyrosine phosphatase N2 (PTPN2) attenuates T cell receptor and cytokine signaling to maintain T cell tolerance. Here, we used CRISPR-Cas9 gene editing or an inhibitor to target PTPN2 in human CAR T cells specific for the Lewis Y (LeY) neoantigen, which is expressed in most epithelial tumors. Targeting PTPN2 increased CAR and cytokine signaling, including interferon signaling, and enhanced the antigen-induced expansion, activation, and cytotoxicity of anti-LeY CAR T cells in vitro and in vivo. The deletion of PTPN2 in CAR T cells repressed the growth of human tumor and patient-derived xenografts in mice, when compared with unedited CAR T cells, and prolonged mouse survival. The administration of inhibitor also enhanced the ability of α-LeY CAR T cells to repress tumor growth. Cellular indexing of transcriptomes and epitopes by sequencing analysis of splenic PTPN2-deficient CD8[+] CAR T cells in tumor-bearing mice revealed that PTPN2 deficiency favored the generation of CD45RA[+] CAR T cells expressing markers of long-lived stem cell memory (SCM) CAR T cells. Flow cytometric analysis reaffirmed that the deletion or inhibition of PTPN2 promoted the intratumoral accumulation of SCM CD8[+] CAR T cells and the overall persistence of CD8[+] CAR T cells. These data support the use of gene editing or small-molecule inhibitors targeting PTPN2 in human CAR T cells to treat solid tumors.},
}

Animals
Humans
*Protein Tyrosine Phosphatase, Non-Receptor Type 2/metabolism/antagonists & inhibitors
*Xenograft Model Antitumor Assays
Mice
*Immunologic Memory
*Receptors, Chimeric Antigen/metabolism/immunology
Immunotherapy, Adoptive
CRISPR-Cas Systems/genetics
Cell Line, Tumor
*T-Lymphocytes/immunology
CD8-Positive T-Lymphocytes/immunology

@article {pmid41160625,
year = {2025},
author = {Bish, LM and Fuss, JL and Panaccione, DG},
title = {Gene editing of the thioester reductase step in the biosynthesis of lysergic acid amides.},
journal = {PloS one},
volume = {20},
number = {10},
pages = {e0334651},
doi = {10.1371/journal.pone.0334651},
pmid = {41160625},
issn = {1932-6203},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Lysergic Acid/analogs & derivatives/metabolism ; *Amides/metabolism ; *Oxidoreductases/genetics/metabolism ; Peptide Synthases/genetics/metabolism ; Fungal Proteins/genetics/metabolism ; },
abstract = {Ergot alkaloids derived from lysergic acid are important in agriculture, as food and feed contaminants, and in medicine, as the foundation of several pharmaceuticals. The fungus Metarhizium brunneum makes several lysergic acid amides, with lysergic acid α-hydroxyethylamide (LAH) being produced in by far the highest concentration. The multifunctional enzyme lysergyl peptide synthetase 3 (Lps3) has multiple domains that play important roles in lysergic acid amide synthesis. We hypothesized a role for the reductase domain of Lps3 in liberating LAH from an enzyme-bound precursor and tested this hypothesis with CRISPR/Cas9-based gene editing experiments. We transformed M. brunneum with a Cas9/single guide RNA complex and a donor DNA that replaced the tyrosine at the active site of the reductase domain of Lps3 with a phenylalanine. Sanger sequencing of edited and wild-type genes demonstrated successful editing of the reductase domain without non-target mutations in Lps3. High performance liquid chromatography of the edited strain showed a significant reduction of LAH and accumulation of the precursor lysergic acid. The phenotype was similar when the edited allele of lpsC was in a wild-type background or in backgrounds with late pathway genes easO or easP knocked out, except no LAH was detectable when the edit was in the easO knockout background. The data demonstrate that the reductase domain plays a key role or roles in formation of LAH. The abundant lysergic acid accumulating in the mutants, as opposed to later pathway intermediates in LAH biosynthesis (such as lysergyl-alanine), indicated severe debilitation of Lps3. The data indicate a requirement for the reductase domain of Lps3 in synthesis of lysergic acid amides and demonstrate the feasibility of the CRISPR/Cas9-based approach for editing genes in Metarhizium species.},
}

*Gene Editing/methods
CRISPR-Cas Systems
*Lysergic Acid/analogs & derivatives/metabolism
*Amides/metabolism
*Oxidoreductases/genetics/metabolism
Peptide Synthases/genetics/metabolism
Fungal Proteins/genetics/metabolism

@article {pmid41160062,
year = {2025},
author = {Yilmaz Çolak, Ç},
title = {Harnessing CRISPR technology for the diagnosis of Bordetella pertussis: advances and implications.},
journal = {Future microbiology},
volume = {},
number = {},
pages = {1-8},
doi = {10.1080/17460913.2025.2581522},
pmid = {41160062},
issn = {1746-0921},
abstract = {Following the discovery of the prokaryotic adaptive immune system known as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins, this technology has revolutionized biotechnology as a multifaceted genome-editing tool with a wide range of applications. CRISPR technology has not only provided novel treatment options, especially for genetic diseases, but also transformed molecular diagnostic platforms. The specific, sensitive, and adaptable nature of the CRISPR-Cas systems has led to the development of innovative solutions for the detection of diseases, including viral and bacterial infections. This review provides an overview of the CRISPR-Cas systems and mainly focuses on the application of CRISPR-based assays for the detection of Bordetella pertussis, which is the main causative agent of a highly infectious disease, whooping cough. The review emphasizes the need for novel diagnostic tools for B. pertussis, along with highlighting some future perspectives, since its diagnosis can be challenging due to nonspecific early symptoms and interference from closely related Bordetella species. In this regard, CRISPR-based diagnostic platforms can offer a promising avenue for rapid and accurate detection of B. pertussis, helping the management of whooping cough.},
}

@article {pmid41159723,
year = {2025},
author = {Duan, Z and Yang, R and Lai, T and Jiang, W and Zhang, J and Chen, B and Liao, L},
title = {Development of a CRISPR/Cas9-induced gene editing system for Pseudoalteromonas fuliginea and its applications in functional genomics.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0177125},
doi = {10.1128/aem.01771-25},
pmid = {41159723},
issn = {1098-5336},
abstract = {Pseudoalteromonas has been used as a model system to study cold adaptation and is of widespread interest in biotechnology and ecology. To explore its physiological responses to extreme cold, uncover functional genes, and clarify their ecological roles, efficient genetic tools are essential. However, existing genetic manipulation methods in Pseudoalteromonas rely on traditional homology-based recombination, which is laborious and time-consuming in this bacterial system. Consequently, improving editing efficiency is crucial for advancing both basic research and applied potential. Here, we established a CRISPR/Cas9 system in Pseudoalteromonas and carried out an extensive investigation of the Type II CRISPR/Cas9 platform for gene editing in Pseudoalteromonas fuliginea, a representative species thriving in the frigid polar oceans. To validate the feasibility of the CRISPR/Cas system in P. fuliginea, multiple genes were selected as targets, and the gene editing effects were confirmed through phenotypic changes or gene expression. We have successfully achieved both gene knockouts and insertions in P. fuliginea, encompassing the deletion of genes such as fliJ, indA, and genes encoding Pf sRNAs, as well as the in vivo insertion of 3×FLAG and the gfp gene. The average CRISPR/Cas9 gene editing efficiency in P. fuliginea exceeded 70%. In summary, we developed an efficient CRISPR/Cas9-based editing system in P. fuliginea, which can be utilized to accelerate the development of Pseudoalteromonas as a model system for addressing fundamental questions related to extreme environmental adaptation and to fulfill its potential biotechnological applications.IMPORTANCEPseudoalteromonas fuliginea is a marine bacterium with great potential for ecological and biotechnological research, yet its genetic manipulation has long been a technical challenge. In this study, we developed a gene editing system based on CRISPR technology that enables efficient and precise genome modification in this organism. Using this system, we successfully deleted, inserted, and tagged multiple genes, including regulatory and non-coding elements, with high success rates. Notably, several of these genes are linked to key traits such as motility and stress response, which contribute to microbial adaptation in polar environments. This tool allows researchers to directly test gene function and study microbial adaptation in cold marine environments. The ability to perform reliable genetic edits in P. fuliginea opens new possibilities for its use as a model organism and will support future advances in microbial ecology, environmental microbiology, and marine biotechnology.},
}

@article {pmid41157107,
year = {2025},
author = {Shpiliukova, K and Kachanov, A and Brezgin, S and Chulanov, V and Ivanov, A and Kostyushev, D and Kostyusheva, A},
title = {m[6]A RNA Modification: Technologies Behind Future Anti-Cancer Therapy.},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {20},
pages = {},
doi = {10.3390/molecules30204091},
pmid = {41157107},
issn = {1420-3049},
support = {075-15-2025-519//Ministry of Science and Higher Education of the Russian Federation (Federal scientific and technical program for the development of genetic technologies for 2019-2030/ ; },
mesh = {Humans ; *Adenosine/analogs & derivatives/metabolism/genetics ; *Neoplasms/genetics/drug therapy/therapy/metabolism ; Methylation ; Animals ; Epigenesis, Genetic ; *Antineoplastic Agents/pharmacology/therapeutic use ; *RNA/genetics/metabolism ; *RNA Processing, Post-Transcriptional ; RNA Methylation ; },
abstract = {N6-methyladenosine (m[6]A) modifications are among the most prevalent epigenetic marks in eukaryotic RNAs, regulating both coding and non-coding RNAs and playing a pivotal role in RNA metabolism. Given their widespread influence, m[6]A modifications are deeply implicated in the pathogenesis of various cancers, including highly aggressive malignancies such as lung cancer, melanoma, and liver cancer. Dysregulation of m[6]A dynamics-marked by an imbalance in methylation and demethylation-can drive tumor progression, enhance metastatic potential, increase aggressiveness, and promote drug resistance, while also exerting context-dependent tumor-suppressive effects. Given this dual role, precise modulation of m[6]A levels and the activity of its regulatory enzymes (writers, erasers, and readers) represent a promising therapeutic avenue. In this review, we highlight recent advances in targeting m[6]A machinery, including small-molecule inhibitors, antisense oligonucleotides, and CRISPR/Cas-based editing tools, capable of both writing and erasing m[6]A marks and altering m[6]A methylation sites per se. By evaluating these strategies, we aim to identify the most effective approaches for restoring physiological m[6]A homeostasis or for strategically manipulating the m[6]A machinery for therapeutic benefit.},
}

Humans
*Adenosine/analogs & derivatives/metabolism/genetics
*Neoplasms/genetics/drug therapy/therapy/metabolism
Methylation
Animals
Epigenesis, Genetic
*Antineoplastic Agents/pharmacology/therapeutic use
*RNA/genetics/metabolism
*RNA Processing, Post-Transcriptional
RNA Methylation

@article {pmid41156655,
year = {2025},
author = {Peláez Sánchez, RG and González Restrepo, J and Pineda, S and Cuartas-López, AM and Martínez Garro, JM and Torres-Castro, M and Urrego, R and López-Rojas, LE and Salazar Florez, JE and Monroy, FP},
title = {Bioinformatic Identification of CRISPR-Cas Systems in Leptospira Genus: An Update on Their Distribution Across 77 Species.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {10},
pages = {},
doi = {10.3390/pathogens14101044},
pmid = {41156655},
issn = {2076-0817},
mesh = {*CRISPR-Cas Systems/genetics ; *Leptospira/genetics/classification ; *Computational Biology/methods ; Genome, Bacterial ; Bacteriophages/genetics ; Leptospirosis/microbiology ; },
abstract = {UNLABELLED: Leptospirosis is a globally distributed zoonotic disease caused by pathogenic bacteria of the Leptospira genus. Genome editing in Leptospira has been difficult to perform. Currently, the functionality of the CRISPR-Cas system has been demonstrated in species such as Leptospira interrogans. However, the different CRISPR-Cas systems present in most of the 77 species are unknown. Therefore, the objective of this study was to identify these arrays across the genomes of all described Leptospira species using bioinformatics tools.

METHODS: a bioinformatics workflow was followed: genomes were downloaded from the NCBI database; Cas protein detection was carried out using the CRISPR-CasFinder and RAST web servers; functional analyses of Cas proteins were performed with InterProScan, ProtParam, Swiss Model, Alphafold3, Swiss PDB Viewer, and Pymol; conservation pattern detection was conducted using MEGA12, and Seqlogos; spacer identification was carried out with the Actinobacteriophages database and BLAST version 1.4.0; and bacteriophage detection was performed using PHASTER, and PHASTEST.

RESULTS: Cas proteins were detected in 36 out of the 77 species of the Leptospira species, including Cas1 to Cas9 and Cas12. These proteins were classified into Class 1 and Class 2 systems, corresponding to types I, II, and V. Direct repeats and spacers were detected in 19 species, with the direct repeats exhibiting two conserved nucleotide motifs. Analysis of spacer sequences revealed 323 distinct bacteriophages. Additionally, three intact bacteriophages were detected in the genomes of four Leptospira species. Notably, two saprophytic species have complete CRISPR-Cas systems.

CONCLUSIONS: The presence of Cas proteins, direct repeats, and spacer sequences with homology to bacteriophage genomes provides evidence for a functional CRISPR-Cas system in at least 19 species.},
}

*CRISPR-Cas Systems/genetics
*Leptospira/genetics/classification
*Computational Biology/methods
Genome, Bacterial
Bacteriophages/genetics
Leptospirosis/microbiology

@article {pmid41155971,
year = {2025},
author = {Serrano, DR and Juste, F and Anaya, BJ and Ramirez, BI and Sánchez-Guirales, SA and Quispillo, JM and Hernandez, EM and Simon, JA and Trallero, JM and Serrano, C and Rawat, S and Lalatsa, A},
title = {Exosome-Based Drug Delivery: A Next-Generation Platform for Cancer, Infection, Neurological and Immunological Diseases, Gene Therapy and Regenerative Medicine.},
journal = {Pharmaceutics},
volume = {17},
number = {10},
pages = {},
doi = {10.3390/pharmaceutics17101336},
pmid = {41155971},
issn = {1999-4923},
support = {PID2024-156769OB-I00//Ministerio de Ciencia, Innovación y Universidades/ ; Innovation in Pharmacology, Nanotechnology, and personalized medicine by 3D printing//Universidad Complutense de Madrid/ ; },
abstract = {Exosomes, naturally derived extracellular vesicles, have emerged as powerful bio-nanocarriers in precision medicine. Their endogenous origin, biocompatibility, and ability to encapsulate and deliver diverse therapeutic payloads position them as transformative tools in drug delivery, gene therapy, and regenerative medicine. This review presents a comprehensive analysis of exosome-based therapeutics across multiple biomedical domains, including cancer, neurological and infectious diseases, immune modulation, and tissue repair. Exosomes derived from stem cells, immune cells, or engineered lines can be loaded with small molecules, RNA, or CRISPR-Cas systems, offering highly specific and low-immunogenic alternatives to viral vectors or synthetic nanoparticles. We explore endogenous and exogenous loading strategies, surface functionalization techniques for targeted delivery, and innovations that allow exosomes to traverse physiological barriers such as the blood-brain barrier. Furthermore, exosomes demonstrate immunomodulatory and regenerative properties in autoimmune and degenerative conditions, with promising roles in skin rejuvenation and cosmeceuticals. Despite their potential, challenges remain in large-scale production, cargo loading efficiency, and regulatory translation. Recent clinical trials and industry efforts underscore the accelerating momentum in this field. Exosomes represent a promising platform in precision medicine, though further standardization and validation are required before widespread clinical use. This review offers critical insights into current technologies, therapeutic mechanisms, and future directions to unlock the full translational potential of exosomes in clinical practice.},
}

@article {pmid41155374,
year = {2025},
author = {Honjo, A and Yako, H and Miyamoto, Y and Yagi, M and Yamamoto, M and Nishi, A and Sakagami, H and Yamauchi, J},
title = {Knocking Down FRMD4A, a Factor Associated with the Brain Development Disorder and a Risk Factor for Alzheimer's Disease, Using RNA-Targeting CRISPR/Cas13 Reveals Its Role in Cell Morphogenesis.},
journal = {International journal of molecular sciences},
volume = {26},
number = {20},
pages = {},
doi = {10.3390/ijms262010083},
pmid = {41155374},
issn = {1422-0067},
mesh = {*Alzheimer Disease/genetics/metabolism/pathology ; Animals ; *CRISPR-Cas Systems ; Neurons/metabolism/cytology ; Mice ; *Morphogenesis/genetics ; *Cytoskeletal Proteins/genetics/metabolism ; *Membrane Proteins/genetics/metabolism ; Gene Knockdown Techniques ; Humans ; *Brain/metabolism ; Cell Line ; },
abstract = {Genetic truncation or mutation of the gene encoding band 4.1, ezrin, radixin, and moesin (FERM) domain protein containing 4A (FRMD4A) is associated with brain developmental diseases, including microcephaly with global developmental delay. It has also been identified as a risk factor for Alzheimer's disease. By analogy with other FERM domain-containing proteins, FRMD4A is believed to regulate cell morphogenesis and/or cell polarization in central nervous system (CNS) cells; however, it remains unclear whether and how dysfunction of FRMD4A and/or its closely homologous protein FRMD4B causes abnormal morphogenesis in neuronal cells. Here, we describe for the first time the roles of FRMD4A and FRMD4B in process elongation in neuronal cells. Knockdown of Frmd4a or Frmd4b using specific RNA-targeting clustered regularly interspaced short palindromic repeat (CRISPR) and Cas13-fitted gRNAs led to decreased process elongation in primary cortical neurons. Similar decreases in neuronal marker expression were observed in the N1E-115 cell line, a model of neuronal differentiation. Furthermore, hesperetin, an aglycone of the citrus flavonoid hesperidin known to promote neuroprotective signaling, recovered the decreased process elongation induced by the knockdown of Frmd4a or Frm4b. Hesperetin also stimulated phosphorylation of mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPKs/ERKs), which could help promote neuronal processes. These results suggest that FRMD4A and FRMD4B regulate process elongation through a possible signaling pathway linked to the sustained phosphorylation of MAPKs/ERKs. Crucially, this study reveals that, at the molecular and cellular levels, hesperetin can restore normal phenotypes when FRMD4A protein or FRMD4B protein is impaired.},
}

*Alzheimer Disease/genetics/metabolism/pathology
Animals
*CRISPR-Cas Systems
Neurons/metabolism/cytology
Mice
*Morphogenesis/genetics
*Cytoskeletal Proteins/genetics/metabolism
*Membrane Proteins/genetics/metabolism
Gene Knockdown Techniques
Humans
*Brain/metabolism
Cell Line

@article {pmid41155368,
year = {2025},
author = {Hossain, MM and Sultana, F and Mostafa, M and Khan, I and Tran, LP and Mostofa, MG},
title = {Reinforced Defenses: R-Genes, PTI, and ETI in Modern Wheat Breeding for Blast Resistance.},
journal = {International journal of molecular sciences},
volume = {26},
number = {20},
pages = {},
doi = {10.3390/ijms262010078},
pmid = {41155368},
issn = {1422-0067},
mesh = {*Triticum/genetics/microbiology/immunology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics/immunology ; *Plant Breeding ; *Plant Immunity/genetics ; *Genes, Plant ; Magnaporthe/pathogenicity ; Host-Pathogen Interactions/genetics/immunology ; },
abstract = {Wheat blast, caused by Magnaporthe oryzae pathotype Triticum (MoT), poses a major threat to wheat (Triticum aestivum) cultivation, particularly in South America and Bangladesh. The rapid evolution and spread of the pathogen necessitate the development of durable and broad-spectrum resistance in wheat cultivars. This review summarizes current insights into the multi-layered defense mechanisms of wheat, encompassing resistance (R) genes, pattern-triggered immunity (PTI), and effector-triggered immunity (ETI) against MoT. The R-genes provide race-specific resistance through ETI, while both ETI and PTI are required to form integral layers of the plant immune system that synergistically reinforce host defense network. Recent advances in genomics, transcriptomics, and molecular breeding have facilitated the discovery and deployment of key R-genes and signaling components involved in PTI and ETI pathways. Integrating these immune strategies through gene pyramiding, marker-assisted selection (MAS), and genome editing offers a promising route towards enhanced and durable resistance in hosts. Harnessing and optimizing these multilayered immune systems will be pivotal to securing wheat productivity amid the growing threat of wheat blast.},
}

*Triticum/genetics/microbiology/immunology
*Disease Resistance/genetics
*Plant Diseases/microbiology/genetics/immunology
*Plant Breeding
*Plant Immunity/genetics
*Genes, Plant
Magnaporthe/pathogenicity
Host-Pathogen Interactions/genetics/immunology

@article {pmid41155240,
year = {2025},
author = {Carbone, F},
title = {Special Issue: Latest Research on Plant Genomics and Genome Editing.},
journal = {International journal of molecular sciences},
volume = {26},
number = {20},
pages = {},
doi = {10.3390/ijms26209946},
pmid = {41155240},
issn = {1422-0067},
mesh = {*Gene Editing/methods ; *Genome, Plant ; *Genomics/methods ; *Plants/genetics ; CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing ; },
abstract = {Over the past ten years, plant science has undergone a remarkable transformation driven by the convergence of next-generation sequencing, increasingly sophisticated bioinformatics tools, and the rise of targeted genome editing platforms [...].},
}

*Gene Editing/methods
*Genome, Plant
*Genomics/methods
*Plants/genetics
CRISPR-Cas Systems
High-Throughput Nucleotide Sequencing

@article {pmid41154642,
year = {2025},
author = {Zhang, H and Yang, Y and Yang, T and Cao, P and Yu, C and Liang, L and Liu, R and Chen, Z},
title = {Engineering a High-Fidelity MAD7 Variant with Enhanced Specificity for Precision Genome Editing via CcdB-Based Bacterial Screening.},
journal = {Biomolecules},
volume = {15},
number = {10},
pages = {},
doi = {10.3390/biom15101413},
pmid = {41154642},
issn = {2218-273X},
support = {2023YFC3402300//National Key R&D Program of China/ ; 22208044//National Natural Science Foundation of China/ ; 22278058//National Natural Science Foundation of China/ ; XLYC2203075//"Xingliao Talent Plan"project/ ; 2024-MSBA-09//Natural Science Foundation of Liaoning Province/ ; 2025JH2/101330156//Natural Science Foundation of Liaoning Province/ ; 2023JJ12SN030//Science and Technology Innovation Foundation of Dalian/ ; DUT24YG131//Fundamental Research Funds for the Central Universities/ ; DUT25LAB105//Fundamental Research Funds for the Central Universities/ ; DUT25RC(3)019//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; *Bacterial Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Endonucleases/genetics/metabolism ; Mutation ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) nucleases enable precise genome editing, but off-target cleavage remains a critical challenge. Here, we report the development of MAD7_HF, a high-fidelity variant of the MAD7 nuclease engineered through a bacterial screening system leveraging the DNA gyrase-targeting toxic gene ccdB. This system couples survival to efficient on-target cleavage and minimal off-target activity, mimicking the transient action required for high-precision editing. Through iterative selection and sequencing validation, we identified MAD7_HF, harboring three substitutions (R187C, S350T, K1019N) that enhanced discrimination between on- and off-target sites. In Escherichia coli assays, MAD7_HF exhibited a >20-fold reduction in off-target cleavage across multiple mismatch contexts while maintaining on-target efficiency comparable to wild-type MAD7. Structural modeling revealed that these mutations stabilize the guide RNA-DNA hybrid at on-target sites and weaken interactions with mismatched sequences. This work establishes a high-throughput bacterial screening strategy that allows the identification of Cas12a variants with improved specificity at a given target site, providing a useful framework for future efforts to develop precision genome-editing tools.},
}

*Gene Editing/methods
CRISPR-Cas Systems/genetics
*Escherichia coli/genetics
*Bacterial Proteins/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
*Endonucleases/genetics/metabolism
Mutation

@article {pmid41153450,
year = {2025},
author = {Nardon, E and Azzalini, E and Paladin, D and Boscarino, D and Bonin, S},
title = {CRISPR/Cas Tools for the Detection of Borrelia sensu lato in Human Samples.},
journal = {Genes},
volume = {16},
number = {10},
pages = {},
doi = {10.3390/genes16101233},
pmid = {41153450},
issn = {2073-4425},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Lyme Disease/diagnosis/microbiology/genetics ; *Borrelia burgdorferi Group/genetics/isolation & purification ; DNA, Bacterial/genetics ; Real-Time Polymerase Chain Reaction/methods ; Sensitivity and Specificity ; Bacterial Outer Membrane Proteins/genetics ; Antigens, Surface ; Bacterial Vaccines ; Lipoproteins ; },
abstract = {BACKGROUND/OBJECTIVES: Lyme disease diagnosis remains challenging due to the limitations of current methods. While PCR-based assays are widely used, their sensitivity can be affected by sample type and the inhibition of host DNA. This study aimed to evaluate the feasibility and sensitivity of a CRISPR/Cas12-based detection system for Borrelia burgdorferi sensu lato, comparing its performance with real-time PCR.

METHODS: DNA from three Borrelia genospecies (B. burgdorferi, B. garinii, and B. afzelii) was amplified targeting the OspA gene. Detection was performed using a Cas12/crRNA system with a fluorescent ssDNA reporter. Sensitivity assays were conducted on serial dilutions of Borrelia DNA, with and without human genomic DNA, and results were compared with qPCR.

RESULTS: Direct detection of Borrelia DNA without amplification was not feasible. However, when combined with PCR, the Cas12/crRNA system reliably detected as few as 5 genome copies per reaction. End-point PCR extended to 60 cycles improved detection robustness for B. garinii and B. afzelii, although sensitivity decreased in the presence of human genomic DNA.

CONCLUSIONS: The Cas12/crRNA-based system offers a sensitive and accessible alternative to qPCR, especially in settings lacking real-time PCR instrumentation. Future developments may include integration with isothermal amplification and microfluidic platforms to enhance direct detection capabilities.},
}

Humans
*CRISPR-Cas Systems/genetics
*Lyme Disease/diagnosis/microbiology/genetics
*Borrelia burgdorferi Group/genetics/isolation & purification
DNA, Bacterial/genetics
Real-Time Polymerase Chain Reaction/methods
Sensitivity and Specificity
Bacterial Outer Membrane Proteins/genetics
Antigens, Surface
Bacterial Vaccines
Lipoproteins

@article {pmid41153439,
year = {2025},
author = {Stunf Pukl, S},
title = {Genetic Therapy of Fuchs Endothelial Corneal Dystrophy: Where Are We? A Review.},
journal = {Genes},
volume = {16},
number = {10},
pages = {},
doi = {10.3390/genes16101222},
pmid = {41153439},
issn = {2073-4425},
mesh = {*Fuchs' Endothelial Dystrophy/therapy/genetics ; Humans ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Collagen Type VIII/genetics ; Mutation ; Transcription Factor 4/genetics ; Animals ; Gene Editing ; },
abstract = {OBJECTIVES: The incidence of Fuchs endothelial corneal dystrophy (FECD) is growing, and with it, the unmet need for a corneal transplant. Among alternative treatment modalities, only genetic therapy represents a causal therapy.

METHODS: Following the SNARA protocol, the PubMed and ClinicalTrials databases were searched using the keywords Fuchs endothelial corneal dystrophy, FECD, genetic therapy, and CRISPR-Cas9.

RESULTS: FECD is polyfactorial disease and mutations or polymorphisms in at least 15 different genes were connected to the disease. For the early-onset form of the disease, exclusive connection to mutations in COL8A2 was confirmed, while for the late-onset form, the most characteristic mutation is the expansion of the CTG18.1 triplet in the TCF4 gene, making these two possible targets. While the CRISPR-Cas9 approach represents the mainstay of genetic therapy development recently, the application of this method to FECD contains several obstacles, studied in preclinical settings. DT-168 and the Ad-Cas9-Col8a2gRNA molecules were developed for FECD treatment and preclinically tested, and phase I and II clinical studies for DT-168 are also already being performed.

CONCLUSIONS: The review of the literature proved that genetic therapy for FECD is at the level of preclinical research and that there are several specific challenges connected to the target genetic mutation as well as the delivery of possible treatment and duration of the effect. Further studies in the field might bring solutions in the future for alternative treatments for this common corneal disease.},
}

*Fuchs' Endothelial Dystrophy/therapy/genetics
Humans
*Genetic Therapy/methods
CRISPR-Cas Systems
Collagen Type VIII/genetics
Mutation
Transcription Factor 4/genetics
Animals
Gene Editing

@article {pmid41153385,
year = {2025},
author = {Boti, MA and Diamantopoulos, MA and Scorilas, A},
title = {RNA-Targeting Techniques: A Comparative Analysis of Modern Approaches for RNA Manipulation in Cancer Research and Therapeutics.},
journal = {Genes},
volume = {16},
number = {10},
pages = {},
doi = {10.3390/genes16101168},
pmid = {41153385},
issn = {2073-4425},
mesh = {Humans ; *Neoplasms/genetics/therapy ; RNA Interference ; CRISPR-Cas Systems ; Oligonucleotides, Antisense/therapeutic use/genetics ; Animals ; *RNA/genetics ; },
abstract = {RNA-targeting techniques have emerged as powerful tools in cancer research and therapeutics, offering precise and programmable control over gene expression at the post-transcriptional level. Once viewed as passive intermediates in the central dogma, RNA molecules are now recognized as dynamic regulators of cellular function, capable of influencing transcription, translation, and epigenetic regulation. Advances in high-throughput sequencing technologies, transcriptomics, and structural RNA biology have uncovered a diverse landscape of coding and non-coding RNAs involved in oncogenesis, drug resistance, and tumor progression. In response, several RNA-targeting strategies have been developed to modulate these transcripts, including antisense oligonucleotides (ASOs), RNA interference (RNAi), CRISPR-Cas13 systems, small molecules, and aptamers. This review provides a comparative analysis of these technologies, highlighting their molecular mechanisms, therapeutic potential, and current limitations. Emphasis is placed on the translational progress of RNA-targeting agents, including recent FDA approvals and ongoing clinical trials for cancer indications. Through a critical comparison of these strategies, this review underscores the growing significance of RNA-targeting technologies as a foundation for next-generation cancer therapeutics and precision oncology.},
}

Humans
*Neoplasms/genetics/therapy
RNA Interference
CRISPR-Cas Systems
Oligonucleotides, Antisense/therapeutic use/genetics
Animals
*RNA/genetics

@article {pmid41152294,
year = {2025},
author = {Nemoto, A and Imaizumi, K and Miya, F and Hiroi, Y and Yamada, M and Ideno, H and Saitoh, S and Kosaki, K and Okuno, H and Okano, H},
title = {Rescue of imprinted genes by epigenome editing in human cellular models of Prader-Willi syndrome.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9442},
pmid = {41152294},
issn = {2041-1723},
mesh = {*Prader-Willi Syndrome/genetics/therapy ; Humans ; *Genomic Imprinting/genetics ; Induced Pluripotent Stem Cells/metabolism ; *Gene Editing/methods ; DNA Methylation/genetics ; CRISPR-Cas Systems ; *Epigenome/genetics ; Hypothalamus/metabolism ; Organoids/metabolism ; Chromosomes, Human, Pair 15/genetics ; Epigenesis, Genetic ; Epigenome Editing ; },
abstract = {Prader-Willi syndrome (PWS) is a genomic imprinting disorder caused by the loss of function of the paternal chromosome 15q11-13, resulting in a spectrum of symptoms associated with hypothalamic dysfunction. PWS patients lack the expression of paternally expressed genes (PEGs) in the 15q11-13 locus but possess an epigenetically silenced set of these genes in the maternal allele. Thus, activation of these silenced genes can serve as a therapeutic target for PWS. Here, we leverage CRISPR-based epigenome editing system to modulate the DNA methylation status of the PWS imprinting control region (PWS-ICR) in induced pluripotent stem cells (iPSCs) derived from PWS patients. Successful demethylation in the PWS-ICR restores the PEG expression from the maternal allele and reorganizes the methylation patterns in other PWS-associated imprinted regions beyond the PWS-ICR. Remarkably, these corrected epigenomic patterns and PEG expression are maintained following the differentiation of these cells into hypothalamic organoids. Finally, the single-cell transcriptomic analysis of epigenome-edited organoids demonstrates a partial restoration of the transcriptomic dysregulation observed in PWS. This study highlights the utility of epigenome editing technology as a therapeutic approach in addressing PWS and potentially other imprinting disorders.},
}

*Prader-Willi Syndrome/genetics/therapy
Humans
*Genomic Imprinting/genetics
Induced Pluripotent Stem Cells/metabolism
*Gene Editing/methods
DNA Methylation/genetics
CRISPR-Cas Systems
*Epigenome/genetics
Hypothalamus/metabolism
Organoids/metabolism
Chromosomes, Human, Pair 15/genetics
Epigenesis, Genetic
Epigenome Editing

@article {pmid41152284,
year = {2025},
author = {Mishal, R and Meléndez-Zajgla, J and Rueda-Zarazúa, B and Labra-Barrios, ML and Castañón-Sánchez, CA and Uribe Carvajal, S and Padierna-Mota, L and Hernández-Hernández, JM and Leon-Avila, G and Pérez Rangel, A and Hernández-Martínez, E and Angeles-Morales, EB and Albalawi, IK and Luna-Arias, JP},
title = {RNA-seq analysis of wild-type and mutated TBPL1 gene in breast cancer cells lines through CRISPR/Cas9 approach reveals novel molecular signatures.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {37578},
pmid = {41152284},
issn = {2045-2322},
support = {Ph.D. fellowship 465228//Consejo Nacional de Humanidades, Ciencia y Tecnologia, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnología, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnología, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnología, Mexico/ ; PhD. fellowship 752614//Consejo Nacional de Humanidades, Ciencia y Tecnología, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnología, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnología, Mexico/ ; },
mesh = {Humans ; *Breast Neoplasms/genetics/pathology ; *CRISPR-Cas Systems ; Female ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; *TATA-Box Binding Protein/genetics/metabolism ; *Mutation ; RNA-Seq ; Transcriptome ; Cell Proliferation/genetics ; Gene Expression Profiling ; Cell Movement/genetics ; },
abstract = {Breast cancer is the leading cause of death among women globally. Several genes have been found to be transcriptionally dysregulated in cancer, according to recent studies. TATA-box binding protein (TBP) and its two paralogs, TBPL1 and TBPL2, play roles in human transcription. The TBPL1 gene is implicated in colorectal carcinomas by suppressing the expression of miR-18a. However, its function in breast cancer remains undisclosed. TBPL1 is distantly related to TBP and possesses a 40% similarity with TBP's core domain. In the present study, we explored the potential role of the TBPL1 gene in transcriptome regulation by knocking out the TBPL1 gene through the CRISPR/Cas9 method. Following the knockout of the TBPL1 gene, we examined the gene transcription patterns and compared them to wild-type cell lines. We observed disparate signatures of upregulated and downregulated genes in wild-type and mutated conditions. Healthy breast MCF-12F, and T47D, SKBR3, and MDA-MB-231 breast cancer cell lines were assessed, as these cancer cells exhibit overexpression of the TBPL1 gene. Next-generation sequencing data revealed distinct marker genes regulated by the TBPL1 gene and their potential involvement in cell migration, proliferation, anti-apoptosis, and metastasis. Additionally, we also discovered novel lncRNAs implicated in the transcriptome analysis of the TBPL1 knocked-out gene. Our investigation indicated that this gene might affect varied stages of breast cancer cell lines' cellular properties, such as cell duplication, morphology, and growth. It might also contribute to tumor formation in more aggressive cell lines like MDA-MB-231 in vivo.},
}

Humans
*Breast Neoplasms/genetics/pathology
*CRISPR-Cas Systems
Female
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
*TATA-Box Binding Protein/genetics/metabolism
*Mutation
RNA-Seq
Transcriptome
Cell Proliferation/genetics
Gene Expression Profiling
Cell Movement/genetics

@article {pmid41152217,
year = {2025},
author = {Song, Z and Guo, J and Fan, Z and Huang, S and Li, G and Zhao, Z and Chen, B and Huang, S and Zheng, W and Wei, Y and Chen, Y and Huang, X and Liu, J and Wu, L and Wang, X},
title = {Noncanonical target-strand cytosine base editing via engineered Un1Cas12f1 platform.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9499},
pmid = {41152217},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; Animals ; *CRISPR-Cas Systems/genetics ; *Cytosine/metabolism ; Mice ; Male ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics ; Protein Engineering ; },
abstract = {CRISPR/Cas-derived base editors harness various deaminase or glycosylase activities to target bases within non-target strand (NTS) of the R-loop, catalyzing base conversions independent of double-strand break formation. To develop miniature BEs compatible with therapeutic viral vectors, we explore the compact Cas12f system. Through computational modeling and mutagenesis, we establish a highly active enUn1Cas12f1 protein, and subsequently construct the derivative cytosine BE (CBE). Remarkably, the engineered CBE exhibits an unexpected activity to also edit the target strand (TS), indicating its substantially expanded editable space. We refine this activity via a focused alanine scan, establishing a nickase-CBE that preferentially install TS edits (TSminiCBE). Further engineering with a non-specific DNA binding domain yields an optimized TS-editing BE that enables in vivo base edits in mice (male). Overall, through extensive engineering of the Cas12f platform and repurposing its intrinsic dynamics, our work establishes a strand-selectable miniature CBE toolkit with strong potential for diverse applications.},
}

*Gene Editing/methods
Animals
*CRISPR-Cas Systems/genetics
*Cytosine/metabolism
Mice
Male
Humans
*CRISPR-Associated Proteins/metabolism/genetics
Protein Engineering

@article {pmid41151616,
year = {2025},
author = {Terhalle, E and Rademacher, J},
title = {[Nontuberculous Mycobacteria: Diagnostic Challenges and Individualized Therapeutic Approaches].},
journal = {Deutsche medizinische Wochenschrift (1946)},
volume = {150},
number = {22},
pages = {1360-1366},
doi = {10.1055/a-2502-6525},
pmid = {41151616},
issn = {1439-4413},
mesh = {Humans ; *Mycobacterium Infections, Nontuberculous/diagnosis/drug therapy/epidemiology/therapy ; *Nontuberculous Mycobacteria/isolation & purification ; Anti-Bacterial Agents/therapeutic use ; Precision Medicine ; },
abstract = {Non-tuberculous mycobacteria (NTM) are increasingly recognized as clinically relevant pathogens, particularly in countries with a low tuberculosis incidence. Recent data from Denmark demonstrate a continuous annual rise in NTM-related pulmonary disease (NTM-PD) of 4.6% over 3 decades, with more than half of the isolates associated with true disease. Structural lung diseases such as bronchiectasis, prior tuberculosis, and chronic pulmonary conditions are major risk factors, alongside immunodeficiencies and immunosuppressive therapies. The diagnosis of NTM-PD requires a combination of clinical symptoms, radiological findings, and the microbiological confirmation. Novel diagnostic tools, such as anti-GPL IgA serology and a CRISPR-Cas-based cfDNA assay, show promise for differentiating colonization from disease and monitoring treatment response, but the sputum culture remains essential for species identification and drug susceptibility testing. Treatment is complex and species-specific, with macrolides forming the backbone of most regimens. Refractory cases, particularly those involving Mycobacterium abscessus, pose therapeutic challenges and often require multidisciplinary management. Inhaled liposomal amikacin (ALIS) has shown benefit in refractory MAC disease. Clinical decision-making must balance efficacy, tolerability, and long-term adherence, highlighting the need for individualized treatment strategies and regular monitoring. This review outlines current evidence and practical recommendations for clinicians managing NTM-PD.},
}

Humans
*Mycobacterium Infections, Nontuberculous/diagnosis/drug therapy/epidemiology/therapy
*Nontuberculous Mycobacteria/isolation & purification
Anti-Bacterial Agents/therapeutic use
Precision Medicine

@article {pmid41149352,
year = {2025},
author = {Qin, Y and Xie, J and Zhen, S},
title = {CRISPR-Cas12a and DNA Tetrahedron Assemblies Amplified Fluorescence Anisotropy for the Sensitive Detection of Hepatitis B Virus DNA.},
journal = {Biosensors},
volume = {15},
number = {10},
pages = {},
pmid = {41149352},
issn = {2079-6374},
support = {22322409//National Natural Science Foundation of China/ ; 21974109//National Natural Science Foundation of China/ ; SWU-KF25011//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Hepatitis B virus/genetics ; *DNA, Viral/analysis ; *Fluorescence Polarization/methods ; Humans ; *CRISPR-Cas Systems ; *Biosensing Techniques ; Limit of Detection ; Hepatitis B ; },
abstract = {Fluorescence anisotropy (FA) has been widely used for analyzing biomolecules due to its high throughput, homogeneous detection, and strong resistance to photobleaching. However, the traditional FA method suffers from low sensitivity when the target molecules are small and rotate rapidly, often producing insignificant changes in the FA value. In this study, by combining double signal amplification through the trans-cleavage of CRISPR-Cas12a and DNA tetrahedron assemblies with a large molecular size, a new, fast, simple and highly sensitive FA method was constructed to achieve the quantitative detection of hepatitis B virus DNA (HBV-DNA). The experimental results showed that the linear range of this method was 0.5-9 nmol/L, and the detection limit (LOD = 3σ/k) was 48 pmol/L. In addition, the method demonstrated excellent selectivity and anti-interference, and it was successfully applied to detect HBV-DNA in human serum, indicating that this method has the potential for clinical diagnosis.},
}

*Hepatitis B virus/genetics
*DNA, Viral/analysis
*Fluorescence Polarization/methods
Humans
*CRISPR-Cas Systems
*Biosensing Techniques
Limit of Detection
Hepatitis B

@article {pmid41149312,
year = {2025},
author = {Zhou, Z and Cho, IH and Kadam, US},
title = {CRISPR-Cas-Based Diagnostics in Biomedicine: Principles, Applications, and Future Trajectories.},
journal = {Biosensors},
volume = {15},
number = {10},
pages = {},
pmid = {41149312},
issn = {2079-6374},
mesh = {*CRISPR-Cas Systems ; Humans ; *Biosensing Techniques ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) systems, originally identified as prokaryotic adaptive immune mechanisms, have rapidly evolved into powerful tools for molecular diagnostics. Leveraging their precise nucleic acid targeting capabilities, CRISPR diagnostics offer rapid, sensitive, and specific detection solutions for a wide array of targets. This review delves into the fundamental principles of various Cas proteins (e.g., Cas9, Cas12a, Cas13a) and their distinct mechanisms of action (cis- and trans-cleavage). It highlights the diverse applications spanning infectious disease surveillance, cancer biomarker detection, and genetic disorder screening, emphasizing key advantages such as speed, high sensitivity, specificity, portability, and cost-effectiveness, particularly for point-of-care (POC) testing in resource-limited settings. The report also addresses current challenges, including sensitivity limitations without pre-amplification, specificity issues, and complex sample preparation, while exploring promising future trajectories like the integration of artificial intelligence (AI) and the development of universal diagnostic platforms to enhance clinical translation.},
}

*CRISPR-Cas Systems
Humans
*Biosensing Techniques

@article {pmid41149303,
year = {2025},
author = {Zhu, Z and Ma, H and Yao, H and Yuan, Y and Miao, X and Su, S},
title = {CRISPR-Enhanced Colorimetric Aptasensor for Adenosine Triphosphate Detection Based on MoS2-Based Nanozymes.},
journal = {Biosensors},
volume = {15},
number = {10},
pages = {},
pmid = {41149303},
issn = {2079-6374},
support = {SYW2025037//the Science and Technology Program of Suzhou/ ; 2024GRFX045//the Visiting Scholar Project for Higher Vocational Colleges in Jiangsu Province/ ; TC2024JCYL23//the Science and Technology Program of Taicang/ ; 2023JXKYTD01//the Innovation Team Funds of Suzhou Chien-shiung Institute of Technology/ ; },
mesh = {Colorimetry/methods ; *Adenosine Triphosphate/analysis ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide ; Molybdenum/chemistry ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Disulfides/chemistry ; CRISPR-Cas Systems ; },
abstract = {As the direct energy source in organisms, accurate and simple detection of adenosine triphosphate (ATP) is of great significance. Herein, a colorimetric aptasensor for ATP determination was designed by integrating the CRISPR/Cas12a system with an aptamer, and with Prussian blue nanocube and gold nanoparticle co-functionalized MoS2 (MoS2-PBNCs-AuNPs) nanozymes. As expected, the introduced CRISPR/Cas12a system and aptamer could efficiently amplify the detection signal and improve the specific recognition ability, respectively. Meanwhile, the catalytic activity of the MoS2-PBNCs-AuNPs nanozymes can be regulated with the concentration of ATP. The high-affinity binding of ATP to the aptamer competitively inhibited aptamer-crRNA hybridization, causing fewer Cas12 proteins to be activated. As a result, the uncleaved single-stranded DNA (ssDNA) adsorbed onto the surface of nanozymes to effectively enhance their catalytic oxidation capability toward 3,3',5,5'-tetramethylbenzidine (TMB). According to this phenomenon, this CRISPR-enhanced colorimetric aptasensor can detect down to 0.14 μM ATP with high selectivity, reproducibility, and stability. In addition, acceptable recoveries and low relative standard deviations of the aptasensor for ATP determination suggest that it is promising for application in early detection of clinical-related diseases.},
}

Colorimetry/methods
*Adenosine Triphosphate/analysis
*Biosensing Techniques/methods
*Aptamers, Nucleotide
Molybdenum/chemistry
Gold/chemistry
Metal Nanoparticles/chemistry
Disulfides/chemistry
CRISPR-Cas Systems

@article {pmid41148810,
year = {2025},
author = {Liu, H and Zhang, P},
title = {Advances in β-Thalassemia Gene Therapy: CRISPR/Cas Systems and Delivery Innovations.},
journal = {Cells},
volume = {14},
number = {20},
pages = {},
pmid = {41148810},
issn = {2073-4409},
mesh = {*beta-Thalassemia/therapy/genetics ; Humans ; *Genetic Therapy/methods ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Animals ; *Gene Transfer Techniques ; beta-Globins/genetics ; },
abstract = {β-thalassemia is an inherited blood disorder caused by mutations in the β-globin (HBB) gene, leading to reduced or absent β-globin production, resulting in chronic anemia. While current therapies, including blood transfusions and hematopoietic stem cell transplantation, offer symptomatic relief, they are limited by complications and their limited accessibility. CRISPR-based gene editing technologies provide new therapeutic avenues by enabling the precise correction of HBB mutations or the reactivation of fetal hemoglobin (HbF) through the targeting of regulatory elements such as BCL11A. These approaches have shown promising preclinical and clinical outcomes. However, efficient and safe delivery remains a major challenge. Viral vectors offer high efficiency but raise concerns about immunogenicity and insertional mutagenesis, whereas non-viral systems such as lipid nanoparticles and engineered exosomes offer lower toxicity and modularity but face targeting limitations. This review highlights recent progress in CRISPR-based therapies for β-thalassemia and emerging delivery strategies to enhance clinical translation.},
}

*beta-Thalassemia/therapy/genetics
Humans
*Genetic Therapy/methods
*CRISPR-Cas Systems/genetics
Gene Editing/methods
Animals
*Gene Transfer Techniques
beta-Globins/genetics

@article {pmid41134871,
year = {2025},
author = {Charlier, J and Sherkatghanad, Z and Makarenkov, V},
title = {Similarity-based transfer learning with deep learning networks for accurate CRISPR-Cas9 off-target prediction.},
journal = {PLoS computational biology},
volume = {21},
number = {10},
pages = {e1013606},
doi = {10.1371/journal.pcbi.1013606},
pmid = {41134871},
issn = {1553-7358},
mesh = {*Deep Learning ; *CRISPR-Cas Systems/genetics ; Neural Networks, Computer ; Computational Biology/methods ; *Gene Editing/methods ; Humans ; Machine Learning ; },
abstract = {Transfer learning has emerged as a powerful tool for enhancing predictive accuracy in complex tasks, particularly in scenarios where data is limited or imbalanced. This study explores the use of similarity-based pre-evaluation as a methodology to identify optimal source datasets for transfer learning, addressing the dual challenge of efficient source-target dataset pairing and off-target prediction in CRISPR-Cas9, while existing transfer learning applications in the field of gene editing often lack a principled method for source dataset selection. We use cosine, Euclidean, and Manhattan distances to evaluate similarity between the source and target datasets used in our transfer learning experiments. Four deep learning network architectures, i.e. Multilayer Perceptron (MLP), Convolutional Neural Networks (CNNs), Feedforward Neural Networks (FNNs), and Recurrent Neural Networks (RNNs), and two traditional machine learning models, i.e. Logistic Regression (LR) and Random Forest (RF), were tested and compared in our simulations. The results suggest that similarity scores are reliable indicators for pre-selecting source datasets in CRISPR-Cas9 transfer learning experiments, with cosine distance proving to be a more effective dataset comparison metric than either Euclidean or Manhattan distances. An RNN-GRU, a 5-layer FNN, and two MLP variants provided the best overall prediction results in our simulations. By integrating similarity-based source pre-selection with machine learning outcomes, we propose a dual-layered framework that not only streamlines the transfer learning process but also significantly improves off-target prediction accuracy. The code and data used in this study are freely available at: https://github.com/dagrate/transferlearning_offtargets.},
}

*Deep Learning
*CRISPR-Cas Systems/genetics
Neural Networks, Computer
Computational Biology/methods
*Gene Editing/methods
Humans
Machine Learning

@article {pmid41117290,
year = {2025},
author = {Ye, T and Xue, M and Xu, Y and Fan, M and Yuan, M and Yu, J and Cao, H and Hao, L and Wu, X and Yin, F and Xu, F},
title = {Inverted Tetrahedral DNA Reporters Enable Label-Free Ratiometric CRISPR Electrochemical Aptasensing of Kanamycin.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {43},
pages = {27694-27702},
doi = {10.1021/acs.jafc.5c06928},
pmid = {41117290},
issn = {1520-5118},
mesh = {*Electrochemical Techniques/methods/instrumentation ; *Kanamycin/analysis ; *Biosensing Techniques/methods/instrumentation ; *DNA/chemistry/genetics ; Milk/chemistry ; CRISPR-Cas Systems ; *Aptamers, Nucleotide/chemistry/genetics ; Animals ; *Anti-Bacterial Agents/analysis ; Limit of Detection ; Food Contamination/analysis ; },
abstract = {Integrating CRISPR technology with electrochemical sensing has promising potential in point-of-care testing applications. However, inappropriate immobilization of the reporter on the heterogeneous surface leads to a poor trans-cleavage efficiency. Additionally, the accuracy and reliability of electrochemical sensing still face challenges. Herein, an inverted tetrahedral DNA reporter was developed for electrochemical CRISPR aptasensing. Thiol-modified single-strand oligonucleotides were self-assembled on the edge of tetrahedral DNA nanostructures (TDNs) as a scaffold, enabling an inverted immobilization of DNA tetrahedra via the Au-S bond. The trans-cleavage activity of CRISPR/Cas12a on the single-stranded oligonucleotides resulted in the TDNs dissociating from the electrode surface. The recovery of electron transfer of potassium ferricyanide on the electrode enhances the electrochemical response, while the signal of adsorbed methylene blue on the skeleton of TDNs decreases, enabling a ratiometric signal output. As a proof of concept, the proposed inverted tetrahedral DNA reporters were employed to develop a label-free ratiometric electrochemical aptasensing method for kanamycin detection. Under the optimal conditions, as low as 0.35 pM kanamycin was detected in 50 min with a 4 orders of magnitude dynamic range from 1 pM to 10 nM. Furthermore, the practical application ability of the proposed method for kanamycin detection in a spiked milk sample was also demonstrated. This work offers a new perspective for electrochemical CRISPR sensing development.},
}

*Electrochemical Techniques/methods/instrumentation
*Kanamycin/analysis
*Biosensing Techniques/methods/instrumentation
*DNA/chemistry/genetics
Milk/chemistry
CRISPR-Cas Systems
*Aptamers, Nucleotide/chemistry/genetics
Animals
*Anti-Bacterial Agents/analysis
Limit of Detection
Food Contamination/analysis

@article {pmid41052905,
year = {2025},
author = {Sun, Y and Hong, Z and Wang, W and Zhang, H and Ren, X and He, X and Kan, T and Fan, Y and Wang, C and Cao, Y and Zhang, H},
title = {Establishment of an efficient and versatile genome editing platform for L. ruthenicum.},
journal = {Plant physiology},
volume = {199},
number = {2},
pages = {},
doi = {10.1093/plphys/kiaf486},
pmid = {41052905},
issn = {1532-2548},
support = {2022BBF01001-03//Key Research & Development Program of Ningxia Hui Autonomous Region/ ; 32170547//National Natural Science Foundation of China/ ; 2024AAC03123//Natural Science Foundation of Ningxia Hui Autonomous Region/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Genome, Plant ; Promoter Regions, Genetic ; },
abstract = {Black goji berry (Lycium ruthenicum Murr.) is a valuable functional food and traditional medicinal plant owing to its rich content of anthocyanins, trace minerals, vitamins, and polysaccharides. However, limited genetic manipulation tools have hindered functional genomic studies and trait improvement in this species. In this study, we optimized the genetic transformation system for L. ruthenicum, achieving a remarkably high transformation efficiency of 95.4%. Based on this system, we developed a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene knockout approach, using the Arabidopsis U6 (AtU6) promoter to drive sgRNA expression and the cauliflower mosaic virus 35S (35S) promoter to drive Cas9 expression, achieving editing efficiencies of 68.8% at the phytoene desaturase (LrPDS) locus and 96.0% at the betaine aldehyde dehydrogenase (LrBADH2) locus. Furthermore, we established an adenine base editing (ABE) system using the ribosomal protein subunit 5A (RPS5A) promoter to drive tRNA adenine deaminase-8e (TadA-8e-nSpCas9) cassette expression, achieving an editing efficiency of 72.2% at the LrPDS locus. To broaden protospacer adjacent motif (PAM) compatibility, we introduced the PAM-relaxed variant SpRY, enabling successful A-to-G editing at an acetolactate synthase (LrALS) target site containing a noncanonical NAN PAM, with an efficiency of 5.3%. Additionally, we developed a multiplex ABE system based on the tRNA-processing strategy, which enabled simultaneous editing at 2 independent loci with an efficiency of 33.3%. Our study establishes a robust genome editing toolkit for L. ruthenicum, offering valuable tools for functional gene analysis and molecular breeding in this economically important species.},
}

*Gene Editing/methods
CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
*Genome, Plant
Promoter Regions, Genetic

@article {pmid39565542,
year = {2025},
author = {Zhou, L and Li, X and Ji, Z and Zhou, C and Yang, L and Li, Y and Fu, C and Gu, L and Zhang, S and Gao, J and Yue, P and Yu, H},
title = {Generation of Ext1 Gene-Edited Mice Model Via Dual sgRNAs/Cas9 System and Phenotypic Analyses.},
journal = {Molecular biotechnology},
volume = {67},
number = {11},
pages = {4233-4246},
pmid = {39565542},
issn = {1559-0305},
support = {32160147//National Natural Science Foundation of China/ ; YCSW2020229//Youth and Middle-aged Scientific and Technological Innovation Leading Talents Program of the Corps/ ; 2020GXNSFAA297097//Natural Science Foundation of Guangxi Zhuang Autonomous Region/ ; },
mesh = {Animals ; *N-Acetylglucosaminyltransferases/genetics/metabolism ; Exostosin 1 ; Mice ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Disease Models, Animal ; Phenotype ; Male ; Female ; *Exostoses, Multiple Hereditary/genetics/pathology ; Frameshift Mutation ; Exons ; Humans ; },
abstract = {Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disease. Genetic linkage analyses have identified that mutations in the exostosin glycosyltransferase (EXT)1 and EXT2 genes are linked to HME pathogenesis, with EXT1 mutation being the most frequent. The aim of this study was to generate a mice model with Ext1 gene editing to simulate human EXT1 mutation and investigate the genetic pathogenicity of Ext1 through phenotypic analyses. We designed a pair of dual sgRNAs targeting exon 1 of the mice Ext1 gene for precise deletion of a 46 bp DNA fragment, resulting in frameshift mutation of the Ext1 gene. The designed dual sgRNAs and Cas9 proteins were injected into mice zygotes cytoplasm. A total of 14 mice were obtained via embryo transfer, among which two genotypic chimera mice had a deletion of the 46 bp DNA fragment in exon 1 of the Ext1 gene. By hybridization and breeding, we successfully generated heterozygous mice with edited Ext1 gene (Ext[+/-]). Off-target effect analysis did not reveal off-target mutations in Ext[+/-] mice caused by the two sgRNAs used. Compared to wild-type mice, Ext[+/-] mice exhibited lower body weights. X-ray imaging showed hyperplastic bone near caudal vertebrae only in male Ext[+/-] mice, with computed tomography values approximately at 200 HU for hyperplastic bone between ribs and spine regions. Furthermore, immunohistochemical analysis revealed fewer articular chondrocytes expressing EXT1 in edited mice compared to wild-type ones. Pathological section analysis demonstrated no structural or morphological abnormalities in heart, liver, lung, or kidney tissues from Ext[+/-] mice. In conclusion, we successfully generated an accurate DNA deletion model for studying Ext1 using dual sgRNAs/Cas9 systems. In conclusion, we successfully generated precise DNA deletions in the Ext1 mice model using the dual sgRNAs/Cas9 system. In conclusion, we observed significant phenotypic changes in Ext[+/-] mice, particularly bone hyperplasia in male individuals; however, no exostosis was detected in the gene-edited mice. The introduction of a frameshift mutation into the Ext1 gene through CRISPR/Cas9 technology resulted in novel phenotypic alterations, highlighting the genetic pathogenicity of Ext1. Therefore, our Ext[+/-] mice serve as a valuable model for further biomedical investigations related to the Ext1 gene.},
}

Animals
*N-Acetylglucosaminyltransferases/genetics/metabolism
Exostosin 1
Mice
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Disease Models, Animal
Phenotype
Male
Female
*Exostoses, Multiple Hereditary/genetics/pathology
Frameshift Mutation
Exons
Humans

@article {pmid41148687,
year = {2025},
author = {Nass, NM and Zaher, KA},
title = {Beyond the Resistome: Molecular Insights, Emerging Therapies, and Environmental Drivers of Antibiotic Resistance.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {14},
number = {10},
pages = {},
doi = {10.3390/antibiotics14100995},
pmid = {41148687},
issn = {2079-6382},
abstract = {Antibiotic resistance remains one of the most formidable challenges to modern medicine, threatening to outpace therapeutic innovation and undermine decades of clinical progress. While resistance was once viewed narrowly as a clinical phenomenon, it is now understood as the outcome of complex ecological and molecular interactions that span soil, water, agriculture, animals, and humans. Environmental reservoirs act as silent incubators of resistance genes, with horizontal gene transfer and stress-induced mutagenesis fueling their evolution and dissemination. At the molecular level, advances in genomics, structural biology, and systems microbiology have revealed intricate networks involving plasmid-mediated resistance, efflux pump regulation, integron dynamics, and CRISPR-Cas interactions, providing new insights into the adaptability of pathogens. Simultaneously, the environmental dimensions of resistance, from wastewater treatment plants and aquaculture to airborne dissemination, highlight the urgency of adopting a One Health framework. Yet, alongside this growing threat, novel therapeutic avenues are emerging. Innovative β-lactamase inhibitors, bacteriophage-based therapies, engineered lysins, antimicrobial peptides, and CRISPR-driven antimicrobials are redefining what constitutes an "antibiotic" in the twenty-first century. Furthermore, artificial intelligence and machine learning now accelerate drug discovery and resistance prediction, raising the possibility of precision-guided antimicrobial stewardship. This review synthesizes molecular insights, environmental drivers, and therapeutic innovations to present a comprehensive landscape of antibiotic resistance. By bridging ecological microbiology, molecular biology, and translational medicine, it outlines a roadmap for surveillance, prevention, and drug development while emphasizing the need for integrative policies to safeguard global health.},
}

@article {pmid41148514,
year = {2025},
author = {Tripathi, A and Vishwakarma, K and Tripathi, S and Jadaun, JS and Nayak, AK},
title = {Utilization of MADS-Box genes for agricultural advancement: current insights and future prospects.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {20},
pmid = {41148514},
issn = {1573-4978},
mesh = {*MADS Domain Proteins/genetics/metabolism ; Gene Expression Regulation, Plant/genetics ; Plant Proteins/genetics/metabolism ; *Crops, Agricultural/genetics ; CRISPR-Cas Systems ; Oryza/genetics ; *Agriculture/methods ; Solanum lycopersicum/genetics ; Phylogeny ; Plants, Genetically Modified/genetics ; Gene Editing/methods ; Genes, Plant ; },
abstract = {MADS-box genes constitute a highly conserved family of transcription factors integral to the regulation of a diverse array of plant developmental processes, encompassing floral organ specification, fruit maturation, root architecture and adaptation to abiotic stresses. These transcription factors encode proteins containing the distinctive MADS (MCM1, AGAMOUS, DEFICIENS, and SRF) domain, which mediates DNA binding and orchestrates interaction with co-regulators, thereby enabling the precise transcriptional control of developmental gene networks. Functional characterization through transgenic approaches including overexpression, knockdown, and CRISPR/Cas9-based mutagenesis-has revealed the capacity of MADS-box gene manipulation to modulate key agronomic traits, such as yield potential, as well as resilience to salinity, drought, and temperature fluctuations. In rice, targeted editing of OsMADS18 using CRISPR/Cas9 generated a substantial quantitative variation in tiller and panicle number, demonstrating the direct contribution of MADS-box gene function to biomass and yield performance. Similarly, CRISPR/Cas9-mediated disruption of the RIPENING INHIBITOR (RIN) gene in tomato (Solanum lycopersicum) underscored its central role in regulating fruit ripening, linking MADS-box gene activity to postharvest quality and development. Phylogenomic studies reveal strong conservation of MADS-box gene lineages in monocot grasses, as evidenced by clustered short internal branches, whereas eudicots, particularly Solanaceae present well-differentiated subclades, reflecting lineage-specific diversification events. Notably, network analysis highlight the high connectivity and central regulatory position of many MADS-box proteins, underlining their roles as master integrators of developmental and environmental signalling involved in both floral and vegetative transitions. A mechanistic understanding of these regulatory circuits offers translational opportunities to engineer crops with improved performance and resilience, reinforcing the pivotal role of MADS-box genes in crop improvement.},
}

*MADS Domain Proteins/genetics/metabolism
Gene Expression Regulation, Plant/genetics
Plant Proteins/genetics/metabolism
*Crops, Agricultural/genetics
CRISPR-Cas Systems
Oryza/genetics
*Agriculture/methods
Solanum lycopersicum/genetics
Phylogeny
Plants, Genetically Modified/genetics
Gene Editing/methods
Genes, Plant

@article {pmid41148377,
year = {2025},
author = {Baars, J and Kurm, V and Scholten, B and Griekspoor, Y and Lavrijssen, B and Steens, JA and Smulders, MJM and van Peer, A},
title = {On site discrimination between two closely related commercial strains of oyster mushroom using a loop-mediated isothermal amplification (LAMP) test.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {17},
pmid = {41148377},
issn = {1573-4978},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Pleurotus/genetics/classification ; DNA, Fungal/genetics ; Molecular Diagnostic Techniques ; },
abstract = {BACKGROUND: Protection of the intellectual property (IP) rights on new crop varieties is important as it allows the breeding company or entity that produced the variety to earn back (part of) the investment. Infringement on the IP rights of mushroom varieties is not uncommon. In order to combat infringement of the IP rights on two strains of Oyster mushroom (SPOPPO and ALLERPO) it is important to be able to readily recognize and discriminate the two strains in commercial practice. This article describes the development of tools for the on-site identification of two closely related sporeless strains of Oyster mushroom.

METHODS AND RESULTS: To develop a reliable method of discriminating between SPOPPO and ALLERPO, we used either the LAMP technique or a modification of that technique. It allows for fast (within 30 min) identification of the commercially used strains SPOPPO and ALLERPO with high specificity. Fast on-site answers on strain identity can be important when experiencing unexpected strain behavior or when strains are of suspect origin. Both strains are discriminated from sporulating strains by a LAMP reaction on the intact version of the msh4 gene; sporeless strains contain a msh4 gene with a large insert that renders the associated protein inactive.

CONCLUSIONS: SPOPPO and ALLERPO are distinguished from each other and other commercially used Pleurotus ostreatus strains by LAMP reactions that target genomic regions with strain specific recombinations. To our knowledge, this is the first time LAMP reactions have been developed to discriminate between Pleurotus ostreatus strains.},
}

*Nucleic Acid Amplification Techniques/methods
*Pleurotus/genetics/classification
DNA, Fungal/genetics
Molecular Diagnostic Techniques

@article {pmid41145060,
year = {2025},
author = {Monteiro Belo Dos Santos, S and Van Tricht, C and Lammertyn, J and Spasic, D},
title = {Zoonotic disease detection at the point-of-care: the best of RPA and CRISPR-Cas.},
journal = {Biosensors & bioelectronics},
volume = {293},
number = {},
pages = {118139},
doi = {10.1016/j.bios.2025.118139},
pmid = {41145060},
issn = {1873-4235},
abstract = {Biosensors are increasingly crucial in detecting biomarkers for emerging zoonotic diseases at the point-of-care (POC). This imminence was recently highlighted by the deficient response during the SARS-CoV-2 pandemic. While polymerase chain reaction (PCR) is the common nucleic acid (NA) testing method for zoonotic diseases in laboratory settings, it is impractical for the POC settings due to the equipment-related cost, lack of portability and user-friendliness. Recent advances in NA amplification introduced isothermal methods, such as recombinase polymerase amplification (RPA), which is known for its low temperature (37-42 °C), short incubation time (5-20 min) and suitability for integration in miniaturized, portable, low-cost, highly sensitive diagnostic platforms. However, RPA susceptibility to false positive results steered to its combination with CRISPR-Cas12/13, leading to the rise of SHERLOCK and DETECTR. This review first explores RPA-CRISPR-Cas bioassay development as either two- or one-step. This is followed by a discussion on the integration of canonical RPA, or its combination with CRISPR-Cas, into different diagnostic platforms towards NA amplification at the POC (e.g., mobile laboratories, centrifugal, or pump-free platforms). Finally, the advantages, limitations, and outlook for POC-based diagnostics of zoonotic diseases with RPA(-CRISPR-Cas) are discussed, highlighting the need for innovative technologies to address global health challenges. While promising, many of these approaches still require further research to achieve streamlined, single-step reactions and seamless integration into diagnostic platforms. Moreover, despite two decades of RPA(-CRISPR-Cas) development, technology readiness is limited, still missing validated platforms, integrated sample preparation, and AI-powered results analysis enabling real time epidemiological monitoring.},
}

@article {pmid41144208,
year = {2026},
author = {Shaw, S and Sateriale, A and Pawlowic, MC and Vinayak, S and Brooks, CF and Byerly, JH and Striepen, B},
title = {Genetic Manipulation of Cryptosporidium parvum.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2978},
number = {},
pages = {173-185},
pmid = {41144208},
issn = {1940-6029},
mesh = {*Cryptosporidium parvum/genetics ; Animals ; Mice ; CRISPR-Cas Systems ; *Cryptosporidiosis/parasitology ; Gene Editing/methods ; Transfection/methods ; Humans ; },
abstract = {Cryptosporidium parvum can be reliably genetically manipulated using CRISPR/Cas9 driven homologous repair coupled to in vivo propagation in immunodeficient mice. This chapter will guide through foundational procedures for excystation, transfection, infection, collection, and purification of transgenic Cryptosporidium parvum. The genetic tools for Cryptosporidium research were expanded significantly over the last 5 years. While we cannot cover all advances in detail, we will highlight novel selection markers, conditional mutagenesis strategies, and genetic crosses.},
}

*Cryptosporidium parvum/genetics
Animals
Mice
CRISPR-Cas Systems
*Cryptosporidiosis/parasitology
Gene Editing/methods
Transfection/methods
Humans

@article {pmid41141466,
year = {2025},
author = {Mohiuddin, M},
title = {Monitoring and Assessment of Circulating Tumor DNA in Cancers Using Ultrarapid Sensitivity as an Innovative Practice.},
journal = {Health science reports},
volume = {8},
number = {10},
pages = {e71409},
pmid = {41141466},
issn = {2398-8835},
abstract = {BACKGROUND: Liquid biopsy with circulating tumor DNA (ctDNA) has rapidly emerged as a new paradigm for assessing tumor burden, genetic heterogeneity, and therapeutic response in a real-time, noninvasive manner. However, ctDNA is often visually low (sometimes < 0.1% of the total circulating cell-free DNA), creating a significant challenge for reliable detection (especially for early-stage disease and minimal residual disease).

DISCUSSION: New technologies for structural variant (SV)-based ctDNA assays, nanomaterial-based electrochemical sensors, magnetic nano-electrode platforms, and fragment-enriched library preparation have improved sensitivity to attomolar concentrations and less in some populations. In some cancers, ctDNA may provide early evidence of recurrence (i.e., > 1 year) before being clinically evident using traditional metrics. These technologies allow for unprecedented opportunities and sensitivity for early detection, monitoring of treatment response, and early detection of molecular recurrence. Nevertheless, a barrier remains for widespread clinical application owing to pre-analytical technique variability, analytical platform variability, cost, and the necessity of large-scale, prospective trials.

CONCLUSION: This study will analyze new innovative technology-based ultrasensitive ctDNA assay detection and future research and clinical applications for breast, colorectal, lung, lymphoid, and gastroesophageal cancers, and studies assessing ctDNA for monitoring treatment. Prospects for ctDNA detection utilizing multiplexed CRISPR-Cas ctDNA assays, microfluidic point-of-care (POC) devices, and AI-based error suppression methods may be the next horizon for ctDNA liquid biopsy technology.},
}

@article {pmid41141388,
year = {2026},
author = {Wang, T and Yu, M and Liu, P and Song, Z and Li, C and Yang, J and Liu, N},
title = {In vivo gene therapy: A strategy for mutations, degenerations, and tumors.},
journal = {Genes & diseases},
volume = {13},
number = {1},
pages = {101808},
pmid = {41141388},
issn = {2352-3042},
abstract = {Gene mutations, organ function degeneration, and carcinogenesis are the primary threats to human health. Gene therapy, which involves the addition, deletion, regulation, and editing of genes, as well as the development of genetic vaccines, can potentially cure genetic mutation disorders, degenerative diseases, and cancers. Ex vivo gene therapy has recently been used to treat monogenetic mutation diseases of the hematopoietic system and cancers. However, in vivo gene therapy remains inapplicable. The primary elements of in vivo gene therapy include deoxyribonucleic acid (DNA) nucleases (e.g., zinc finger nucleases, transcription activator-like effector nucleases), CRISPR-Cas system, base editors, prime editors, and delivery vectors (e.g., viral and non-viral vehicles). According to the development of DNA nucleases and delivery vectors, in vivo gene therapy can be made available for future clinical use. The current review summarizes the development of DNA nucleases and delivery vectors for in vivo gene therapy, emphasizing recent progress.},
}

@article {pmid41139369,
year = {2025},
author = {Wei, H and Li, D and Xie, K and Lou, S and Dong, G and Guo, F and Lian, G and Pan, X and Zeng, Z and Han, N and Gao, Z and Bian, H},
title = {Creation of new rice germplasm with cross-resistance to auxin herbicides picloram and dicamba by genome editing of OsAFB4.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {138},
number = {11},
pages = {282},
pmid = {41139369},
issn = {1432-2242},
support = {LGN21C130006//Natural Science Foundation of Zhejiang Province/ ; 32201834//Innovative Research Group Project of the National Natural Science Foundation of China/ ; },
mesh = {*Oryza/genetics/drug effects/growth & development ; *Herbicide Resistance/genetics ; *Gene Editing ; *Picloram/pharmacology ; *Herbicides/pharmacology ; *Dicamba/pharmacology ; Indoleacetic Acids ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Mutation ; Plant Breeding ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; },
abstract = {Genome Editing of OsAFB4 in rice cultivar Jiahe212 confers cross-resistance to synthetic auxin herbicides picloram and dicamba without reducing grain yield, providing a novel germplasm for herbicide-resistant rice breeding. Synthetic auxin herbicides (SAHs) are widely employed in global agriculture owing to low toxicity and high efficiency in weed management. Mutations in OsAFB4, an AUXIN SIGNALING F-BOX gene, led to specific resistance to SAH picloram in rice. However, potential application of OsAFB4 modification for resistance to other SAHs and its effect on agronomic traits in rice remain uncharacterized. In this study, we employed CRISPR/Cas9-mediated genome editing to generate OsAFB4 mutations in the elite commercial rice cultivar Jiahe212 (JH212). Hydroponic-culture experiments showed that Osafb4 mutant lines exhibited significant resistance to both SAHs: picloram and dicamba. Promoter activity assays using a pOsAFB4:GUS reporter line revealed that SAH treatments induced significant upregulation of GUS expression specifically in shoot apices. Under picloram or dicamba treatment, expressions of OsIAA1, OsIAA3, OsIAA9 and OsIAA20 were significantly upregulated in wild-type plants, while remarkably suppressed in Osafb4 mutants, revealing a critical role for OsAFB4 in regulating auxin-responsive IAA genes during OsAFB4-mediated sensitivity to SAH. Selected for field trials, the Cas9-free Osafb4-13 mutant line exhibited robust resistance to both picloram and dicamba without reducing grain yield compared to untreated controls. This study provides a new cross-herbicide-resistant rice germplasm without reducing grain yield by targeted editing of OsAFB4.},
}

*Oryza/genetics/drug effects/growth & development
*Herbicide Resistance/genetics
*Gene Editing
*Picloram/pharmacology
*Herbicides/pharmacology
*Dicamba/pharmacology
Indoleacetic Acids
*Plant Proteins/genetics/metabolism
CRISPR-Cas Systems
Mutation
Plant Breeding
Gene Expression Regulation, Plant
Plants, Genetically Modified

@article {pmid41104823,
year = {2025},
author = {Li, X and Ma, J and Luo, Y and Zhu, D and Wang, L and Su, S},
title = {Enhancing Performance in Electrochemical Early Diagnosis of African Swine Fever Based on CRISPR-Responsive DNA Nanoflowers.},
journal = {Analytical chemistry},
volume = {97},
number = {42},
pages = {23401-23408},
doi = {10.1021/acs.analchem.5c04188},
pmid = {41104823},
issn = {1520-6882},
mesh = {*African Swine Fever Virus/genetics/isolation & purification ; *African Swine Fever/diagnosis ; Animals ; *Electrochemical Techniques/methods ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Swine ; *Nanostructures/chemistry ; *DNA, Viral/analysis/genetics ; Early Diagnosis ; Limit of Detection ; Nucleic Acid Amplification Techniques ; *DNA/chemistry ; },
abstract = {Accurate, ultrasensitive, and fast detection of the African swine fever virus (ASFV) can efficiently prevent its spread and reduce the losses. Herein, an electrochemical biosensor was designed for high-performance detection of ASFV DNA by coupling with CRISPR technology and signal amplification technology. Porous DNA nanoflowers (DNFs) were prepared by rolling circle amplification (RCA), which was preconjugated Cas12a-crRNA complex to improve detection sensitivity, shorten detection time, and simplify detection steps. In the presence of ASFV DNA, the trans-cleavage activity of Cas12a was activated, degrading DNFs into DNA fragments and causing a significant electrochemical signal change. Combined with the CRISPR-Cas12a system, the detection limit of the DNF-amplified biosensor (3.57 aM) is 3 orders of magnitude lower than that of the conventional RCA-amplified biosensor (2.90 fM). Moreover, the designed electrochemical biosensor showed excellent reproducibility, storage stability, and practical analysis ability, suggesting that it has a promising application in the early diagnosis of African swine fever (ASF).},
}

*African Swine Fever Virus/genetics/isolation & purification
*African Swine Fever/diagnosis
Animals
*Electrochemical Techniques/methods
*Biosensing Techniques/methods
*CRISPR-Cas Systems
Swine
*Nanostructures/chemistry
*DNA, Viral/analysis/genetics
Early Diagnosis
Limit of Detection
Nucleic Acid Amplification Techniques
*DNA/chemistry

@article {pmid41084819,
year = {2025},
author = {Gao, Z and Yang, X and Ren, X and Ma, H and Wu, D and Du, Y and Fan, Q and Ma, Q and Wei, Q},
title = {Ultrasensitive Detection of m[6] A-Modified RNA Using CRISPR/Cas12a-Integrated Iontronic Biosensor with Hydrophobized Nanochannels: Toward Early Cancer Diagnosis by Machine Learning.},
journal = {Analytical chemistry},
volume = {97},
number = {42},
pages = {23609-23621},
doi = {10.1021/acs.analchem.5c05377},
pmid = {41084819},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; Humans ; *Machine Learning ; *Adenosine/analogs & derivatives/analysis/chemistry ; *CRISPR-Cas Systems ; *Early Detection of Cancer/methods ; *RNA, Long Noncoding/genetics/analysis ; Hydrophobic and Hydrophilic Interactions ; *Neoplasms/diagnosis ; Limit of Detection ; Aluminum Oxide/chemistry ; },
abstract = {N[6] -methyladenosine (m[6] A), the most prevalent internal modification in eukaryotic RNAs, has emerged as a focal point of intensive research in recent years owing to its pivotal regulatory roles in carcinogenesis, progression, and metastasis. However, conventional methods for site-specific detection of m[6] A modifications are plagued by operational complexity, pose challenges for quantitative assessment of methylation levels, and exhibit elevated false-positive rates, severely limiting their utility in clinical and mechanistic studies. In this study, we engineered an ultrasensitive iontronic biosensor leveraging a hydrophobized anodic aluminum oxide (AAO) nanochannel platform, synergistically integrating the precise target recognition capability of the CRISPR/Cas12a system with the efficient signal amplification of the clamped hybridization chain reaction (CHCR). This integration enables ultrasensitive and specific detection of m[6] A-modified RNA with a low detection limit of 32 aM. Validation experiments targeting MALAT1 and HOTAIR lncRNAs demonstrated that the sensor achieves exceptional specificity in qualitative analysis of m[6] A modifications. Furthermore, combinatorial detection of these two lncRNAs enables robust discrimination between cancer patients and healthy individuals. Through in-depth mining of latent data patterns via machine learning, the random forest (RF) model yielded a cancer diagnostic accuracy of 96.7%. This study establishes a novel and potent paradigm for early cancer diagnosis, with far-reaching implications for epitranscriptomic research and clinical translation.},
}

*Biosensing Techniques/methods
Humans
*Machine Learning
*Adenosine/analogs & derivatives/analysis/chemistry
*CRISPR-Cas Systems
*Early Detection of Cancer/methods
*RNA, Long Noncoding/genetics/analysis
Hydrophobic and Hydrophilic Interactions
*Neoplasms/diagnosis
Limit of Detection
Aluminum Oxide/chemistry

@article {pmid41081763,
year = {2025},
author = {Wang, X and Feng, S and Chen, H and Zhou, B and Fan, T and Qin, Y and Zhao, L and Jiang, Y and Chen, Y},
title = {Development of an Aptamer/CRISPR-Cas12a-Based Dual-Modal Biosensor for Fusobacterium nucleatum Detection in Non-Invasive Colorectal Cancer Screening.},
journal = {Analytical chemistry},
volume = {97},
number = {42},
pages = {23360-23369},
doi = {10.1021/acs.analchem.5c04132},
pmid = {41081763},
issn = {1520-6882},
mesh = {*Fusobacterium nucleatum/isolation & purification ; *Colorectal Neoplasms/diagnosis/microbiology ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Aptamers, Nucleotide/chemistry ; *Early Detection of Cancer/methods ; Feces/microbiology ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Colorectal cancer (CRC) is the third most common cancer and leading cause of cancer-related deaths worldwide. However, current CRC screening methods are complex, invasive, and tend to exhibit low sensitivity. Recent evidence has highlighted gut microbiota dysbiosis, especially elevated Fusobacterium nucleatum levels, as a promising biomarker for CRC. In this study, a sensitive and specific detection platform was developed for F. nucleatum by combining a highly specific aptamer with rolling circle amplification (RCA) and the CRISPR/Cas12a technology. The aptamer enables specific target recognition, while RCA amplifies the target signal, and the Cas12a-mediated cleavage of a fluorescence-quenching substrate generates a quantifiable fluorescence or grayscale signal. Using a microplate reader, this assay achieved a limit of detection (LOD) of 3.68 CFU/mL; furthermore, by incorporating smartphone-assisted ImageJ grayscale analysis, it elevated the LOD to 4.30 CFU/mL, thereby enabling a dual-mode output along with on-site applicability. Additionally, the strong correlation between the two signals allowed for mutual validation. Upon application to clinical fecal samples, the developed method sensitively distinguished CRC patients from healthy controls, and its results correlated with the quantitative polymerase chain reaction results. This triple-synergistic platform, integrating aptamer specificity, RCA amplification, and CRISPR/Cas12a sensitivity, enables the noninvasive, ultrasensitive detection of F. nucleatum, supporting early CRC screening, prognosis monitoring, and microbiome-targeted therapy. Moreover, this approach overcomes the challenges of detecting low-abundance bacteria in early stage CRC and advances the precision of microbiome-based diagnostics for CRC.},
}

*Fusobacterium nucleatum/isolation & purification
*Colorectal Neoplasms/diagnosis/microbiology
*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems
*Aptamers, Nucleotide/chemistry
*Early Detection of Cancer/methods
Feces/microbiology
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40905611,
year = {2025},
author = {Hu, Y and Yan, H and Zhang, Y and Yu, Q and Xue, T and Zhang, X and Zeng, Q and Yang, H and Xia, X and Xu, Y and Deng, R and Li, J},
title = {In-Field Molecular Diagnostics of Plant Pathogens Using Bioluminescent CRISPR-Guided Caspase Assay.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {64},
number = {44},
pages = {e202508870},
doi = {10.1002/anie.202508870},
pmid = {40905611},
issn = {1521-3773},
support = {2023YFB3208302//National Key Research and Development Program of China/ ; //New Cornerstone Investigator Program/ ; 2025NSFTD0001//Sichuan Science and Technology/ ; 22522408//National Natural Science Foundation of China/ ; 22074100//National Natural Science Foundation of China/ ; },
mesh = {*Plant Diseases/microbiology ; *Caspases/metabolism ; *Luminescent Measurements/methods ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {In-field molecular diagnostics of plant pathogens are critical for crop disease management and precision agriculture, but tools are still lacking. Herein, we present a bioluminescent molecular diagnostic assay capable of detecting viable pathogens directly in minimally processed plant samples, enabling rapid and precise in-field crop disease diagnosis. The assay, called bioluminescent craspase diagnostics (BioCrastics), leverages newly discovered RNA-activated protease of CRISPR (Craspase) with enzymatic luminescence to generate a cascaded amplification, thus bypasses nucleic acid purification and amplification while achieving sub-nanogram sensitivity for fungal pathogens. Using wheat stripe rust as a proof of concept, we demonstrate direct pathogen detection in crude leaf homogenates within 40 min, early identification of infections 6 days prior to symptom emergence. Notably, the assay, via targeting pathogenic RNAs, specifically quantifies viable fungi, overcoming false positives from dead pathogens-a limitation of PCR-based methods that impairs disease risk assessment. Featuring simplified sample processing, portable detection, and species-specific accuracy, BioCrastics establishes a field-deployable tool that bridges the gap between laboratory-level precision and on-farm diagnostic needs for crop disease management.},
}

*Plant Diseases/microbiology
*Caspases/metabolism
*Luminescent Measurements/methods
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41138779,
year = {2025},
author = {Afresham, S and Khan, MK and Mughal, MAS and Mehmood, MS and Ali, S and Bashir, M and Abbas, Z and Azeem, A and Ahmed, W and Imran, M and Abbas, RZ and Sindhu, ZU and Sajid, MS},
title = {Recent Advancements in the Diagnosis of Parasitic Diseases.},
journal = {Molecular and biochemical parasitology},
volume = {},
number = {},
pages = {111706},
doi = {10.1016/j.molbiopara.2025.111706},
pmid = {41138779},
issn = {1872-9428},
abstract = {Parasitic infections present a significant health risk to the public, affecting millions of people, particularly in underdeveloped and developing countries. In developing countries, these infections are also responsible for causing significant economic challenges due to elevated healthcare expenditure. Accurate diagnosis and effective treatment methods are essentially required to combat this global issue. For decades, traditional diagnostic methods such as microscopy, serological testing, histopathology, and culturing have been used for the diagnosis of these parasitic infections. While these methods can be effective and helpful in many ways, they often consume a lot of time, require an elevated level of expertise, and have limited applications particularly in endemic regions having issues like poor infrastructure and limited access to healthcare facilities. This review aims to highlight the urgent need for a revolution to replace these conventional techniques with more affordable, quick, and field-adjustable tools such as rapid diagnostic tests (RDTs) and molecular methods and provides a comprehensive picture of advanced diagnostic tools used in the identification of parasites. With the advancements in science and technology, molecular methods such as Polymerase chain reaction, Next generation sequencing, and isothermal loop-mediated amplification have remarkably enhanced the sensitivity and accuracy of parasite detection and identification. The range of these diagnostic methods has further extended by advanced serological methods, imaging techniques, and immunological methods. Moreover, the innovations in nanotechnology, CRISPR-Cas methods, and multi-omics techniques for identification of parasite DNA, antigens, metabolites, and host responses are invaluable for diagnostic accuracy, comprehensive understanding of parasite biology, and for the discovery of new therapeutic targets and diagnostic biomarkers. However, further research and developments are required for an effective and long-lasting impact of these advancements.},
}

@article {pmid41138228,
year = {2025},
author = {Hodge, CA and Donegan, NP and Armstrong, DA and Hayden, MS and Howell, AL},
title = {Enhanced cleavage of genomic CCR5 using CASX2[Max].},
journal = {RNA biology},
volume = {22},
number = {1},
pages = {1-18},
doi = {10.1080/15476286.2025.2577449},
pmid = {41138228},
issn = {1555-8584},
mesh = {Humans ; *CRISPR-Cas Systems ; *Receptors, CCR5/genetics/metabolism/chemistry ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Development of novel CRISPR/Cas systems enhances opportunities for gene editing to treat infectious diseases, cancer, and genetic disorders. CasX2 (PlmCas12e) belongs to the class II CRISPR system derived from Planctomycetes, a non-pathogenic bacterium present in aquatic and terrestrial soils and offers several advantages as a potential therapeutic CRISPR system over Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9). These advantages include its smaller size, distinct protospacer adjacent motif (PAM) requirements, staggered cleavage cuts that promote homology-directed repair, and the absence of pre-existing immunity in humans. We compared the cleavage efficiency and double-stranded break repair characteristics between CasX2 and CasX2[Max], a recently generated CasX2 variant with three amino acid substitutions, for targeting CCR5, a gene that encodes the CCR5 receptor important for HIV-1 infection. Two single guide RNAs (sgRNAs) were designed that flank the 32 bases deleted in the natural CCR5 ∆32 mutation. Nanopore sequencing demonstrated that CasX2 using sgRNAs with spacers of 17 nucleotides (nt), 20 nt or 23 nt in length were ineffective at cleaving genomic CCR5. In contrast, CasX2[Max] using sgRNAs with 20 nt and 23 nt spacer lengths, enabled cleavage of genomic CCR5. Structural modelling indicated that two of the CasX2[Max] amino acid substitutions enhanced sgRNA-DNA duplex stability, while the third improved DNA strand alignment within the catalytic site. These structural changes likely underlie the increased activity of CasX2[Max] in cellular gene excision. In sum, CasX2[Max] consistently outperformed native CasX2 across all assays and represents a superior gene-editing platform for therapeutic applications.},
}

Humans
*CRISPR-Cas Systems
*Receptors, CCR5/genetics/metabolism/chemistry
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
HEK293 Cells

@article {pmid41137488,
year = {2025},
author = {Kang, YW and Park, HH},
title = {The anti-CRISPR protein AcrIE8.1 inhibits the type I-E CRISPR-Cas system by directly binding to the Cascade subunit Cas11.},
journal = {FEBS letters},
volume = {},
number = {},
pages = {},
doi = {10.1002/1873-3468.70201},
pmid = {41137488},
issn = {1873-3468},
support = {2024//Chung-Ang University/ ; RS-2025-02316334//National Research Foundation of Korea (NRF)/ ; },
abstract = {CRISPR-Cas systems provide adaptive immunity to bacteria by recognizing and destroying foreign genetic elements. The type I-E CRISPR-Cas system utilizes a multi-subunit Cascade complex to detect target DNA and recruit the Cas3 nuclease for degradation. To overcome this defense, bacteriophages have evolved anti-CRISPR (Acr) proteins that inhibit various steps of the CRISPR interference pathway. Here, we determined the crystal structure of AcrIE8.1, an uncharacterized Acr, revealing it binds to Cas11, a Cascade subunit, to disrupt function. AcrIE8.1 has a compact fold with a defined Cas11-binding interface, suggesting a unique inhibitory mechanism among AcrIE proteins. These findings highlight Cas11 as a critical target for Acr-mediated immune evasion. Impact statement Through a combination of structural and biochemical analyses, we demonstrate that AcrIE8.1 directly binds to the Cas11 subunit of the Cascade complex to inhibit the CRISPR-Cas system. This represents a novel inhibitory strategy not previously observed among AcrIE proteins.},
}

@article {pmid41137484,
year = {2025},
author = {Lee, SY and Park, HH},
title = {Investigating the molecular mechanisms underlying the anti-CRISPR function of AcrIIA13b protein.},
journal = {The FEBS journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/febs.70304},
pmid = {41137484},
issn = {1742-4658},
support = {RS-2025-02316334//NRF-Korea/ ; },
abstract = {The CRISPR-Cas systems of adaptive immunity in bacteria and archaea provide resistance against phages and other mobile genetic elements. Counteractive anti-CRISPR (Acr) proteins in phages and archaeal viruses impede these CRISPR-Cas systems. Although CRISPR-Cas systems have revolutionized genome editing, potential off-target events remain a safety concern. Hence, a thorough comprehension of the structural and molecular basis of diverse Acrs is imperative to unravel the fundamental mechanisms governing CRISPR-Cas regulation. Here, we present the structure of AcrIIA13b from Staphylococcus haemolyticus and analyze its structural and functional features to reveal the molecular basis underlying the inhibition of Cas9 by AcrIIA13b. Our structural analysis shows that AcrIIA13b eliminates the cleavage activity of Staphylococcus aureus Cas9 (SauCas9) by blocking the PAM-binding region of Cas9 so that Cas9 cannot recognize the target DNA. In addition, we demonstrate that the 15 amino acid residues at the N terminus of AcrIIA13b, which were revealed to be important for its dimerization, are critical for its inhibitory activity against Cas9. Our findings shed light on the molecular basis of AcrIIA13b-mediated CRISPR-Cas inhibition and provide valuable insights into the arms race between bacteria and phages.},
}

@article {pmid41136894,
year = {2025},
author = {Liu, H and Zhu, G and Chen, L and Ye, H and Zhang, Y and Han, G},
title = {Machine learning prediction of bacterial optimal growth temperature from protein domain signatures reveals thermoadaptation mechanisms.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {957},
pmid = {41136894},
issn = {1471-2164},
support = {32172769//National Natural Science Foundation of China/ ; },
abstract = {UNLABELLED: Cultivating the vast majority of uncultured microbes requires knowledge of their physiological preferences, particularly optimal growth temperature (OGT). We present a machine learning approach that utilizes protein domain frequencies from bacterial genomes to predict OGT across a wide continuous range (1–83 °C). Our Random Forest model, trained on a dataset of 1,498 genomes, achieved high predictive accuracy (R[2]=0.853 on test data, 82.4% of predictions within a ± 10 °C error margin), substantially advancing current capabilities and offering a practical tool to guide cultivation experiments. Analysis of the model identified key protein domain signatures associated with thermal adaptation. The enrichment of domains related to polyamine metabolism, the tRNA methyltransferase family, and CRISPR-Cas systems was positively correlated with higher OGTs, providing genomic evidence for their roles in thermotolerance. Conversely, domains involved in redox homeostasis, transport, and nucleic acid binding were more abundant at lower temperatures. These findings not only facilitate targeted cultivation efforts but also deepen our understanding of the molecular strategies bacteria employ to thrive across diverse thermal niches.

SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-025-12162-z.},
}

@article {pmid40618724,
year = {2025},
author = {Botkin, JR and Curtin, SJ},
title = {CRISPR-Cas9 Mutagenesis and Gene Overexpression to Enhance Resistance to Ascochyta medicaginicola in Medicago truncatula.},
journal = {Molecular plant-microbe interactions : MPMI},
volume = {38},
number = {5},
pages = {751-761},
doi = {10.1094/MPMI-05-25-0053-R},
pmid = {40618724},
issn = {0894-0282},
mesh = {*Medicago truncatula/microbiology/genetics/immunology ; *CRISPR-Cas Systems/genetics ; *Plant Diseases/microbiology/immunology/genetics ; *Ascomycota/physiology ; *Disease Resistance/genetics ; Mutagenesis ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; },
abstract = {Alfalfa (Medicago sativa), the most widely cultivated forage legume globally, is vulnerable to Ascochyta medicaginicola, the fungus causing spring black stem and leaf spot (SBS) disease, which significantly reduces yield. SBS disease also affects Medicago truncatula, a diploid model legume with extensive genetic resources, including susceptible and resistant accessions. Using comparative genomics, four candidate genes for disease resistance were identified, MtTCAR1, MtPHO2A, MtCPR1-like, and MtPAM16. CRISPR/Cas9 mutagenesis was applied to generate independent mutant plants in the R108 accession, and disease resistance was evaluated by a detached leaf qPCR-based pathogen assay. MtCPR1-like mutant plants exhibited a 34% reduction in pathogen biomass, along with variable constitutive expression of pathogenesis-related genes. Additionally, a fifth candidate gene, MtKCS12, identified through transcriptomic analysis, was overexpressed in transformed plants, resulting in a 71.4 to 80.9% reduction in pathogen biomass compared with wild-type segregants. This study validates gene editing and transgenic approaches for improving SBS disease resistance in M. truncatula, and future research will focus on applying these strategies to enhance resistance in economically important alfalfa. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.},
}

*Medicago truncatula/microbiology/genetics/immunology
*CRISPR-Cas Systems/genetics
*Plant Diseases/microbiology/immunology/genetics
*Ascomycota/physiology
*Disease Resistance/genetics
Mutagenesis
Gene Expression Regulation, Plant
Plant Proteins/genetics/metabolism
Plants, Genetically Modified

@article {pmid41136797,
year = {2025},
author = {Basarali, MK and Daemi, A and Tahiraga, RG and Özbolat, G and Hooshiar, MH and Shirazi, MSR and Döğüş, Y},
title = {Artificial intelligence-driven epigenetic CRISPR therapeutics: a structured multi-domain meta-analysis of therapeutic efficacy, off-target prediction, and gRNA optimization.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {223},
pmid = {41136797},
issn = {1438-7948},
mesh = {*Artificial Intelligence ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Epigenesis, Genetic ; *Genetic Therapy/methods ; },
abstract = {CRISPR-based epigenetic editing enables reversible regulation of gene expression without permanent DNA modification. The integration of artificial intelligence (AI) enhances guide RNA (gRNA) design, off-target prediction, and delivery optimization. We conducted a systematic review and meta-analysis (2015-2025) in accordance with PRISMA 2020 guidelines to evaluate the impact of AI on the precision, safety, and therapeutic efficacy of epigenetic CRISPR tools. From 540 screened records, 58 studies met inclusion criteria, of which 41 provided extractable quantitative data for meta-analysis and 17 contributed to qualitative synthesis. Random-effects models, subgroup analyses, and bias assessments were applied. Pooled analyses demonstrated strong positive effects across three domains: therapeutic efficacy (SMD = 1.67), gRNA optimization (SMD = 1.44), and off-target prediction (AUC = 0.79). Publication bias was minimal, and subgroup analyses indicated the strongest impact in therapeutic applications. Deep learning models were consistently associated with higher effect sizes. Qualitative synthesis revealed trends in interpretable AI, omics integration, and delivery innovations, underscoring AI's role in safer and more precise CRISPR editing. Overall, AI significantly improves the precision and therapeutic performance of CRISPR-based epigenetic tools, with the strongest effects observed in therapeutic efficacy, supporting their potential for personalized gene editing.},
}

*Artificial Intelligence
Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
*Epigenesis, Genetic
*Genetic Therapy/methods

@article {pmid41136394,
year = {2025},
author = {Chow, JT and Desjardins, A and Lee, DKC and Grigore, IA and Lee, L and Fu, NJ and Chau, S and Lee, BY and Gabra, MM and Salmena, L},
title = {A microRNA CRISPR screen reveals microRNA-483-3p as an apoptotic regulator in prostate cancer cells.},
journal = {Cell death & disease},
volume = {16},
number = {1},
pages = {752},
pmid = {41136394},
issn = {2041-4889},
mesh = {*MicroRNAs/genetics/metabolism ; Humans ; *Prostatic Neoplasms/genetics/pathology/metabolism ; Male ; *Apoptosis/genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cell Survival/genetics ; },
abstract = {The development of traditional protein-targeted cancer therapies is a slow and arduous process, often taking years or even decades. In contrast, RNA-based therapies targeting crucial microRNA (miRNA) offer a faster alternative due to the sequence-specific nature of miRNA inhibitor binding. This, combined with the capacity of individual miRNA to influence multiple cellular pathways, makes these small RNA attractive targets for cancer therapy. While miRNA are known to be dysregulated in prostate cancer (PCa), identifying their individual contributions to disease progression and the identification of therapeutically actionable miRNA targets in PCa has been challenging due to limited profiling and lack of screening tools. To address this need, we developed miRKOv2, a miRNA-only CRISPR knockout library enabling systematic, genome-wide loss-of-function screens to identify miRNA essential for PCa cell survival. Our screens uncovered 70 potential essential miRNA candidates, with miR-483 demonstrating the most significant impact on PCa cell viability. Functional characterization revealed that miR-483 disruption potentiated apoptosis in PCa cell lines. Mechanistically, we uncovered a novel regulatory axis wherein miR-483-3p directly modulates a BCLAF1/PUMA/BAK1 apoptotic signaling network, highlighting its critical role in maintaining PCa cell survival. Our findings provide novel insights into the complex regulatory role of miRNA in PCa progression and offer a potential therapeutic strategy for targeting miRNA-mediated pathways in metastatic disease.},
}

*MicroRNAs/genetics/metabolism
Humans
*Prostatic Neoplasms/genetics/pathology/metabolism
Male
*Apoptosis/genetics
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Cell Survival/genetics

@article {pmid41135510,
year = {2025},
author = {Yang, Q and Sun, Y and Sun, L and Chi, T and Chen, Z},
title = {Cryo-EM structure of the RfxCas13d-crRNA-off-target-RNA complex.},
journal = {Structure (London, England : 1993)},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.str.2025.09.010},
pmid = {41135510},
issn = {1878-4186},
abstract = {The CRISPR-Cas system is crucial for the adaptive immune response of prokaryotes and has been widely applied for genetic engineering. Cas13d, a type VI-D CRISPR-Cas effector, functions as RNA-guided ribonuclease and has been engineered for programmable RNA editing, which is a commonly used, active, and well-characterized small type VI editor. Here, we determined cryoelectron microscopy (cryo-EM) structures of Ruminococcus flavefaciens Cas13d in a RfxCas13d-crRNA-off-target-RNA ternary complex and RfxCas13d-crRNA binary complex at 3.10 and 3.13 Å resolution. The ternary complex consists of RfxCas13d, crRNA, and a captured short off-target ssRNA at a complex state of binding proximal mismatched RNA. RfxCas13d undergoes conformational changes with or without the off-target RNA, but the catalytic sites remain unchanged. Mg[2+] aids in stabilizing the crRNA repeat region structure, which may be crucial for RNA binding. This discovery provides the foundation for developing RfxCas13d as a mature tool and offers a framework for advancing transcriptome engineering.},
}

@article {pmid41134772,
year = {2025},
author = {Xu, X and Zhu, L and Xu, X and Chen, J and Du, X and Zhu, L and Yu, S and Qian, L and Jiang, X and Zhou, L and Dong, Y and Wang, Y and Huang, Y and Wang, Y},
title = {TSSKL is essential for sperm mitochondrial morphogenesis and male fertility in moths.},
journal = {PLoS genetics},
volume = {21},
number = {10},
pages = {e1011914},
pmid = {41134772},
issn = {1553-7404},
mesh = {Animals ; Male ; *Spermatozoa/metabolism ; *Moths/genetics ; *Mitochondria/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; Fertility/genetics ; Bombyx/genetics ; Female ; Infertility, Male/genetics ; Morphogenesis/genetics ; Testis/metabolism ; CRISPR-Cas Systems ; },
abstract = {Sperm deliver male genomic DNA to the ovum, playing a pivotal role in sexual reproduction across the animal kingdom. The molecular regulation of sperm morphogenesis has consequently become a focal point of genetic research, with dual implications for both reproductive medicine and sustainable agriculture. Here, we characterize the functional role of the testis-specific serine/threonine protein kinase-like (TSSKL) gene in the model lepidopteran insect Bombyx mori and the globally destructive crop pest Plutella xylostella. RNA-seq and qPCR analyses revealed TSSKL's testis-specific expression pattern. Using CRISPR/Cas9-mediated mutagenesis, we demonstrate that TSSKL knockout induces complete male sterility, while female fertility remains unaffected compared to wild-type. Fluorescence microscopy and ultrastructural analyses revealed that TSSKL deletion leads to severe morphological defects in both eupyrene and apyrene sperm, accompanied by impaired mitochondrial dynamics and aberrant autophagy. Comparative transcriptome and functional analyses linked these phenotypes to dysregulated energy metabolism pathways. Crucially, this sterility phenotype is conserved in P. xylostella, recapitulating the findings in B. mori. Our study demonstrates that TSSKL is crucial for male fertility, coordinating both structural and metabolic aspects of sperm development. Beyond advancing fundamental knowledge of insect reproductive biology, this work also identifies TSSKL as an ideal target for lepidopteran pest control through precision sterility induction.},
}

Animals
Male
*Spermatozoa/metabolism
*Moths/genetics
*Mitochondria/genetics/metabolism
*Insect Proteins/genetics/metabolism
Fertility/genetics
Bombyx/genetics
Female
Infertility, Male/genetics
Morphogenesis/genetics
Testis/metabolism
CRISPR-Cas Systems

@article {pmid41133737,
year = {2025},
author = {Devine, R and Noble, K and Wilkinson, B and Hutchings, M},
title = {Microbe Profile: Streptomyces formicae KY5: an ANT-ibiotic factory.},
journal = {Microbiology (Reading, England)},
volume = {171},
number = {10},
pages = {},
pmid = {41133737},
issn = {1465-2080},
mesh = {*Streptomyces/genetics/metabolism/isolation & purification/classification ; *Ants/microbiology ; Multigene Family ; Anti-Bacterial Agents/biosynthesis ; Animals ; Antifungal Agents/metabolism/pharmacology ; Genome, Bacterial ; Secondary Metabolism/genetics ; Biosynthetic Pathways/genetics ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {Streptomyces formicae KY5 was isolated from a Tetraponera penzigi plant-ant nest. It is primarily known for its production of the formicamycins, antibiotics with potent activity against Gram-positive pathogens including methicillin-resistant Staphylococcus aureus, and additionally produces an antifungal compound that inhibits multi-drug-resistant fungal pathogens including Lomentospora prolificans. S. formicae is genetically tractable using CRISPR-Cas9 gene editing, allowing for detailed analysis of the formicamycin biosynthetic gene cluster. AntiSMASH analysis predicts the genome to encode at least 45 secondary metabolite biosynthetic gene clusters, many of which appear to encode novel compounds. Current research efforts are focussing on characterising the regulation of secondary metabolism at a global level in order to switch on pathways that are not typically expressed under standard laboratory conditions with the aim of identifying novel antimicrobials.},
}

*Streptomyces/genetics/metabolism/isolation & purification/classification
*Ants/microbiology
Multigene Family
Anti-Bacterial Agents/biosynthesis
Animals
Antifungal Agents/metabolism/pharmacology
Genome, Bacterial
Secondary Metabolism/genetics
Biosynthetic Pathways/genetics
CRISPR-Cas Systems
Gene Editing

@article {pmid41132368,
year = {2025},
author = {Morais, C and Costa, SS and Hanke, D and Santos, A and Krüger-Haker, H and Pomba, C and Feßler, AT and Schwarz, S and Couto, I},
title = {Genomic analysis of the Staphylococcus pseudintermedius mobilome associated with antimicrobial resistance.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1640322},
pmid = {41132368},
issn = {1664-302X},
abstract = {The increasing antimicrobial resistance (AMR) in Staphylococcus pseudintermedius causing skin and soft-tissue infections (SSTIs) in companion animals is a public health concern. The aim of this study was to verify if mobile genetic elements (MGEs), in particular plasmids, are related to the carriage of AMR genes among circulating and clinically relevant S. pseudintermedius. In total, 56 S. pseudintermedius, representing predominant and emerging clonal lineages associated with SSTIs in dogs and cats collected in Lisbon (Portugal), were subjected to plasmid DNA extraction and digestion with EcoRI and XbaI. Each unique restriction pattern was assigned to a plasmid profile. A subset of 17 strains was further selected for hybrid whole genome sequencing (WGS) on Oxford Nanopore MinION and Illumina MiSeq platforms. Thirty-one of the 56 S. pseudintermedius strains carried one or more plasmid(s), mostly of small or medium sizes, corresponding to eight plasmid profiles. Two of the identified plasmids carried AMR determinants; plasmid pSP-G3C4, isolated from ST71 strains, carried the tetracycline resistance gene tet(K) and plasmid pSP5912, isolated from a ST2061 strain, harbored the qacG biocide resistance gene. Other AMR determinants were detected as part of MGEs integrated into the bacterial chromosomal DNA, namely Tn552, Tn552-like, Tn553, Tn916, Tn5405-like, Tn5801, Tn5801-like GI6287 and pRE25-like elements. In addition, a new chromosomal cassette, carrying fusC, was identified in a ST1183 strain. The 12 methicillin-resistant S. pseudintermedius studied carried staphylococcal cassette chromosome mec (SCCmec) type III (n = 5), SCCmec type IVg (n = 3), SCCmec NA45 (n = 1), ΨSCCmec 57395 (n = 1), the recently described cassettes SCCmec 7017-61515 (n = 1), or SCCmec type V(T)SL/154 (n = 1). Most strains carried intact prophages without AMR determinants. Intact restriction-modification systems were detected in 12 out of the 17 strains and CRISPR/Cas in five strains, four of which were methicillin-susceptible. The results of this study suggest that the AMR content in S. pseudintermedius is mainly related to MGEs integrated into the chromosomal DNA rather than located on plasmids. These results provide important insights that may lead to a better understanding of multidrug resistance in S. pseudintermedius towards improved SSTIs treatment in companion animals.},
}

@article {pmid41129856,
year = {2025},
author = {Zhou, J and Ren, XM and Gao, J and Wu, RP and Chen, L and Li, Z},
title = {Amplification-free detection of mycoplasma pneumoniae via CRISPR-Cas12a and deep learning-optimized crRNAs on a lateral flow platform.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {268},
number = {},
pages = {117196},
doi = {10.1016/j.jpba.2025.117196},
pmid = {41129856},
issn = {1873-264X},
abstract = {Accurate and rapid diagnosis of Mycoplasma pneumoniae infection is essential for reducing its significant health burden. An amplification-free CRISPR-Cas12a-mediated detection platform has been developed, incorporating a deep learning-optimized crRNA library (CCDLCL) targeting conserved regions of the MP P1 gene. The system enables visual readout via lateral flow strips, supporting its potential as a point-of-care testing (POCT) nucleic acid testing strategy. Through computational design and screening, 16 highly active crRNAs were identified from an initial set of over 50 candidates. Combinatorial use of these crRNAs demonstrated synergistic enhancement of fluorescence signal intensity and reaction kinetics. Compared to single-crRNA assays, the multiplexed crRNA library improved sensitivity by 16.8-fold, achieving a limit of detection (LOD) of 0.15 pM, and reduced time to signal saturation by 30 %. When deployed on lateral flow strips, the assay exhibited a tenfold increase in visual detection sensitivity, with a LOD of 100 pM. Clinical evaluations confirmed high specificity-showing no cross-reactivity with SARS-CoV-2, hepatitis B virus (HBV), or human genomic DNA-and over 95 % agreement with standard clinical results without target pre-amplification, delivering outcomes within 45 min. This study establishes a deep learning-facilitated crRNA design framework and a novel crRNA library-based detection system, offering a feasible approach for POCT nucleic acid testing in resource-limited settings and paving the way for streamlined clinical translation of CRISPR-Cas diagnostics.},
}

@article {pmid41125583,
year = {2025},
author = {Yang, J and Li, H and Li, M and Song, R and Shen, T and Wang, G and Xu, D and Hao, M and Jia, A and Rehman, SU and Hua, L and Liang, Y and Chi, C and Lan, C and Deng, XW and Dubcovsky, J and Song, B and Wang, X and Chen, S},
title = {Genome-assisted identification of wheat leaf rust resistance gene Lr.ace-4A/Lr30.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9339},
pmid = {41125583},
issn = {2041-1723},
mesh = {*Triticum/genetics/microbiology/immunology ; *Plant Diseases/microbiology/genetics/immunology ; *Disease Resistance/genetics ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Plant Leaves/microbiology/genetics ; Basidiomycota ; Genome, Plant ; *Genes, Plant ; CRISPR-Cas Systems ; Puccinia ; },
abstract = {Leaf rust is a devastating disease of wheat. Growing rust-resistant wheat varieties is the best strategy to mitigate this threat. Here, we generate a 10.51-gigabase chromosome-scale assembly of the durum wheat landrace PI 192051. Using mutagenesis and transcriptome sequencing, we identify the leaf rust resistance gene Lr.ace-4A within a recombination-sparse region of PI 192051 and demonstrate that Lr.ace-4A is identical to the previously designated Lr30 gene in hexaploid wheat. Lr.ace-4A/Lr30 encodes a non-canonical coiled-coil nucleotide-binding leucine-rich repeat receptor, featuring tandem nucleotide-binding domains. This gene is both necessary and sufficient to confer resistance to leaf rust, as demonstrated by CRISPR/Cas9-induced mutations and transgenic complementation. Lr.ace-4A provides near-immunity resistance in durum wheat, though its effectiveness is diminished in hexaploid wheat. Two amino acid polymorphisms differentiate the resistant and susceptible Lr.ace-4A haplotypes, with transgenic plants carrying either susceptible variant showing susceptibility. The cloning of Lr.ace-4A will accelerate its deployment in wheat breeding programs.},
}

*Triticum/genetics/microbiology/immunology
*Plant Diseases/microbiology/genetics/immunology
*Disease Resistance/genetics
*Plant Proteins/genetics/metabolism
Plants, Genetically Modified
Plant Leaves/microbiology/genetics
Basidiomycota
Genome, Plant
*Genes, Plant
CRISPR-Cas Systems
Puccinia

@article {pmid41125159,
year = {2025},
author = {Zhou, C and Zhu, S and Luo, C and Wang, W and Fan, H and Gao, Y and Xu, X and Wang, Q and You, Y and Xie, T},
title = {From IscB to Cas9: Engineering and advances in the next generation of miniature gene editing tools.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108743},
doi = {10.1016/j.biotechadv.2025.108743},
pmid = {41125159},
issn = {1873-1899},
abstract = {The CRISPR-Cas system, distinguished by its inherent modularity and broad programmability, has catalyzed a paradigm shift in genome engineering due to its unprecedented accuracy, specificity, and on-target efficiency, now serving as the cornerstone of modern genome manipulation. The efficient delivery of gene editing tools remains a major technical hurdle to clinical application, primarily due to the lack of compact editors. The recent identification of the transposon-associated nuclease IscB as an evolutionary ancestor of Cas9 has provided important insights into the molecular evolution of the CRISPR-Cas9 system. Notably, IscB is a highly compact nuclease, approximately one-third the size of Cas9, capable of precise nucleic acid cleavage in eukaryotic cells under the guidance of ωRNA. These features make it a promising candidate for the development of next-generation miniaturized genome editors. However, natural IscB exhibits limited editing performance in eukaryotic systems. This review first outlines the biochemical function of the transposon IscB and briefly traces the evolutionary origin of the Cas9 system. It then describes and compares the structural characteristics and cleavage mechanisms of OgeuIscB and Cas9. Subsequent sections summarize various engineering strategies for current IscB systems, including the development of base editors and recent advances in their application. Finally, the limitations of existing systems are discussed, and potential directions for future optimization are proposed, aiming to provide new insights and facilitate the advancement of IscB-based miniaturized editors.},
}

@article {pmid41124242,
year = {2025},
author = {Lu, X and Zhu, Y and Wei, C and Cheng, L and Goodier, KD and Kong, J and Gao, X and Yu, D and Liu, X and Long, Y and Lin, J and Ma, J and Su, Y and Mao, HQ},
title = {A multistep platform identifies spleen-tropic lipid nanoparticles for in vivo T cell-targeted delivery of gene-editing proteins.},
journal = {Science advances},
volume = {11},
number = {43},
pages = {eady5579},
pmid = {41124242},
issn = {2375-2548},
mesh = {Animals ; *Gene Editing/methods ; *Spleen/metabolism/cytology ; Mice ; *T-Lymphocytes/metabolism/immunology ; *Nanoparticles/chemistry ; *Lipids/chemistry ; *Gene Transfer Techniques ; Humans ; Receptors, CCR5/genetics ; CRISPR-Cas Systems ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) are a promising nonviral delivery system for gene-editing proteins, but optimal formulations remain underexplored. Unlike messenger RNA-based approaches, ribonucleoprotein delivery enables immediate genome editing without relying on endogenous translation. However, intracellular delivery remains a major challenge due to protein size, charge variability, and susceptibility to denaturation and degradation. Here, we present a multistep screening platform to optimize LNP formulations for gene-editing protein delivery, focusing on in vivo T cell targeting. Through in vitro screening of a composition library, we identified top-performing candidates. In vivo screening in Ai9 mice revealed a spleen-tropic LNP formulation that preferentially targets T cells, enabling efficient gene editing in vivo. Using this LNP formulation, we achieved targeted knockout of CCR5 and PD-1 in splenic T cells, supporting potential applications in HIV resistance and cancer immunotherapy. Furthermore, a machine learning-guided mechanistic study revealed key design principles for LNP-based protein delivery, highlighting unexplored opportunities for in vivo genome-editing therapies.},
}

Animals
*Gene Editing/methods
*Spleen/metabolism/cytology
Mice
*T-Lymphocytes/metabolism/immunology
*Nanoparticles/chemistry
*Lipids/chemistry
*Gene Transfer Techniques
Humans
Receptors, CCR5/genetics
CRISPR-Cas Systems
Liposomes

@article {pmid41123979,
year = {2025},
author = {Wang, S and Wei, Z and Feng, Y and Gan, Z and Yu, L and Cheng, H and Xiao, Y},
title = {Rationally Trapped Polycrystalline Perovskite in Lanthanide MOF Cages for Ammonia-Mediated Nucleic Acid Intelligent Visualization.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {},
number = {},
pages = {e07214},
doi = {10.1002/smll.202507214},
pmid = {41123979},
issn = {1613-6829},
support = {82273895//National Natural Science Foundation of China/ ; 82304440//National Natural Science Foundation of China/ ; 82073811//National Natural Science Foundation of China/ ; 2024M763727//China Postdoctoral Science Foundation/ ; },
abstract = {Perovskites nanoparticles (PNPs), promising materials in fluorescence biosensing, have has their practical applications stymied by poor stability in polar solvents. Integrating PNPs into metal-organic frameworks (MOFs) offers a solution by enhancing their compatibility with various environments. Lanthanide MOFs (Ln-MOFs) are particularly advantageous due to their customizable structure, enhanced stability, and intrinsic fluorescence. Herein, the mechanism by which hybrid materials achieve a balance between physical and fluorescence properties is elucidated. Through theoretical calculations, 2,2'-bipyridine-5,5'-dicarboxylic acid is selected as the ligand to sensitize Eu[3+] and facilitate Pb[2+] chemisorption. In addition, 4-bromobutyric acid is not only employed to create hierarchical Eu-MOFs and optimize the framework for in situ growth of polycrystalline perovskites but also utilized to generate zwitterionic ligands through an SN2 reaction with MOF cages restricted n-octylamine, ensuring the stable dispersion of hybrid materials in ethyl acetate (polar solvent). The prepared PNPs@Ln-MOF exhibits significantly enhanced fluorescence lifetime (50-fold) and stability in polar solvents. Besides, the highly sensitive fluorescence color shift of PNPs@Ln-MOF in response to ammonia offers a generalizable strategy for ammonia-mediated biosensing device. Supported by CRISPR/Cas technology, this device allows for precise on-site nucleic acid assay (LOD = 200 fM), pioneering advanced applications of perovskite-based hybrid materials in biosensing.},
}

@article {pmid41065718,
year = {2025},
author = {Zeng, B and Sheng, A and Zhang, X and Wang, X and Bao, Y and Huang, Y and Huang, Y and Shan, L and Xu, X and Qin, Y and Yang, Y and Deng, Y and Tian, L and Wang, J and Ma, L},
title = {CRISPR/Cas12a Integrated with a Microfluidic System Enhanced Analysis of Programmed Cell Death Ligand 1 Expression in Circulating Tumor Cells from Non-Small Cell Lung Cancer Patients.},
journal = {ACS sensors},
volume = {10},
number = {10},
pages = {7388-7402},
doi = {10.1021/acssensors.5c01152},
pmid = {41065718},
issn = {2379-3694},
mesh = {Humans ; *Carcinoma, Non-Small-Cell Lung/blood/pathology/metabolism ; *Neoplastic Cells, Circulating/metabolism/pathology ; *B7-H1 Antigen/genetics/metabolism/blood ; *Lung Neoplasms/blood/pathology/metabolism ; *CRISPR-Cas Systems ; *Lab-On-A-Chip Devices ; *Microfluidic Analytical Techniques/methods ; Cell Line, Tumor ; },
abstract = {The detection of programmed cell death ligand 1 (PD-L1) positive circulating tumor cells (CTCs) in peripheral blood has significant clinical value for predicting and evaluating the efficacy of immunotherapy in patients with non-small cell lung cancer (NSCLC). However, traditional methods remain limited by low sensitivity and the precise quantification remains a challenge. A dual-mode microfluidic analysis chip was constructed here that included clustered regularly interspaced short palindromic repeats/Cas12a quantification and immunofluorescence visualization. Quantification of the PD-L1 protein on the surface of CTCs (20 to 10[7] cell/mL) was achieved selectively and sensitively by amplifying the nucleic acid target to generate a strong fluorescent signal, even with very low levels of target cells. The system effectively detected PD-L1[+] CTCs expression in peripheral blood samples from patients with NSCLC and monitored the efficacy of PD-1/PD-L1 targeted immune checkpoint inhibitors in real time. It exhibited excellent performance for clinical applications in monitoring the prognosis in patients with NSCLC.},
}

Humans
*Carcinoma, Non-Small-Cell Lung/blood/pathology/metabolism
*Neoplastic Cells, Circulating/metabolism/pathology
*B7-H1 Antigen/genetics/metabolism/blood
*Lung Neoplasms/blood/pathology/metabolism
*CRISPR-Cas Systems
*Lab-On-A-Chip Devices
*Microfluidic Analytical Techniques/methods
Cell Line, Tumor

@article {pmid41123685,
year = {2025},
author = {Yang, S and Liu, Y and Zhang, J and Xu, J and Li, T and Huang, H and Zhu, Z and Li, M and Wang, H and Yang, C},
title = {An integrated lab-in-a-tube platform for point-of-care detection of blaKPC in urinary tract infections.},
journal = {Mikrochimica acta},
volume = {192},
number = {11},
pages = {748},
pmid = {41123685},
issn = {1436-5073},
support = {82472374//National Natural Science Foundation of China/ ; YG2024LC02//Cross-Research Fund of Biomedical Engineering of Shanghai Jiaotong University/ ; 2024ZY008//The Clinical and Excellence Program at the Institute of Molecular Medicine, Shanghai Jiao Tong University School of Medicine/ ; },
mesh = {Humans ; *Urinary Tract Infections/microbiology/diagnosis/urine ; *Klebsiella pneumoniae/enzymology/genetics/drug effects/isolation & purification ; *beta-Lactamases/genetics/urine ; *Bacterial Proteins/genetics/urine ; *Point-of-Care Systems ; Klebsiella Infections/urine/diagnosis/microbiology ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; },
abstract = {Carbapenem-resistant Klebsiella pneumoniae (CRKP), predominantly mediated by the blaKPC carbapenemase gene, poses a critical therapeutic challenge for urinary tract infections (UTIs). To enable rapid identification at the point of need, we engineered TubeCARE (tube-integrated platform for carbapenem antimicrobial resistance evaluation), an integrated, disposable lab-in-a-tube system for direct blaKPC detection from urine samples. This self-contained system uniquely combines urine processing, nucleic acid extraction, recombinase polymerase amplification (RPA), and CRISPR/Cas12a reaction within a single sealed unit, offering dual-modality outputs: smartphone-based real-time fluorescence or naked-eye lateral flow strip interpretation. The full workflow delivers "sample-in, result-out" in 40 min, eliminating external multi-step nucleic acid extraction and reducing contamination risks. Both detection modes demonstrated laboratory-grade sensitivity (1 CFU/mL) with 100% specificity against non-target carbapenemase genes. Clinical validation using 24 urine samples (20 blaKPC-positive, 4 negative) showed 100% concordance with quantitative PCR. In a representative UTI case, TubeCARE enabled 40-min bedside blaKPC detection; early implementation would circumvent 72-h diagnostic delays, facilitating timely carbapenem avoidance and preventing bloodstream complications. Featuring integrated lab-in-a-tube operation, cost-effective manufacturing, user-friendly workflow, and laboratory-grade accuracy, TubeCARE provides actionable antimicrobial resistance surveillance in diverse clinical and resource-limited settings.},
}

Humans
*Urinary Tract Infections/microbiology/diagnosis/urine
*Klebsiella pneumoniae/enzymology/genetics/drug effects/isolation & purification
*beta-Lactamases/genetics/urine
*Bacterial Proteins/genetics/urine
*Point-of-Care Systems
Klebsiella Infections/urine/diagnosis/microbiology
Nucleic Acid Amplification Techniques
CRISPR-Cas Systems

@article {pmid40938646,
year = {2025},
author = {Lee, JH and Lee, E-S and Kyung, SM and Xiang, X-R and Park, H-E and Shin, M-K and Yoo, HS},
title = {Functional analysis of the intracellular survival of Mycobacterium avium subsp. paratuberculosis in THP-1 cells using CRISPR interference.},
journal = {Journal of bacteriology},
volume = {207},
number = {10},
pages = {e0024425},
doi = {10.1128/jb.00244-25},
pmid = {40938646},
issn = {1098-5530},
support = {//National Institute of Wildlife Disease Control and Prevention/ ; },
mesh = {Humans ; *Mycobacterium avium subsp. paratuberculosis/genetics/physiology/pathogenicity ; *Macrophages/microbiology ; THP-1 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Virulence/genetics ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; *Microbial Viability ; CRISPR-Cas Systems ; Paratuberculosis/microbiology ; },
abstract = {UNLABELLED: Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne's disease in ruminants and a potential zoonotic agent linked with Crohn's disease in humans. Despite the possible risk to public health, few studies have focused on the virulence of MAP against human macrophages. Therefore, a functional analysis of mycobacterial genes associated with virulence, especially the intracellular survival of MAP, was performed after infection of MAP CRISPR interference (CRISPRi) mutants in the human THP-1 macrophages. MAP mutants were targeted to four genes (mdh, pknG, MAP1981c, and icl). The optimal concentration of anhydrotetracycline (ATc) was determined to be 5 µg/mL by measuring the survival of the cells and the downregulation of gene expression levels in the cells up to Day 3. The clump formation and intracellular survival of MAP were investigated using transmission electron microscopy and the colony-forming units, respectively. The clump formation of MAP mutants induced by CRISPRi was decreased in THP-1 macrophages at 24 and 72 h post-infection. The survival rates of the MAP mutants significantly decreased with increasing ATc concentration and time course of infection in MAP-mdhKD, MAP1981cKD, and MAP-iclKD. Conversely, the survival rate of THP-1 macrophages increased with increasing ATc concentration. Our results suggest that these genes might be closely related to MAP virulence along with intracellular survival in THP-1 macrophages. These data can provide novel insights into the utilization of CRISPRi in further research on MAP virulence by exploring intracellular survival using mycobacterial genes related to the virulence of MAP during host infection.

IMPORTANCE: Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP) is a worldwide issue in the dairy industry and has a possible connection to Crohn's disease (CD) in humans. Despite its potential contribution to the etiology of CD, there have been few studies focusing on the virulence of MAP against human macrophages. In the current study, we investigated MAP virulence along with intracellular survival in human THP-1 macrophages using functional analysis of MAP CRISPR interference (CRISPRi) mutants at the knockdown of genes associated with mycobacterial virulence. The identified potential genes represent novel candidate classes that could be necessary for MAP virulence by exploring intracellular survival during host infection and could provide novel insights for future studies on the utilization of CRISPRi.},
}

Humans
*Mycobacterium avium subsp. paratuberculosis/genetics/physiology/pathogenicity
*Macrophages/microbiology
THP-1 Cells
*Clustered Regularly Interspaced Short Palindromic Repeats
Virulence/genetics
Bacterial Proteins/genetics/metabolism
Gene Expression Regulation, Bacterial
*Microbial Viability
CRISPR-Cas Systems
Paratuberculosis/microbiology

@article {pmid40919935,
year = {2025},
author = {Alberts, ME and Kurtz, MP and Müh, U and Bernardi, JP and Bollinger, KW and Dobrila, HA and Duncan, L and Laster, HM and Orea, AJ and Pannullo, AG and Rivera-Rosado, JG and Torres, FV and Ellermeier, CD and Weiss, DS},
title = {Analysis of essential genes in Clostridioides difficile by CRISPRi and Tn-seq.},
journal = {Journal of bacteriology},
volume = {207},
number = {10},
pages = {e0022025},
doi = {10.1128/jb.00220-25},
pmid = {40919935},
issn = {1098-5530},
support = {DBI-1852070//National Science Foundation/ ; R01 AI155492/AI/NIAID NIH HHS/United States ; R21 AI159071/AI/NIAID NIH HHS/United States ; },
mesh = {*Clostridioides difficile/genetics/metabolism ; *Genes, Essential ; *DNA Transposable Elements ; Mutagenesis, Insertional ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Essential genes are interesting in their own right and as potential antibiotic targets. To date, only one report has identified essential genes on a genome-wide scale in Clostridioides difficile, a problematic pathogen for which treatment options are limited. That foundational study used large-scale transposon mutagenesis to identify 404 protein-encoding genes as likely to be essential for vegetative growth of the epidemic strain R20291. Here, we revisit the essential genes of strain R20291 using a combination of CRISPR interference (CRISPRi) and transposon insertion site sequencing (Tn-seq). First, we targeted 181 of the 404 putatively essential genes with CRISPRi. We confirmed essentiality for >90% of the targeted genes and observed morphological defects for >80% of them. Second, we conducted a new Tn-seq analysis, which identified 346 genes as essential, of which 283 are in common with the previous report and might be considered a provisional essential gene set that minimizes false positives. We compare the list of essential genes to those of other bacteria, especially Bacillus subtilis, highlighting some noteworthy differences. Finally, we used fusions to red fluorescent protein (RFP) to identify 18 putative new cell division proteins, 3 of which are conserved in Bacillota but of largely unknown function. Collectively, our findings provide new tools and insights that advance our understanding of C. difficile.IMPORTANCEClostridioides difficile is an opportunistic pathogen for which better antibiotics are sorely needed. Most antibiotics target pathways that are essential for viability. Here, we use saturation transposon mutagenesis and gene silencing with CRISPR interference to identify and characterize genes required for growth on laboratory media. Comparison to the model organism Bacillus subtilis revealed many similarities and a few striking differences that warrant further study and may include opportunities for developing antibiotics that kill C. difficile without decimating the healthy microbiota needed to keep C. difficile in check.},
}

*Clostridioides difficile/genetics/metabolism
*Genes, Essential
*DNA Transposable Elements
Mutagenesis, Insertional
*Clustered Regularly Interspaced Short Palindromic Repeats
Bacterial Proteins/genetics/metabolism
CRISPR-Cas Systems

@article {pmid40897994,
year = {2025},
author = {Kumar, N},
title = {Genome Editing for Fertility: Unlocking the Promise of CRISPR/Cas9 in Addressing Male Infertility - A Narrative Review.},
journal = {Reproductive sciences (Thousand Oaks, Calif.)},
volume = {32},
number = {10},
pages = {3221-3239},
pmid = {40897994},
issn = {1933-7205},
mesh = {*Gene Editing/methods ; Humans ; Male ; *CRISPR-Cas Systems ; *Infertility, Male/genetics/therapy ; Animals ; *Fertility/genetics ; *Genetic Therapy/methods ; },
abstract = {Male infertility remains a significant global reproductive health challenge, frequently attributed to genetic mutations impairing spermatogenesis and sperm function. This narrative review aims to explore the genetic and molecular underpinnings of male infertility and evaluate the emerging role of Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein 9 (CRISPR/Cas9) genome editing as a diagnostic and therapeutic tool, while addressing its associated ethical, technical, and safety considerations. A Comprehensive literature search was conducted across PubMed, Scopus, Web of Science databases, covering studies published between September 1992 and April 2025. Keywords included "male infertility," "genetic causes of male infertility," "genome editing," "CRISPR/Cas9 and male infertility," "genome editing in male reproduction," "ethical concerns of CRISPR," and "future fertility treatments." Eligible studies focused on genetic correction strategies, spermatogonial stem cell applications, off-target effects, mosaicism, and ethical implications of gene editing. The review synthesizes current knowledge on genetic and epigenetic etiologies of male infertility. It discusses the therapeutic potential of CRISPR/Cas9 in correcting these defects and restoring fertility in preclinical models. Critical challenges, including off-target gene editing, germline mosaicism, long-term safety, and ethical debates surrounding human germline modification, were examined. The review also considers future advancements in genome editing and artificial sperm development. CRISPR/Cas9 represents a transformative platform in reproductive medicine with promising implications for treatment of genetically linked male infertility. However, its clinical translation demands rigorous validation, transparent ethical deliberation, and robust regulatory frameworks. Future innovations combining genome editing, regenerative biology, and precision diagnostics may revolutionize fertility care, but must proceed with caution to ensure safety, efficacy, and ethical integrity.},
}

*Gene Editing/methods
Humans
Male
*CRISPR-Cas Systems
*Infertility, Male/genetics/therapy
Animals
*Fertility/genetics
*Genetic Therapy/methods

@article {pmid40782836,
year = {2025},
author = {Agboola, OE and Agboola, SS and Odeghe, OB and Olaiya, OE and Ayinla, ZA and Akinsanya, PO and Ilesanmi, OS and Ibrahim, TK and Adegbuyi, TA and Kolawole, OA and Omotuyi, IO and Oyinloye, BE},
title = {Computational genome engineering through AI-CRISPR-precision medicine integration in modern therapeutics.},
journal = {Annales pharmaceutiques francaises},
volume = {83},
number = {6},
pages = {1073-1085},
doi = {10.1016/j.pharma.2025.08.001},
pmid = {40782836},
issn = {2772-803X},
mesh = {Humans ; *Precision Medicine/methods ; *Gene Editing/methods ; *Artificial Intelligence ; Drug Discovery ; *CRISPR-Cas Systems ; *Genetic Engineering/methods ; Machine Learning ; },
abstract = {The convergence of precision medicine strategies, CRISPR gene editing technologies, and artificial intelligence (AI) is causing a revolutionary change in the pharmaceutical industry in recent times. Latest trends and future directions of these integrated technologies in pharmaceutical science and molecular biology are presented in the present exhaustive review. With more than 250 gene-editing clinical trials being tracked internationally as of February 2025, the recent clinical successes point toward the therapeutic potency of CRISPR-based therapeutics. In parallel, AI-based drug discovery platforms are recording fantastic hit rates; compared to conventional industry benchmarks, AI-emerging drugs reflect 80-90% Phase I trial success rates. Therapeutic development paradigms are being transformed by the intersection of machine learning algorithms, multi-omics technologies, and precision medicine paradigms. The review provides insights into the revolutionary potential of these converging approaches in addressing unmet medical requirements and optimizing therapeutic benefits through syntheses of existing evidence from clinical trials, regulatory matters, and technological innovations.},
}

Humans
*Precision Medicine/methods
*Gene Editing/methods
*Artificial Intelligence
Drug Discovery
*CRISPR-Cas Systems
*Genetic Engineering/methods
Machine Learning

@article {pmid41123679,
year = {2025},
author = {Rocha, DC and Omoregbee, MO and Contiliani, DF and Mandlik, R and Li, G and Mascoveto, J and Coleman, G and Culver, JN and Leal, DR and de Souza, AA and Qi, Y},
title = {Transgene-free genome editing in citrus and poplar trees using positive and negative selection markers.},
journal = {Plant cell reports},
volume = {44},
number = {11},
pages = {244},
pmid = {41123679},
issn = {1432-203X},
support = {IOS-2132693//Division of Integrative Organismal Systems/ ; 2224203//Division of Integrative Organismal Systems/ ; IOS-2428015//Division of Integrative Organismal Systems/ ; 2021-67013-34554//National Institute of Food and Agriculture/ ; 2020-33522-32274//National Institute of Food and Agriculture/ ; 2024-33522-42755//National Institute of Food and Agriculture/ ; 2020-70029- 33161//National Institute of Food and Agriculture/ ; DE-SC0023011//Department of Energy/ ; 2023/09068-9//FAPESP/ ; },
mesh = {*Populus/genetics ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; *Citrus/genetics ; Transgenes/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant ; Genetic Markers ; Herbicide Resistance/genetics ; },
abstract = {Transgene-free genome editing of the gene of interest in citrus and poplar has been achieved by co-editing the ALS gene via transient transgene expression of an efficient cytosine base editor. CRISPR-Cas genome editing systems have been widely used in plants. However, such genome-edited plants are nearly always transgenic in the first generation when Agrobacterium-mediated transformation is used. Transgene-free genome-edited plants are valuable for genetic analysis and breeding as well as simplifying regulatory approval. It can be challenging to generate transgene-free genome-edited plants in vegetatively propagated or perennial plants. To advance transgene-free genome editing in citrus and poplar, we investigated a co-editing strategy using an efficient cytosine base editor (CBE) to edit the ALS gene to confer herbicide resistance combined with transient transgene expression and potential mobile RNA-based movement of CBE transcripts to neighboring, non-transgenic cells. An FCY-UPP based cytotoxin system was used to select non-transgenic plants that survive after culturing on 5-FC containing medium. While the editing efficiency is higher in poplar than in citrus, our results show that the CBE-based co-editing strategy works in both citrus and poplar, albeit with low efficiency for biallelic edits. Unexpectedly, the addition of the TLS mobile RNA sequence reduced genome editing efficiency in both transgenic and non-transgenic plants. Although a small fraction of escaping plants is detected in both positive and negative selection processes, our data demonstrate a promising approach for generating transgene-free base-edited plants.},
}

*Populus/genetics
*Gene Editing/methods
Plants, Genetically Modified/genetics
*Citrus/genetics
Transgenes/genetics
CRISPR-Cas Systems/genetics
Genome, Plant
Genetic Markers
Herbicide Resistance/genetics

@article {pmid41123065,
year = {2025},
author = {Sohail, M and Ma, S and Mushtaq, B and Haider, MU and Li, B and Zhang, X and Huang, H},
title = {Fabricating Four-Element Doped Carbon Dots-Based Fluorescent Ratiometric Reporter Platform for CRISPR/Cas-Driven Precise Sensing of Nucleic Acids.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c03228},
pmid = {41123065},
issn = {1520-6882},
abstract = {Conventional CRISPR/Cas sensing platforms exhibit poor efficiency concerning reporter-based demerits, including their interference-labile nature, photobleaching, low robustness due to a single output signal, and probe-concentration dependence. Herein, a carbon dots (CDs)-based dual-emissive fluorescent ratiometric CRISPR/Cas reporter platform was fabricated for biosensing and other analytical applications to bottleneck the demerits of conventional reporters, integrating the benefits of a ratiometric strategy and four-element doped carbon dots (4D CDs) as a transducer. Briefly, doping enhances the optical and physicochemical traits of CDs and minimizes the effect of the interfering species. A series of state-of-the-art N, P, S, and Cu codoped CDs (4D CDs) were synthesized using the hydrothermal approach and statistical tools, such as Box-Behnken design, analysis of variance, and others, enhancing photophysical traits, surface features, and sensitivity of CDs. The red-emissive CDs were prepared by using the same procedure but different precursors. The optimum 4D CDs (blue-emissive) and red-emissive CDs were used to unleash the principle of the fluorescent ratiometric CRISPR/Cas reporter system for diverse applications. Finally, the designed 4D CDs-based CRISPR/Cas biosensor was applied for nucleic acid monitoring, such as the COVID-19 nucleic acid. This project disclosed the controlled-doping principle to synthesize 4D CDs and unleashed the mechanism of ratiometric dual-emissive CRISPR/Cas-powered reporters for precise sensing applications. We anticipate the implementation of this technology in commercial analytical, biosensing, point-of-care, and other applications.},
}

@article {pmid41122064,
year = {2025},
author = {Balobaid, A and Waterworth, WM and Vila Nova, SF and Causier, B and Sharma, V and Park, MR and Pandey, MK and West, CE},
title = {Arabidopsis thaliana FANCONI ANAEMIA I (FANCI) has roles in the repair of interstrand crosslinks and CRISPR-Cas9 induced DNA double strand breaks.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {2},
pages = {e70533},
doi = {10.1111/tpj.70533},
pmid = {41122064},
issn = {1365-313X},
support = {//King Saud University/ ; BB/S002081/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/Y010426/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Arabidopsis/genetics/metabolism ; *Arabidopsis Proteins/metabolism/genetics ; *DNA Repair/genetics ; *DNA Breaks, Double-Stranded ; CRISPR-Cas Systems/genetics ; Mitomycin/pharmacology ; Cross-Linking Reagents ; Mutation ; },
abstract = {DNA repair is crucial for genome stability, in particular for plants which are exposed to high levels of damage arising from UV irradiation, soil pollutants and reactive oxygen species. Damage that affects both strands of the DNA duplex is harder to repair due to both the lack of a template strand and the potential for physical separation of fragmented chromosomes. As such, DNA double-strand breaks (DSBs) and interstrand DNA crosslinks (ICL) are particularly cytotoxic forms of damage. Here we report the functions of FANCONI ANAEMIA I (FANCI), an Arabidopsis thaliana homologue of the mammalian ICL repair protein. We show that in plant cells, as in mammals, FANCI forms a nuclear localised complex with FANCD2. Genetic analysis of plants lacking FANCI displays significant hypersensitivity to the DNA crosslinking reagent mitomycin C. Furthermore, mutation of FANCI in combination with mutations in a second ICL repair factor, METHYL METHANESULFONATE AND UV-SENSITIVE PROTEIN 81 (MUS81), results in increased levels of programmed cell death compared to the corresponding single mutants, revealing roles in maintaining plant genome stability. Sequence analysis of mutational repair of CRISPR-Cas9-induced DSBs revealed that FANCI promotes single nucleotide insertions and reduces longer deletions. This pattern of mutations may reflect roles for FA proteins in replication-coupled repair of a subset of DSBs. Taken together, this analysis finds evidence for multiple roles for FANCI in the maintenance of plant genome stability.},
}

*Arabidopsis/genetics/metabolism
*Arabidopsis Proteins/metabolism/genetics
*DNA Repair/genetics
*DNA Breaks, Double-Stranded
CRISPR-Cas Systems/genetics
Mitomycin/pharmacology
Cross-Linking Reagents
Mutation

@article {pmid41121375,
year = {2025},
author = {Amieva, R and Rico-San Román, L and Pastor-Fernández, I and Hemphill, A and Boubaker, G and Collantes-Fernández, E and Ortega-Mora, LM and Horcajo, P},
title = {Loss of NcBPK1 impairs bradyzoite differentiation and enhances virulence in Neospora caninum.},
journal = {Parasites & vectors},
volume = {18},
number = {1},
pages = {422},
pmid = {41121375},
issn = {1756-3305},
support = {PRE2020-092101//Spanish Ministry of Science, Innovation and Universities/ ; },
mesh = {*Neospora/pathogenicity/genetics/growth & development ; Animals ; Virulence ; Mice ; *Coccidiosis/parasitology/veterinary ; *Protozoan Proteins/genetics/metabolism ; Female ; Cattle ; Macrophages/parasitology ; Pregnancy ; Virulence Factors/genetics ; Cattle Diseases/parasitology ; CRISPR-Cas Systems ; Host-Parasite Interactions ; Disease Models, Animal ; Gene Deletion ; },
abstract = {BACKGROUND: Neospora caninum is an apicomplexan parasite responsible for bovine neosporosis, a disease that leads to substantial economic losses in cattle due to abortion and reduced productivity. The pathogenesis of N. caninum is shaped by complex host-parasite interactions, and virulence is known to vary between strains. BPK1 (Bradyzoite pseudokinase 1), a pseudokinase previously identified as a potential virulence factor in Toxoplasma gondii, has not yet been functionally characterized in N. caninum.

METHODS: To investigate the role of NcBPK1 in parasite virulence, a knockout strain (NcΔBPK1) was generated using CRISPR/Cas9 genome editing. The virulence of the mutant was evaluated in a pregnant mouse model by assessing neonatal survival and parasite burden in dam tissues. In vitro assays were conducted to examine parasite replication in bovine macrophages and to analyze the expression of stage-specific genes.

RESULTS: Deletion of NcBpk1 resulted in enhanced parasite virulence in vivo, as shown by a decrease in neonatal survival and higher parasite loads in maternal brain tissue. The NcΔBPK1 mutant also displayed enhanced replication in bovine macrophages and reduced expression of bradyzoite-specific genes, suggesting a defect in stage conversion.

CONCLUSIONS: These findings indicate that NcBPK1 is crucial for regulating the balance between acute replication and chronic persistence. Its absence promotes rapid tachyzoite proliferation and worsens disease outcomes. This study sheds light on the molecular mechanisms underlying N. caninum virulence. Further research is needed to elucidate the signaling pathways and protein interactions involving NcBPK1.},
}

*Neospora/pathogenicity/genetics/growth & development
Animals
Virulence
Mice
*Coccidiosis/parasitology/veterinary
*Protozoan Proteins/genetics/metabolism
Female
Cattle
Macrophages/parasitology
Pregnancy
Virulence Factors/genetics
Cattle Diseases/parasitology
CRISPR-Cas Systems
Host-Parasite Interactions
Disease Models, Animal
Gene Deletion

@article {pmid41121349,
year = {2025},
author = {Safarzadeh Kozani, P and Safarzadeh Kozani, P},
title = {Preventing secondary primary malignancies (SPMs) in CAR-T cell therapy through site-specific transgene integration into genomic safe harbors (GSHs).},
journal = {Journal of translational medicine},
volume = {23},
number = {1},
pages = {1155},
pmid = {41121349},
issn = {1479-5876},
mesh = {Humans ; *Transgenes/genetics ; *Neoplasms, Second Primary/prevention & control/genetics ; *Receptors, Chimeric Antigen/metabolism ; Animals ; *Immunotherapy, Adoptive/adverse effects ; Gene Editing ; *Genomics ; CRISPR-Cas Systems ; },
abstract = {Chimeric antigen receptor (CAR)-T cell therapy has revolutionized oncology by achieving durable remissions in refractory hematologic malignancies. However, emerging reports link this therapy to second primary malignancies, including CAR+ lymphomas and leukemias, driven by insertional mutagenesis from semi-random viral vector integration near oncogenes or tumor suppressor loci. These rare but serious complications underscore the dual challenge of eradicating primary tumors while mitigating delayed genotoxic risks. Conventional CAR-T cell manufacturing, reliant on gamma-retroviral or lentiviral vectors, introduces genomic instability through integration into fragile sites or transcriptionally active regions. CRISPR/Cas9-mediated genome editing further amplifies risks via off-target double-strand breaks and chromosomal rearrangements. This review evaluates genomic safe harbors (GSHs)-such as AAVS1, TRAC, CCR5, ROSA26 and CLYBL-as loci validated for stable, high-level CAR transgene expression without oncogenic disruption. GSHs meet stringent criteria: distal from cancer-related genes, resistant to epigenetic silencing, and transcriptionally permissive. Preclinical studies demonstrate that site-directed CAR integration into GSHs preserves antitumor efficacy while eliminating malignant transformation risks. Challenges persist in optimizing homology-directed repair efficiency, mitigating residual dsDNA toxicity, and standardizing regulatory frameworks for long-term genomic surveillance. Emerging technologies-base/prime editing, hybrid nucleases, and rigorous monitoring-promise enhanced precision and safety. By reconciling therapeutic innovation with genomic integrity, GSH-engineered CAR-T cells herald a paradigm shift toward precision immunotherapies, offering curative potential while preempting secondary oncogenesis. Collaborative efforts to refine manufacturing, harmonize global standards, and prioritize patient-specific risk stratification will be critical to advancing this transformative approach.},
}

Humans
*Transgenes/genetics
*Neoplasms, Second Primary/prevention & control/genetics
*Receptors, Chimeric Antigen/metabolism
Animals
*Immunotherapy, Adoptive/adverse effects
Gene Editing
*Genomics
CRISPR-Cas Systems

@article {pmid41121307,
year = {2025},
author = {Li, C and Peng, W and Zhong, Z and Zhang, C and Wang, X and Qin, R and Lei, Q and Lv, J and Liu, F and Zhao, Y and Lv, Z and Li, C and Yang, S and Zhang, H and Tao, Z and Sun, C},
title = {CRISPR/Cas9 library screening reveals that STK19 has synergistic antitumor effects when combined with cisplatin on tongue squamous cell carcinoma.},
journal = {Journal of translational medicine},
volume = {23},
number = {1},
pages = {1142},
pmid = {41121307},
issn = {1479-5876},
support = {82360568, 81960543//Natural Science Foundation of China/ ; 82160452//Natural Science Foundation of China/ ; 202401AY070001-137//Applied Basic Research Foundation of Yunnan Province/ ; 202301AY070001-247//Yunnan Fundamental Research Projects/ ; 2023YJKTB01//Basic Research Program of the First People's Hospital of Qujing/ ; 202305AF150091//Yunnan Province Academician Expert Workstation/ ; },
mesh = {*Cisplatin/pharmacology/therapeutic use ; *Tongue Neoplasms/drug therapy/genetics/pathology ; Humans ; Cell Line, Tumor ; *CRISPR-Cas Systems/genetics ; Animals ; Drug Synergism ; *Carcinoma, Squamous Cell/drug therapy/genetics/pathology ; *Protein Serine-Threonine Kinases/metabolism/genetics ; Xenograft Model Antitumor Assays ; *Antineoplastic Agents/pharmacology/therapeutic use ; DNA Damage/drug effects ; Mice ; Mice, Nude ; Cell Proliferation/drug effects ; },
abstract = {BACKGROUND: Tongue squamous cell carcinoma (TSCC) is a common oral cancer that has a high propensity for recurrence and metastasis. Therefore, TSCC has a 50% 5-year survival rate. Platinum-based chemotherapy is an effective treatment for squamous cell carcinoma, however, chemotherapy resistance remains a major issue. Therefore, innovative and effective drug combinations are needed to improve TSCC patient prognosis.

METHODS: In this study, we conducted an in vitro CRISPR/Cas9 library screen using two TSCC cell lines (Tscca and Cal27) to identify specific genes that, when inhibited, synergize with cisplatin to effectively suppress tumor growth.

RESULTS: We identified STK19 as a potential drug target. Inhibition of STK19 enhances the response of TSCC to cisplatin. Through genetic and pharmacological methods, it has been demonstrated that reducing STK19 activity enhances cisplatin-induced DNA damage. The mechanism involves the depletion of MGMT with STK19 inhibition, leading to conditional lethality and synergistic reduction of tumors in vivo when combined with cisplatin. Overall, in this study, unbiased genetic testing was used to successfully identify synthetic lethal drug combinations for TSCC.

CONCLUSION: STK19 was identified as a promising target that could enhance the killing effects of cisplatin on tongue squamous carcinoma cells, offering a novel therapeutic option for individuals who are insensitive to conventional treatment methods.},
}

*Cisplatin/pharmacology/therapeutic use
*Tongue Neoplasms/drug therapy/genetics/pathology
Humans
Cell Line, Tumor
*CRISPR-Cas Systems/genetics
Animals
Drug Synergism
*Carcinoma, Squamous Cell/drug therapy/genetics/pathology
*Protein Serine-Threonine Kinases/metabolism/genetics
Xenograft Model Antitumor Assays
*Antineoplastic Agents/pharmacology/therapeutic use
DNA Damage/drug effects
Mice
Mice, Nude
Cell Proliferation/drug effects

@article {pmid41121296,
year = {2025},
author = {Shi, X and Lu, S and Tang, Q and Zhao, X},
title = {Targeted modification of cis-elements in the CUL3 gene to restore exon 9 inclusion for treating Gordon syndrome.},
journal = {Human genomics},
volume = {19},
number = {1},
pages = {119},
pmid = {41121296},
issn = {1479-7364},
support = {ZR2022MH300//the Shandong Province Natural Science Foundation/ ; 82271540//the National Natural Science Foundation of China/ ; QDFY+X2024131//the Natural Science Foundation of the Affiliated Hospital of Qingdao University/ ; },
mesh = {*Cullin Proteins/genetics ; Humans ; *Exons/genetics ; RNA Splice Sites/genetics ; Introns/genetics ; RNA Splicing/genetics ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {BACKGROUND: The weak splice acceptor site (AS) of exon 9 underlies almost all pathogenic variants of Cullin3 (CUL3) causing exon 9 skipping in Gordon syndrome, emphasizing the need for splicing-targeted therapeutic strategies. This study explored universal therapeutic targets to modulate AS and investigated their potential and mechanisms for restoring normal splicing.

RESULTS: Through bioinformatic prediction, minigene assays, EMSA, CRISPR/Cas9-mediated construction of mutant cell lines and RIP, three rescue sites in the polypyrimidine (Py) tract of intron 8 were identified, including A(-9)T, A(-10)T and AA(-9, -10)TT, with AA(-9, -10)TT most effectively promoting exon inclusion by extending the Py-tract to increase U2AF2 binding. Additionally, previous candidate target A18G was confirmed to rescue exon 9 skipping by weakening hnRNP A1 splicing inhibition in endogenous cell models.

CONCLUSIONS: Our findings highlight the therapeutic potential of AA(-9, -10)TT and A18G in CUL3-related Gordon syndrome, suggesting the targeted modification of cis-elements could be an ideal and universal strategy to develop treatments for splicing-related diseases.},
}

*Cullin Proteins/genetics
Humans
*Exons/genetics
RNA Splice Sites/genetics
Introns/genetics
RNA Splicing/genetics
CRISPR-Cas Systems/genetics
HEK293 Cells

@article {pmid41120304,
year = {2025},
author = {Wang, Z and Liu, F and Chen, N and Wu, J and Li, X and Fang, M and Yan, M and Zhang, J and Deng, B and Wang, L and Wang, X and Liu, M and Zeng, D and Zou, Z and Wang, B and Songyang, Z and He, B and Liu, Q},
title = {Chromatin looping-based CRISPR screen identifies TLK2 as chromatin loop formation regulator in cancer stemness plasticity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9288},
pmid = {41120304},
issn = {2041-1723},
support = {82321003//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82341020//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82473123//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82173367//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82403653//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82302929//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82303009//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2022A1515010915//Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)/ ; },
mesh = {Humans ; *Chromatin/metabolism/genetics ; Animals ; *Neoplastic Stem Cells/metabolism/pathology ; Female ; Mice ; Kruppel-Like Factor 4 ; *Breast Neoplasms/genetics/pathology/metabolism ; Cell Line, Tumor ; *Protein Serine-Threonine Kinases/metabolism/genetics ; CCCTC-Binding Factor/metabolism/genetics ; Cell Plasticity/genetics ; Kruppel-Like Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; Cell Cycle Proteins/metabolism ; Chromosomal Proteins, Non-Histone/metabolism ; Lung Neoplasms/secondary/genetics ; },
abstract = {Targeting cancer cell plasticity through chromatin organization is an emerging research area, yet the molecular mechanisms that govern chromatin loop formation remain unclear. Here, we develop a CRISPR screen based on our engineered live-cell CTCF-cohesin contact reporters to identify regulators of chromatin loops. Our findings reveal that tousled-like kinase 2 (TLK2) functions as a key regulator of chromatin loop formation during the cancer stemness transition. Mechanistically, TLK2 phosphorylates DYNLL1, enhancing its interaction with CTCF to promote CTCF-cohesin hub formation at the KLF4 locus. Suppressing TLK2 impairs cancer stemness plasticity, sensitizes cancer cells to cytotoxic stress in vitro, and reduces lung metastases and enhances immunotherapy response in breast cancer mouse models. Clinically, elevated TLK2 expression correlates with poor prognosis in breast cancer patients. Collectively, these findings identify TLK2 as a potential therapeutic target for mitigating cancer stemness plasticity, highlighting chromatin loop-targeting therapy as a promising strategy to eradicate cancer stem cells.},
}

Humans
*Chromatin/metabolism/genetics
Animals
*Neoplastic Stem Cells/metabolism/pathology
Female
Mice
Kruppel-Like Factor 4
*Breast Neoplasms/genetics/pathology/metabolism
Cell Line, Tumor
*Protein Serine-Threonine Kinases/metabolism/genetics
CCCTC-Binding Factor/metabolism/genetics
Cell Plasticity/genetics
Kruppel-Like Transcription Factors/genetics/metabolism
CRISPR-Cas Systems
Gene Expression Regulation, Neoplastic
Cell Cycle Proteins/metabolism
Chromosomal Proteins, Non-Histone/metabolism
Lung Neoplasms/secondary/genetics

@article {pmid41118579,
year = {2025},
author = {Hanai, Y and Hilario, PLL and Shiraishi, Y and Yoshida, N and Murakami, S and Shimizu, Y and Kano, N and Kojima, M and Murai, K and Kawai, T and Okamura, K},
title = {The knock-in atlas: a web resource for targeted protein trap by CRISPR/Cas9 in human and mouse cell lines.},
journal = {Nucleic acids research},
volume = {53},
number = {19},
pages = {},
doi = {10.1093/nar/gkaf1050},
pmid = {41118579},
issn = {1362-4962},
support = {//NAIST for the Creation of Innovation in Science and Technology/ ; 24KJ1692//JSPS Research Fellowship/ ; 25KJ1828//NAIST Granite Program/ ; 17K20145//JSPS Found for the Promotion of Joint International Research/ ; //Academic Assistant Grant by Office for Gender Equality at NAIST/ ; //Takeda Science Foundation/ ; 20H03468//KAKENHI/ ; 23K14546//JSPS/ ; },
mesh = {Humans ; Animals ; *CRISPR-Cas Systems/genetics ; Mice ; *Gene Knock-In Techniques/methods ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics ; Internet ; Cell Line ; RNA-Binding Proteins/genetics ; HeLa Cells ; *Databases, Genetic ; },
abstract = {Various cell engineering techniques have been developed by leveraging the CRISPR-Cas9 technology, but large-scale resources for targeted gene knock-in are still limited. Here we introduce the Knock-in Atlas, a web resource for gene tagging by fluorescent proteins by inserting artificial exons in target gene introns. To produce knock-in cells efficiently and reproducibly, we carefully chose and catalogued guide RNAs (gRNAs) for targeting genes in the human and mouse genomes by taking the gRNA efficacy scores and protein structures around the insertion sites into account. As of August 2025, we have characterized knock-in cell lines for 350 proteins, with a focus on RNA binding proteins, by flow cytometry and confocal microscopy. The transfection and flow cytometry protocols were optimized for several cell lines including HEK293T, eHAP1, HeLa, THP-1, Neuro2a, mouse embryonic fibroblast (MEF) and mouse embryonic stem cell (mESC). A website has been launched to organize the results of initial characterization including flow cytometry data after transfection, confocal microscopy, and western blot results for the genes for which knock-in HEK293T cell lines were already made. The site also provides a database to organize the information of pre-designed gRNAs for the human and mouse genomes. .},
}

Humans
Animals
*CRISPR-Cas Systems/genetics
Mice
*Gene Knock-In Techniques/methods
HEK293 Cells
RNA, Guide, CRISPR-Cas Systems/genetics
Internet
Cell Line
RNA-Binding Proteins/genetics
HeLa Cells
*Databases, Genetic

@article {pmid41118576,
year = {2025},
author = {Shu, WJ and Ma, Z and Jia, L and Guo, B and Tian, X and He, C and Wang, F},
title = {MiR-ON-CRISPR: a microRNA-activated CRISPR-dCas9 system for precise gene therapy in living cells and mouse models of sepsis.},
journal = {Nucleic acids research},
volume = {53},
number = {19},
pages = {},
doi = {10.1093/nar/gkaf1037},
pmid = {41118576},
issn = {1362-4962},
support = {32271512//National Natural Science Foundation of China/ ; 82572281//National Natural Science Foundation of China/ ; 32201199//National Natural Science Foundation of China/ ; 2023-JC-ZD-43//Natural Science Basic Research Program of Shaanxi/ ; 2025JC-YBQN-1129//Natural Science Basic Research Program of Shaanxi/ ; 2022JC-56//Natural Science Basic Research Program of Shaanxi/ ; 2024SF-YBXM-138//Key Research and Development Program of Shaanxi/ ; 0959202513035//Young Talent Fund of Xi'an Association for Science and Technology/ ; 24YXYJ0031//Xi'an Science and Technology Research Program/ ; },
mesh = {Animals ; *MicroRNAs/genetics/metabolism ; *Sepsis/therapy/genetics/pathology ; *CRISPR-Cas Systems ; Mice ; Disease Models, Animal ; *Genetic Therapy/methods ; Humans ; Gene Editing/methods ; Endoplasmic Reticulum Stress/genetics ; Mice, Inbred C57BL ; RNA, Guide, CRISPR-Cas Systems/genetics ; NF-E2-Related Factor 2/genetics/metabolism ; },
abstract = {The CRISPR-dCas9 technology is a powerful tool for manipulating the expression of target genes in a variety of biomedical applications. Nevertheless, it is imperative that the activity of the CRISPR-dCas9 system be tightly controlled to improve its safety and applicability. In this study, we successfully designed a microRNA-activated CRISPR-dCas9 system, termed miR-ON-CRISPR, in which the core components (dCas9 and sgRNA) are both regulated by endogenous miRNA. Our findings demonstrated that the miR-ON-CRISPR system can regulate firefly luciferase reporter gene expression to faithfully visualize miRNA activity and image the differentiation status of neural cells. Moreover, the miR-ON-CRISPR was designed as an AND/OR gate system, thereby enabling the simultaneous detection of two distinct miRNAs. Furthermore, the system was adapted to achieve cell type-specific killing by activating the exogenous DTA genes or endogenous BAX genes. Finally, in mouse models of sepsis, the miR-ON-CRISPR system was shown to alleviate the sepsis-induced liver injury as well as the associated oxidative stress damage and endoplasmic reticulum stress via activating the nuclear erythroid 2-related factor 2 gene. In conclusion, this proof-of-concept study demonstrates the feasibility of the miR-ON-CRISPR system for cell type-specific control of CRISPR-dCas9 activity and its therapeutic applications in the treatment of genetic diseases.},
}

Animals
*MicroRNAs/genetics/metabolism
*Sepsis/therapy/genetics/pathology
*CRISPR-Cas Systems
Mice
Disease Models, Animal
*Genetic Therapy/methods
Humans
Gene Editing/methods
Endoplasmic Reticulum Stress/genetics
Mice, Inbred C57BL
RNA, Guide, CRISPR-Cas Systems/genetics
NF-E2-Related Factor 2/genetics/metabolism

@article {pmid41118570,
year = {2025},
author = {Braithwaite, J and Cannon, C and Chalmers, R and Edwards, H},
title = {Single-colony resolution of CRISPR-Cas adaptation in E. coli reveals altered spacer-source bias during solid-phase growth.},
journal = {Nucleic acids research},
volume = {53},
number = {19},
pages = {},
doi = {10.1093/nar/gkaf1044},
pmid = {41118570},
issn = {1362-4962},
support = {RPG-2020-079//Leverhulme Trust/ ; //University of Nottingham/ ; },
mesh = {*Escherichia coli/genetics/growth & development ; *CRISPR-Cas Systems ; Plasmids/genetics ; Escherichia coli Proteins/genetics/metabolism ; CRISPR-Associated Proteins/genetics/metabolism ; Lac Operon ; Adaptation, Physiological/genetics ; *DNA, Intergenic/genetics ; },
abstract = {CRISPR-Cas systems provide adaptive immunity by integrating short DNA fragments from mobile genetic elements into host arrays. While the core biochemical mechanism of adaptation is well defined, its modulation by physiological contexts is less well understood. Here, we present a visual papillation assay that enables single-colony detection of CRISPR-Cas adaptation in Escherichia coli. Spacer acquisition restores the reading frame of a disrupted lacZ gene, forming blue papillae on lactose X-gal plates. The assay is semi-quantitative, highly sensitive, capable of detecting single events among 109 cells, and responds predictably to Cas1-Cas2 expression levels. Spacer mapping revealed a major shift in source bias: in liquid culture, 64% of spacers were plasmid-derived, but on solid medium this dropped to ∼9%. Adjusting inducer concentration to match liquid conditions did not restore plasmid bias, indicating a physiological basis linked to colony growth. Accounting for the molar excess of chromosomal DNA, the 9% plasmid share reflects near-neutral DNA source sampling rather than plasmid overrepresentation. These findings suggest that the spatial and metabolic structure of colonies strongly shapes the adaptation landscape. The assay provides a scalable platform for dissecting condition-specific features of CRISPR-Cas adaptation, including spacer origin, sequence features, and growth context.},
}

*Escherichia coli/genetics/growth & development
*CRISPR-Cas Systems
Plasmids/genetics
Escherichia coli Proteins/genetics/metabolism
CRISPR-Associated Proteins/genetics/metabolism
Lac Operon
Adaptation, Physiological/genetics
*DNA, Intergenic/genetics

@article {pmid41118394,
year = {2025},
author = {Gouin, Y and Wilcockson, A and Chan, AM and Suttle, CA and Zhong, KX},
title = {The genome of Gallaecimonas pentaromativorans strain 10A, isolated from a Pacific oyster, sheds light on an environmentally widespread genus with remarkable metabolic potential.},
journal = {PloS one},
volume = {20},
number = {10},
pages = {e0334406},
pmid = {41118394},
issn = {1932-6203},
mesh = {Animals ; *Genome, Bacterial ; Phylogeny ; *Ostreidae/microbiology ; },
abstract = {Bacteria in the genus Gallaecimonas are known for their ability to breakdown complex hydrocarbons, making them of particular ecological and biotechnological significance. However, few species have been isolated to date, and their ecological distribution has yet to be examined. Here, we report a novel strain of G. pentaromativorans, designated as strain 10A, which was isolated from a Pacific oyster (Magallana gigas, a.k.a. Crassostrea gigas) collected from a farm experiencing a mass mortality event in British Columbia (BC), Canada. Gallaecimonas pentaromativorans strain 10A is a rod-shaped, motile bacterium and has a circular genome of 4,322,156 bp encoding 3,928 protein-coding sequences (CDS). Phylogenetic analysis showed that strain 10A is closely related to members of G. pentaromativorans. Like other Gallaecimonas members, strain 10A is predicted to harbor specific pathways involved in degrading xenobiotic compounds including polycyclic aromatic hydrocarbons (PAHs), producing biosurfactants, and assimilating nitrate and sulfate; however, it is uniquely equipped with an additional 166 genes belonging to 147 protein families, including a putative higB-higA that likely contributes to enhanced stress response. Strain 10A also possesses Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-associated (Cas) system (CRISPR-Cas), prevalent in Gallaecimonas (detected in three out of four species), implying a potential defense mechanism against exogenous mobile genetic elements such as plasmids and viruses. We also mined publicly available databases to establish the widespread distribution of bacteria in the genus Gallaecimonas in seawater, sediments, and freshwater across latitude, suggesting its versatility and importance to environmental processes. Ultimately, this study demonstrates that the genome of G. pentaromativorans strain 10A, isolated from a Pacific oyster, may encode a suite of putative functions, including xenobiotic breakdown, biosurfactant production, and CRISPR-Cas defense. This plasticity and breadth in metabolic function help to explain the cosmopolitan distribution of members of this genus.},
}

Animals
*Genome, Bacterial
Phylogeny
*Ostreidae/microbiology

@article {pmid41068553,
year = {2025},
author = {Wang, YM and Xu, T and Duan, JQ and Zhao, L and You, D and Lai, SY and Qing, Y and Ge, LP and Liu, ZH and Sun, J and Zeng, X and Xu, ZW and Zhu, L},
title = {An Integrated One-Tube RPA-CRISPR/Cas13d Assay Coupled with Lateral Flow for Rapid PRRSV-1 Detection.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {42},
pages = {27080-27088},
doi = {10.1021/acs.jafc.5c07919},
pmid = {41068553},
issn = {1520-5118},
mesh = {*CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; Animals ; *Porcine respiratory and reproductive syndrome virus/genetics/isolation & purification ; Swine ; *Porcine Reproductive and Respiratory Syndrome/virology/diagnosis ; RNA, Viral/genetics ; },
abstract = {Rapid and scalable diagnostic technologies are essential for controlling infectious diseases. We present STEP (Single-Tube Extraction-free Platform for CRISPR/Cas13d detection), a streamlined, equipment-minimal CRISPR-based platform enabling rapid, sensitive, and accurate viral RNA detection. STEP integrates isothermal RPA amplification with CRISPR-based nucleic acid cleavage, providing multimodal readouts including lateral flow strips, in-tube fluorescence, and fluorescence quantification. Lab-free extraction reagents and lyophilized formulations enhance user friendliness of STEP and stability for point-of-care testing (POCT), reduce cost, and eliminate cold-chain requirements. Optimization of baseline time and the ability to operate at both ambient and body temperatures minimize temporal and equipment constraints. Clinical evaluation showed 100% sensitivity and specificity versus RT-qPCR, delivering a sample-to-answer workflow within 35 min. STEP provides a robust platform for decentralized infectious disease diagnostics and rapid public health response, combining speed, user friendliness, and minimal instrumentation requirements.},
}

*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods/instrumentation
Animals
*Porcine respiratory and reproductive syndrome virus/genetics/isolation & purification
Swine
*Porcine Reproductive and Respiratory Syndrome/virology/diagnosis
RNA, Viral/genetics

@article {pmid41015034,
year = {2025},
author = {Fontana, L and Martinucci, P and Amistadi, S and Felix, T and Mombled, M and Tachtsidi, A and Corre, G and Chalumeau, A and Hardouin, G and Martin, J and Romano, O and Amendola, M and Antoniou, P and Miccio, A},
title = {Multiplex base editing of BCL11A regulatory elements to treat sickle cell disease.},
journal = {Cell reports. Medicine},
volume = {6},
number = {10},
pages = {102376},
doi = {10.1016/j.xcrm.2025.102376},
pmid = {41015034},
issn = {2666-3791},
mesh = {*Anemia, Sickle Cell/genetics/therapy ; *Gene Editing/methods ; Humans ; Fetal Hemoglobin/metabolism/genetics ; Animals ; *Repressor Proteins/genetics ; CRISPR-Cas Systems/genetics ; Mice ; Hematopoietic Stem Cells/metabolism ; *Regulatory Sequences, Nucleic Acid/genetics ; DNA Breaks, Double-Stranded ; *Carrier Proteins/genetics/metabolism ; },
abstract = {Sickle cell disease (SCD) is a genetic anemia caused by the production of an abnormal adult hemoglobin. Elevated levels of fetal hemoglobin (HbF) in adulthood reduce disease severity. A promising therapy involves the treatment of hematopoietic stem/progenitor cells (HSPCs) with CRISPR-Cas9 to downregulate the HbF repressor BCL11A via generation of double-strand breaks (DSBs) in the +58-kb enhancer. To improve safety and HbF induction, we use base editors to target both the +58-kb and +55-kb enhancers without generating DSBs. We dissect key DNA motifs recognized by transcriptional activators and identify critical nucleotides. Multiplex base editing efficiently disrupts these sites, reactivating HbF to levels exceeding those achieved with CRISPR-Cas9-induced editing, while minimizing DSBs and genomic rearrangements. Base editing is effective in long-term repopulating HSPCs and results in robust HbF reactivation in vivo. These findings demonstrate that multiplex base editing of BCL11A enhancers is a safe, efficient, and durable strategy to treat SCD.},
}

*Anemia, Sickle Cell/genetics/therapy
*Gene Editing/methods
Humans
Fetal Hemoglobin/metabolism/genetics
Animals
*Repressor Proteins/genetics
CRISPR-Cas Systems/genetics
Mice
Hematopoietic Stem Cells/metabolism
*Regulatory Sequences, Nucleic Acid/genetics
DNA Breaks, Double-Stranded
*Carrier Proteins/genetics/metabolism

@article {pmid40993398,
year = {2025},
author = {Datlinger, P and Pankevich, EV and Arnold, CD and Pranckevicius, N and Lin, J and Romanovskaia, D and Schaefer, M and Piras, F and Orts, AC and Nemc, A and Biesaga, PN and Chan, M and Neuwirth, T and Artemov, AV and Li, W and Ladstätter, S and Krausgruber, T and Bock, C},
title = {Systematic discovery of CRISPR-boosted CAR T cell immunotherapies.},
journal = {Nature},
volume = {646},
number = {8086},
pages = {963-972},
pmid = {40993398},
issn = {1476-4687},
mesh = {Humans ; Animals ; Mice ; *Receptors, Chimeric Antigen/immunology/genetics/metabolism ; *T-Lymphocytes/immunology/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Immunotherapy, Adoptive/methods ; Female ; Gene Editing ; Gene Knockout Techniques ; rho GTP-Binding Proteins/genetics/deficiency ; Xenograft Model Antitumor Assays ; Male ; Leukemia/therapy/immunology/genetics ; *Immunotherapy/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Chimeric antigen receptor (CAR) T cell therapy has shown remarkable success in treating blood cancers, but CAR T cell dysfunction remains a common cause of treatment failure[1]. Here we present CELLFIE, a CRISPR screening platform for enhancing CAR T cells across multiple clinical objectives. We performed genome-wide screens in human primary CAR T cells, with readouts capturing key aspects of T cell biology, including proliferation, target cell recognition, activation, apoptosis and fratricide, and exhaustion. Screening hits were prioritized using a new in vivo CROP-seq[2] method in a xenograft model of human leukaemia, establishing several gene knockouts that boost CAR T cell efficacy. Most notably, we discovered that RHOG knockout is a potent and unexpected CAR T cell enhancer, both individually and together with FAS knockout, which was validated across multiple in vivo models, CAR designs and sample donors, and in patient-derived cells. Demonstrating the versatility of the CELLFIE platform, we also conducted combinatorial CRISPR screens to identify synergistic gene pairs and saturation base-editing screens to characterize RHOG variants. In summary, we discovered, validated and biologically characterized CRISPR-boosted CAR T cells that outperform standard CAR T cells in widely used benchmarks, establishing a foundational resource for optimizing cell-based immunotherapies.},
}

Humans
Animals
Mice
*Receptors, Chimeric Antigen/immunology/genetics/metabolism
*T-Lymphocytes/immunology/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Immunotherapy, Adoptive/methods
Female
Gene Editing
Gene Knockout Techniques
rho GTP-Binding Proteins/genetics/deficiency
Xenograft Model Antitumor Assays
Male
Leukemia/therapy/immunology/genetics
*Immunotherapy/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid40993381,
year = {2025},
author = {Knudsen, NH and Escobar, G and Korell, F and Kienka, T and Nobrega, C and Anderson, S and Cheng, AY and Zschummel, M and Armstrong, A and Bouffard, A and Kann, MC and Goncalves, S and Pope, HW and Pezeshki, M and Rojas, A and Suermondt, JSMT and Phillips, M and Berger, TR and Park, S and Salas-Benito, D and Darnell, EP and Birocchi, F and Leick, MB and Larson, RC and Doench, JG and Sen, D and Yates, KB and Manguso, RT and Maus, MV},
title = {In vivo CRISPR screens identify modifiers of CAR T cell function in myeloma.},
journal = {Nature},
volume = {646},
number = {8086},
pages = {953-962},
pmid = {40993381},
issn = {1476-4687},
mesh = {*Multiple Myeloma/therapy/immunology/genetics/pathology ; Animals ; Humans ; Mice ; *Receptors, Chimeric Antigen/immunology/metabolism/genetics ; *T-Lymphocytes/immunology/cytology/metabolism ; *CRISPR-Cas Systems/genetics ; Female ; *Immunotherapy, Adoptive ; Male ; Cell Proliferation ; Cell Line, Tumor ; B-Cell Maturation Antigen/immunology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Chimeric antigen receptor (CAR) T cells are highly effective in haematological malignancies[1]. However, progressive loss of CAR T cells contributes to relapse in many patients[2-4]. Here we performed in vivo loss-of-function CRISPR screens in CAR T cells targeting B cell maturation antigen to investigate genes that influence CAR T cell persistence and function in a human multiple myeloma model. We tracked the expansion and persistence of CRISPR library-edited T cells in vitro and at early and late time points in vivo to track the performance of gene-modified CAR T cells from manufacturing to survival in tumours. The screens revealed context-specific regulators of CAR T cell expansion and persistence. Ablation of RASA2 and SOCS1 enhanced T cell expansion in vitro, whereas loss of PTPN2, ZC3H12A and RC3H1 conferred early growth advantages to CAR T cells in vivo. Notably, we identified cyclin-dependent kinase inhibitor 1B (encoded by CDKN1B), a cell cycle regulator, as the most important factor limiting CAR T cell fitness at late time points in vivo. CDKN1B ablation increased CAR T cell proliferation and effector function, significantly enhancing tumour clearance and overall survival. Our findings reveal differing effects of gene perturbation on CAR T cells over time and in different environments, highlight CDKN1B as a promising target to generate highly effective CAR T cells for multiple myeloma and underscore the potential of in vivo screening for identifying genes to enhance CAR T cell efficacy.},
}

*Multiple Myeloma/therapy/immunology/genetics/pathology
Animals
Humans
Mice
*Receptors, Chimeric Antigen/immunology/metabolism/genetics
*T-Lymphocytes/immunology/cytology/metabolism
*CRISPR-Cas Systems/genetics
Female
*Immunotherapy, Adoptive
Male
Cell Proliferation
Cell Line, Tumor
B-Cell Maturation Antigen/immunology
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid40993380,
year = {2025},
author = {Mittler, E and Tse, AL and Tran, PT and Florez, C and Janer, J and Varnaite, R and Kasikci, E and Mv, VK and Loomis, M and Christ, W and Cazares, E and Bakken, RR and Martin, CK and Zeng, X and Raymond, JL and Shahsavani, M and Khanal, S and Wilkinson, ER and Oktavia, RM and Slough, MM and Haslwanter, D and Han, J and Berrigan, J and Rosendal, E and Kielian, M and Manicassamy, B and Överby, AK and Falk, A and Barba-Spaeth, G and Rey, FA and Klingström, J and Gavathiotis, E and Herbert, AS and Chandran, K and Gredmark-Russ, S},
title = {LRP8 is a receptor for tick-borne encephalitis virus.},
journal = {Nature},
volume = {646},
number = {8086},
pages = {945-952},
pmid = {40993380},
issn = {1476-4687},
mesh = {Animals ; Mice ; Humans ; *Encephalitis Viruses, Tick-Borne/physiology/metabolism/pathogenicity ; Reelin Protein ; *LDL-Receptor Related Proteins/metabolism/genetics ; *Encephalitis, Tick-Borne/virology/prevention & control/metabolism ; *Receptors, Virus/metabolism/genetics ; Female ; Cell Line ; Virus Internalization ; Viral Envelope Proteins/metabolism ; Male ; Neurons/virology/metabolism ; Brain/metabolism/virology ; Virus Attachment ; HEK293 Cells ; CRISPR-Cas Systems/genetics ; },
abstract = {Tick-borne encephalitis virus (TBEV) causes tick-borne encephalitis (TBE), a severe and sometimes life-threatening disease characterized by viral invasion of the central nervous system with symptoms of neuroinflammation[1,2]. As with other orthoflaviviruses-enveloped, arthropod-borne RNA viruses-host factors required for TBEV entry remain poorly defined. Here we used a genome-scale CRISPR-Cas9-based screen to identify LRP8, an apolipoprotein E and reelin receptor with high expression in the brain, as a TBEV receptor. LRP8 downregulation reduced TBEV infection in human cells, and its overexpression enhanced infection. LRP8 bound directly to the TBEV E glycoprotein and mediated viral attachment and internalization into cells. An LRP8-based soluble decoy blocked infection of human cell lines and neuronal cells and protected mice from lethal TBEV challenge. LRP8's role as a TBEV receptor has implications for TBEV neuropathogenesis and the development of antiviral countermeasures.},
}

Animals
Mice
Humans
*Encephalitis Viruses, Tick-Borne/physiology/metabolism/pathogenicity
Reelin Protein
*LDL-Receptor Related Proteins/metabolism/genetics
*Encephalitis, Tick-Borne/virology/prevention & control/metabolism
*Receptors, Virus/metabolism/genetics
Female
Cell Line
Virus Internalization
Viral Envelope Proteins/metabolism
Male
Neurons/virology/metabolism
Brain/metabolism/virology
Virus Attachment
HEK293 Cells
CRISPR-Cas Systems/genetics

@article {pmid40963013,
year = {2025},
author = {Tamura, S and Nelson, AD and Spratt, PWE and Hamada, EC and Zhou, X and Kyoung, H and Li, Z and Arnould, C and Barskyi, V and Krupkin, B and Young, K and Zhao, J and Holden, SS and Sahagun, A and Keeshen, CM and Lu, C and Ben-Shalom, R and Taloma, SE and Schamiloglu, S and Li, YC and Min, L and Jenkins, PM and Pan, JQ and Paz, JT and Sanders, SJ and Matharu, N and Ahituv, N and Bender, KJ},
title = {CRISPR activation for SCN2A-related neurodevelopmental disorders.},
journal = {Nature},
volume = {646},
number = {8086},
pages = {983-991},
pmid = {40963013},
issn = {1476-4687},
mesh = {Animals ; Mice ; *NAV1.2 Voltage-Gated Sodium Channel/genetics/deficiency ; Humans ; *Neurodevelopmental Disorders/genetics/therapy/physiopathology ; Haploinsufficiency/genetics ; Dependovirus/genetics ; Male ; Female ; *CRISPR-Cas Systems/genetics ; Seizures/genetics/therapy/chemically induced ; Pyramidal Cells/metabolism/pathology ; Gene Knock-In Techniques ; Phenotype ; Synapses/metabolism ; Disease Models, Animal ; Proof of Concept Study ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Genetic Therapy/methods ; },
abstract = {Most neurodevelopmental disorders with single gene diagnoses act via haploinsufficiency, in which only one of the two gene copies is functional[1]. SCN2A haploinsufficiency is one of the most frequent causes of neurodevelopmental disorder, often presenting with autism spectrum disorder, intellectual disability and, in a subset of children, refractory epilepsy[2]. Here, using SCN2A haploinsufficiency as a proof-of-concept, we show that upregulation of the existing functional gene copy through CRISPR activation (CRISPRa) can rescue neurological-associated phenotypes in Scn2a haploinsufficient mice. We first show that restoring Scn2a expression in adolescent heterozygous Scn2a conditional knock-in mice rescues electrophysiological deficits associated with Scn2a haploinsufficiency (Scn2a[+/-]). Next, using an adeno-associated virus CRISPRa-based treatment in adolescent mice, we show that we can correct intrinsic and synaptic deficits in neocortical pyramidal cells, a major cell type that contributes to neurodevelopmental disorders and seizure aetiology in SCN2A haploinsufficiency. Furthermore, we find that systemic delivery of CRISPRa protects Scn2a[+/-] mice against chemoconvulsant-induced seizures. Finally, we also show that adeno-associated virus CRISPRa treatment rescues excitability in SCN2A haploinsufficient human stem-cell-derived neurons. Our results showcase the potential of this therapeutic approach to rescue SCN2A haploinsufficiency and demonstrates that rescue even at adolescent stages can ameliorate neurodevelopmental phenotypes.},
}

Animals
Mice
*NAV1.2 Voltage-Gated Sodium Channel/genetics/deficiency
Humans
*Neurodevelopmental Disorders/genetics/therapy/physiopathology
Haploinsufficiency/genetics
Dependovirus/genetics
Male
Female
*CRISPR-Cas Systems/genetics
Seizures/genetics/therapy/chemically induced
Pyramidal Cells/metabolism/pathology
Gene Knock-In Techniques
Phenotype
Synapses/metabolism
Disease Models, Animal
Proof of Concept Study
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Genetic Therapy/methods

@article {pmid40919940,
year = {2025},
author = {Le, Y and Liu, X and Zhou, S and Wu, P and Zhang, M and Sun, J and Ni, J and Wang, H},
title = {A thermostable Cas9-based genome editing system for thermophilic acetogenic bacterium Thermoanaerobacter kivui.},
journal = {Applied and environmental microbiology},
volume = {91},
number = {10},
pages = {e0117025},
doi = {10.1128/aem.01170-25},
pmid = {40919940},
issn = {1098-5336},
support = {32470130//National Natural Science Foundation of China/ ; BK20231326//Natural Science Foundation of Jiangsu Province/ ; 2020YFA0906800//National Key Research and Development Program of China/ ; M2022-10//State Key Laboratory of Microbial Technology Open Projects Fund/ ; },
mesh = {*Thermoanaerobacter/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Ethanol/metabolism ; Alcohol Dehydrogenase/genetics/metabolism ; Metabolic Engineering ; Genome, Bacterial ; *CRISPR-Associated Protein 9/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Hot Temperature ; },
abstract = {Thermoanaerobacter kivui is a thermophilic acetogenic bacterium capable of thriving at elevated temperatures up to 66°C. It metabolizes carbohydrates such as glucose, mannose, and fructose and can also grow lithotrophically utilizing hydrogen (H2) and carbon dioxide (CO2) or carbon monoxide (CO), with acetate serving as its main product. A simple and efficient genome editing system for T. kivui would not only facilitate the understanding of the physiological function of enzymes involved in energy and carbon metabolism but also enable metabolic engineering. To address this issue, we developed a thermostable Cas9-based genome editing system for targeted gene knockout and gene integration into the T. kivui genome. Gene knockout assays were conducted on the adh gene, responsible for encoding alcohol dehydrogenase, and the ldh gene, encoding lactate dehydrogenase. Furthermore, the adhE gene from Thermoanaerobacter ethanolicus, which encodes a bifunctional aldehyde/alcohol dehydrogenase enzyme, was successfully integrated into the T. kivui genome. As a result, the engineered strain was able to produce ethanol. Following a liquid culturing period with kanamycin sulfate for about 72 hours, the efficiency of gene editing was enhanced, resulting in a ratio of mutants out of all colonies obtained of 90%. The results confirm the validity and efficiency of the thermostable Cas9-based genome editing system in T. kivui for gene editing.IMPORTANCEThermophilic acetogenic microorganisms represent an emerging metabolic engineering platform for the production of various biochemicals from hydrogen and carbon dioxide, or synthesis gas, under conditions of high-temperature fermentation. Gas fermentation has gained significant research interest due to its excellent thermodynamics, economic feasibility, and multisubstrate utilization. However, a major obstacle to the use of thermophilic acetogenic microorganisms as metabolic engineering platforms is the scarcity of genetic tools. This study demonstrates a proof of concept for a thermostable Cas9-based genome editing of the thermophilic acetogenic bacterium T. kivui. The system is an important expansion to the genetic toolbox of T. kivui, enabling a better understanding of key enzyme functions and the construction of cell factories for the biotechnological conversion of carbon dioxide and organic substrates into value-added products.},
}

*Thermoanaerobacter/genetics/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems
Ethanol/metabolism
Alcohol Dehydrogenase/genetics/metabolism
Metabolic Engineering
Genome, Bacterial
*CRISPR-Associated Protein 9/genetics/metabolism
Bacterial Proteins/genetics/metabolism
Hot Temperature

@article {pmid40891847,
year = {2025},
author = {Li, L and Luo, K and Zhang, S and Wang, X and Wang, S and Liu, X and Zang, S and Liu, Y and Zhou, C and Luo, C},
title = {A three-plasmid-containing CRISPR-Cas9 platform to engineer Bacillus velezensis 916 as an efficient biocontrol agent.},
journal = {Applied and environmental microbiology},
volume = {91},
number = {10},
pages = {e0138925},
doi = {10.1128/aem.01389-25},
pmid = {40891847},
issn = {1098-5336},
mesh = {*Bacillus/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Plasmids/genetics ; *Biological Control Agents ; Plant Diseases/prevention & control/microbiology ; Lipopeptides/genetics ; Bacterial Proteins/genetics ; Peptide Synthases/genetics ; Oryza/microbiology ; },
abstract = {Bacillus velezensis (Bv) is a widely used biocontrol agent against plant diseases, mainly because its genome contains numerous non-ribosomal peptide synthetases (NRPS) gene clusters for the synthesis of various cyclic lipopeptides (CLPs). The domesticated strain Bv916, capable of co-producing four CLPs, has been successfully applied for green control of rice sheath blight and angular leaf spot. To enhance Bv916's biological control efficacy while maintaining environmental safety, it is essential to establish a food-grade gene editing platform in Bv916. Here, a three-plasmid CRISPR-Cas9 platform for Bv916 was constructed using the thermosensitive origin pET194ts, constitutive P43 promoters for Cas9, the specific promoter Psrf for single guide RNAs (sgRNAs), and three resistance gene expression cassettes. By replacing the native promoters of ComX and RecA in Bv916 with the strong promoters P43 and PrepU, respectively, this platform achieved a single-gene editing efficiency of 96%, while the simultaneous dual-gene editing efficiency reached 61%, with each round completed within five business days. Furthermore, this gene editing platform is used to replace promoters of four NRPS gene clusters (loc, srf, bl, and fen) in Bv916 with strong constitutive promoters (PB, PA, P43, and PrepU), generating the derivative BvLSBF. Compared to Bv916, BvLSBF showed 6.8-fold, 5.9-fold, 10.9-fold, and 6.2-fold increases in locillomycin, surfactin, bacillomycin L, and fengycin, respectively. Its antagonistic activity against plant pathogens was also significantly enhanced. This system enables further development of Bv916 as a cell factory and integration of multiple biocontrol factors, offering significant potential for sustainable agriculture.IMPORTANCEIn this study, a food-grade three-plasmid CRISPR-Cas9 platform for Bv916 was established by incorporating the optimized BvCas9 under the constitutive promoter P43, single guide RNAs (sgRNAs), and homologous recombination fragments into three thermosensitive shuttle vectors. This gene editing system was used to achieve gene insertion, deletion, and replacement in Bv916, particularly by editing four non-ribosomal peptide synthetase (NRPS) gene clusters. This resulted in increased production of four cyclic lipopeptides and significantly enhanced antibacterial and antifungal activity.},
}

*Bacillus/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Plasmids/genetics
*Biological Control Agents
Plant Diseases/prevention & control/microbiology
Lipopeptides/genetics
Bacterial Proteins/genetics
Peptide Synthases/genetics
Oryza/microbiology

@article {pmid41118255,
year = {2025},
author = {Ong, CJN and Elesho, OE and Bramwell, BB and Cabuhat, KSP and Bacalzo, GD and Nuevo, JJM and Fortaleza, JAG},
title = {Staphylococcus aureus: Antimicrobial resistance, quorum sensing, and antibiofilm approaches.},
journal = {European journal of microbiology & immunology},
volume = {},
number = {},
pages = {},
doi = {10.1556/1886.2025.00050},
pmid = {41118255},
issn = {2062-509X},
abstract = {Staphylococcus aureus is a clinically important bacterial pathogen causing infections from superficial skin lesions to life-threatening systemic diseases. The emergence of methicillin-resistant S. aureus (MRSA) has compounded the global health burden, particularly in low- and middle-income countries, as its quorum-sensing (QS) mediated mechanisms contribute to its persistence, resistance, and evasion from host immune responses and antimicrobial treatments. Thus, these features compromise the effectiveness of conventional antibiotics, urging the need for alternative therapeutic approaches. To resolve these issues, several non-antibiotic antibiofilm approaches have been developed. Bacteriophages and phage-derived enzymes show promising specificity in lysing bacterial cells and disrupting biofilms. Antimicrobial peptides (AMPs), with their broad-spectrum activity, destabilize bacterial membranes and modulate immune responses. Monoclonal antibodies can neutralize toxins or inhibit adhesion molecules within biofilms. Phytochemicals have demonstrated activity against QS pathways and efflux pumps. Metal ion chelators like deferiprone interfere with iron acquisition, which is essential for biofilm stability. Nanoparticles (NPs), ranging from metallic and polymeric to lipid-based and cyclodextrin-based systems, enhance drug delivery and biofilm penetration. CRISPR-Cas systems provide precise genome editing to target resistance genes and virulence factors. Rhamnolipids disrupt biofilm matrix integrity, while enzymes such as dispersin B degrade extracellular polymeric substances. Photodynamic and laser therapies offer localized disruption of biofilm structures through oxidative stress. Collectively, this review offers a transformative complementary approach to traditional antibiotics, enhancing treatment efficacy while potentially reducing the emergence of resistance. Continued research on delivery systems, safety profiles, and synergistic combinations will be pivotal for their clinical translation against S. aureus infections.},
}

@article {pmid41117937,
year = {2025},
author = {Menge, S and Segura, I and Hartmann, M and Decker, L and Kiran, S and Danzer, KM and Iben, S and Harbauer, AB and Oeckl, P and Freischmidt, A},
title = {Comparing loss of individual fragile X proteins suggests strong links to cellular senescence and aging.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {82},
number = {1},
pages = {358},
pmid = {41117937},
issn = {1420-9071},
mesh = {Humans ; *Cellular Senescence/genetics ; *Fragile X Mental Retardation Protein/genetics/metabolism ; *Aging/genetics/metabolism ; *RNA-Binding Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Autophagy ; Cell Line, Tumor ; Proteomics/methods ; Mitochondria/metabolism ; Fragile X Syndrome/metabolism/genetics/pathology ; Neurodegenerative Diseases/metabolism/genetics/pathology ; },
abstract = {Members of the fragile X protein (FXP) family (FMR1, FXR1 and FXR2) are differentially expressed in most types of cancer and major neurodegenerative diseases. While increased expression of FXR1 in cancer has been linked to senescence evasion and consequently tumor initiation and progression, decreased expression of FXPs in neurodegeneration may contribute to pathogenic protein aggregation and death of vulnerable neurons. However, due the causal role in fragile x syndrome, most data are available about loss of FMR1 in neurons while functions of FXR1 and especially FXR2 remain largely unexplored. To address this knowledge gap, and to directly compare functions of the FXPs, we used proteomics of CRISPR/Cas9 edited HAP1 cells carrying knockouts of the individual FXPs for identification of cellular mechanisms associated with these proteins. Further exploration of proteomic findings suggests roles of the FXPs in ribosome biogenesis, autophagy and mitochondrial health linked to organismal aging, and cellular senescence. Validation of FXP induced defects relevant for neurodegenerative diseases in neuroblastoma cell line SH-SY5Y upon FXP knockdown revealed high cell type specificity of individual FXP functions. Overall, we provide a comprehensive overview and comparison of cellular mechanisms related to the individual FXPs, as well as starting points for further studying this protein family in respective cell types of FXP associated diseases, and in aging in general.},
}

Humans
*Cellular Senescence/genetics
*Fragile X Mental Retardation Protein/genetics/metabolism
*Aging/genetics/metabolism
*RNA-Binding Proteins/metabolism/genetics
CRISPR-Cas Systems
Autophagy
Cell Line, Tumor
Proteomics/methods
Mitochondria/metabolism
Fragile X Syndrome/metabolism/genetics/pathology
Neurodegenerative Diseases/metabolism/genetics/pathology

@article {pmid41117602,
year = {2025},
author = {Keshry, SS and Nayak, U and Mamidi, P and Mohanty, S and Ghorai, U and Swain, RK and Chattopadhyay, S},
title = {CRISPR-Induced Mutations of mk2b and mk3 Host Proteins Enhance Chikungunya Virus Susceptibility and Modulate Host Immune Responses in Zebrafish.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {39},
number = {20},
pages = {e71112},
doi = {10.1096/fj.202501236RR},
pmid = {41117602},
issn = {1530-6860},
support = {113-2334-6704/2K23/1//CSIR | Human Resource Development Centre, Council of Scientific And Industrial Research (HRDC)/ ; IF180156//Department of Science and Technology, Ministry of Science and Technology, India (DST)/ ; BT/PR42322/TRM/120/525/2021//Department of Biotechnology, Ministry of Science and Technology, India (DBT)/ ; },
mesh = {Animals ; *Zebrafish/genetics/virology/immunology ; *Chikungunya virus/immunology ; *Chikungunya Fever/immunology/genetics/virology ; *Protein Serine-Threonine Kinases/genetics/metabolism ; *Zebrafish Proteins/genetics/metabolism ; *Intracellular Signaling Peptides and Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Mutation ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Host factors are essential at every stage of the viral life cycle and therefore represent attractive and potentially effective targets for the development of antiviral therapeutics. This study highlights the crucial roles of host factors, specifically mitogen-activated protein kinase 2 (mk2) and mitogen-activated protein kinase 3 (mk3), both of which are stress-stimulated serine/threonine kinases. The roles of mk2 and mk3 were investigated by generating single (mk2b-/- and mk3-/-) and double knockouts (mk2b-/-mk3-/-) in a zebrafish model using the CRISPR-Cas9 technique, followed by chikungunya virus (CHIKV) infection. All knockout lines exhibited significantly higher CHIKV titers and severe phenotypes compared to the WT control, with mk3[-]/[-] showing the greatest susceptibility. After CHIKV infection, expression levels of TNF-α changed across all knockout models. Notably, mk2b[-]/[-] and mk2b[-]/[-]mk3[-]/[-] double knockout larvae exhibited reduced TNF-α expression, suggesting that higher levels of TNF-α may be associated with viral clearance via the p38-MK2-TNF-α signaling axis. In contrast, mk3-/- zebrafish exhibited increased vulnerability to CHIKV through alternative, yet unidentified, pathways. Furthermore, an increase in viral titer corresponded with an enhanced host immune response, as indicated by significantly higher expression levels of ifnɸ1 and rsad2 in all knockout groups. In conclusion, this study confirms that the mk2b and mk3 host proteins are essential in controlling CHIKV infection at the organism level. These findings might have implications towards designing strategies for future antiviral therapeutics. Furthermore, the knockout model of mk2b and mk3 in zebrafish could serve as a valuable tool for studying their roles in other viral infections.},
}

Animals
*Zebrafish/genetics/virology/immunology
*Chikungunya virus/immunology
*Chikungunya Fever/immunology/genetics/virology
*Protein Serine-Threonine Kinases/genetics/metabolism
*Zebrafish Proteins/genetics/metabolism
*Intracellular Signaling Peptides and Proteins/genetics/metabolism
CRISPR-Cas Systems
*Mutation
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41116722,
year = {2025},
author = {Lin, Y and Jiang, D and Dong, X and Li, Y and Wu, X and Li, R and Li, F and Sun, D and Yu, Y},
title = {A label-free orthogonal dual-channel CRISPR-Cas platform for simultaneous detection of Mycobacterium tuberculosis and respiratory syncytial virus.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc05269f},
pmid = {41116722},
issn = {1364-548X},
abstract = {A label-free, orthogonal dual-channel CRISPR-Cas platform is developed for the simultaneous detection of diverse pathogens. Cas12a is reported by a G4-PPIX complex, and Cas13 by a split Broccoli-DFHBI aptamer, which ensure exclusive channel specificity and eliminate optical cross-talk. Clinical tests on 50 samples show 100% concordance with PCR.},
}

@article {pmid41115973,
year = {2025},
author = {Mladenov, E and Kallies, M and Stuschke, M and Gkika, E and Iliakis, G},
title = {CRISPR/Cas9 generated DSB clusters mimic complex lesions induced by high-LET radiation and shift repair from c-NHEJ to mutagenic repair pathways.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {36480},
pmid = {41115973},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Humans ; *DNA Breaks, Double-Stranded/radiation effects ; *DNA End-Joining Repair/radiation effects/genetics ; *Linear Energy Transfer ; Hypoxanthine Phosphoribosyltransferase/genetics ; Animals ; *DNA Repair ; Cell Line ; Poly (ADP-Ribose) Polymerase-1/genetics/metabolism ; Mutagenesis ; },
abstract = {DNA double-strand break (DSB) clusters are a hallmark of high-linear energy transfer (high-LET) radiation and are associated with pronounced biological effects, including reduced cell survival and elevated genomic instability. Our previous work in Chinese hamster cells, engineered with variably designed clusters of I-SceI recognition sites, integrated at multiple genomic locations, revealed that DSB clusters suppress classical non-homologous end-joining (c-NHEJ) and induce chromosomal translocations that ultimately increase cell lethality. Here, we extend this line of investigation to human cell lines and generate DSB clusters using alternative approaches that do not require prior genetic manipulation of the test cell lines. We employ CRISPR/Cas9-technology to generate DSB clusters of specific design at a selected genomic locus and examine their consequences on locus integrity. We target Exon 3 of the human HPRT (hHPRT) gene and introduce single DSBs or DSB clusters of varying numbers and inter-DSB distances. Alterations at the locus reflecting hHPRT gene inactivation, are quantified as mutations causing resistance to 6-thioguanine (6TG). Our results show that DSB clusters are markedly more potent inducers of mutations than single DSBs and that DSBs spaced within ~ 600 base pairs synergize in mutation induction. Mechanistic analyses using small-molecule inhibitors and engineered gene knockout cell lines reveal that the increased mutagenicity of clustered DSBs is primarily mediated by DNA end resection and PARP1-dependent alternative end-joining (alt-EJ) pathways. These findings reinforce the biological relevance of DSB clusters as a severe form of complex DNA damage and provide mechanistic insights into high-LET radiation-induced increased cell killing and genomic instability.},
}

*CRISPR-Cas Systems
Humans
*DNA Breaks, Double-Stranded/radiation effects
*DNA End-Joining Repair/radiation effects/genetics
*Linear Energy Transfer
Hypoxanthine Phosphoribosyltransferase/genetics
Animals
*DNA Repair
Cell Line
Poly (ADP-Ribose) Polymerase-1/genetics/metabolism
Mutagenesis

@article {pmid41114873,
year = {2025},
author = {Su, CW and Tsai, LC and Hsu, YC and Lu, YS and Lee, JC and Linacre, A and Hsieh, HM},
title = {Saliva identification by RT-LAMP integrated with CRISPR-Cas and LFA.},
journal = {Forensic science, medicine, and pathology},
volume = {},
number = {},
pages = {},
pmid = {41114873},
issn = {1556-2891},
support = {MOST 110-2320-B-015-001 and MOST 111-2320-B-015-001-MY2//National Science and Technology Council/ ; },
abstract = {Saliva is a frequently encountered body fluid at crime scenes, however currently there are no definite means to rapidly identify a body fluid as being saliva. In this study, a novel detection method for saliva using a modified Loop-mediated Isothermal Amplification (LAMP) integrated with CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated protein) and LFA (Lateral Flow Assay) was developed to detect the expression of a saliva-specific gene: follicular dendric cell secreted protein (FDCSP). To determine the specificity of the assay, RNA from saliva plus other commonly encountered body fluids was tested (peripheral blood, semen, vaginal fluid, and menstrual blood): positive results were only observed from RNA extracted from known saliva samples and RNA from all the other body fluids exhibited a negative result. To assess the reproducibility, triplicates were used from one saliva sample, and the assay was performed on three different days: positive results were observed from all triplicates. The limit of detection was 2[-6] (0.3906 ng RNA) or 2[-7] (0.1953 ng RNA). This preliminary study for the identification of saliva requires no complex equipment and is easy to perform, offering an alternative means for body fluid identification.},
}

@article {pmid41113410,
year = {2025},
author = {Feng, L and Huang, Y and Zhao, R and Zhang, K and Yang, W},
title = {[Effect of Different Caenorhabditis elegans U6 Promoters on the Efficiency of CRISPR/Cas9-Mediated Gene Editing].},
journal = {Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition},
volume = {56},
number = {4},
pages = {1038-1044},
pmid = {41113410},
issn = {1672-173X},
mesh = {Animals ; *Caenorhabditis elegans/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Promoter Regions, Genetic/genetics ; *RNA, Small Nuclear/genetics ; Plasmids/genetics ; Caenorhabditis elegans Proteins/genetics ; },
abstract = {OBJECTIVE: To investigate the effects of Caenorhabditis elegans (C. elegans) endogenous U6 promoters on dpy-10 gene editing efficiency.

METHODS: We screened endogenous U6 small nuclear RNA (snRNA) genes of C. elegans from the WormBase database and constructed 14 editing plasmids targeting dpy-10 by replacing the U6 r07e5.16 promoter in the pSX524 plasmid (Peft-3::cas9::tbb-2 terminator::U6 r07e5.16::dpy-10 sgRNA) through molecular cloning. Gene editing was performed in wild-type C. elegans using a standardized microinjection protocol. Gene editing efficiency and the high-efficiency gene editing index were quantified based on the screening of dpy-10 mutant phenotypes in the F1 progeny.

RESULTS: A total of 15 U6 snRNA genes (r07e5.16, f35c11.9, t20d3.13, k09b11.15, k09b11.16, w05b2.8, c28a5.7, f54c8.9, k09b11.11, k09b11.12, k09b11.14, t20d3.12, f54c8.8, f54c8.10, and k09b11.13) were identified from the WormBase database. Based on the editing efficiency and high-efficiency gene editing index, the activity of these promoters was evaluated, and 4 U6 promoters (w05b2.8, c28a5.7, f54c8.9, and k09b11.11) were found to have significantly enhanced gene editing success rates, outperforming other promoters, including U6 r07e5.16 and U6 k09b11.12 , which are commonly used in the C. elegans research community. Notably, the gRNA[F+E] scaffold did not show superior editing efficiency over the gRNA scaffold when paired with the optimal U6w05b2.8 promoter.

CONCLUSION: In this study, U6 promoters that significantly improve gene editing efficiency in C. elegans are identified and the critical role of promoter optimization in CRISPR-Cas9 systems is highlighted. These findings provide a valuable foundation for improving genome editing strategies and offer new ideas for optimizing the CRISPR technology applied in nematode research.},
}

Animals
*Caenorhabditis elegans/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Promoter Regions, Genetic/genetics
*RNA, Small Nuclear/genetics
Plasmids/genetics
Caenorhabditis elegans Proteins/genetics

@article {pmid41112309,
year = {2025},
author = {Cruz-Cárdenas, JA and López-Arredondo, A and Cázares-Preciado, JA and Rodríguez-Gonzalez, M and Palomares, LA and Brunck, MEG},
title = {Development of CRISPR/Cas9-mediated CD16b[-/-] and CD32a[-/-] promyelocytic cell lines to study FcγR signaling in human neutrophils.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1633609},
pmid = {41112309},
issn = {1664-3224},
mesh = {Humans ; *Receptors, IgG/genetics/metabolism/immunology ; *Neutrophils/immunology/metabolism ; *CRISPR-Cas Systems ; *Signal Transduction/immunology ; HL-60 Cells ; Phagocytosis ; GPI-Linked Proteins/genetics ; Reactive Oxygen Species/metabolism ; Cytokines/metabolism ; },
abstract = {INTRODUCTION: Neutrophils use Fc gamma receptors (FcγRs) to recognize IgG-opsonized pathogens, triggering antimicrobial functions including phagocytosis, ROS production, and cytokine release. CD16b, the most abundant FcγR on neutrophils, plays a key role in initiating these responses, while CD32a is another abundant FcγR on neutrophils that contributes to modulating immune functions. CD16b lacks an intracellular domain and its signaling mechanisms remain unclear. The prevalence of the CD16b-deficient phenotype on donor neutrophils is estimated at <1% of the global population, which complicates its study. To address this, we employed CRISPR/Cas9 to generate HL-60-derived neutrophil-like cells deficient for CD16b or CD32a, that facilitate investigation of their respective roles in neutrophil biology.

METHODS: We disrupted the FCGR3B or FCGR2A genes using CRISPR/Cas9 in the HL-60 cell line and differentiated clones into neutrophil-like cells using 1.3% DMSO. Functional assays were performed, including phagocytosis, ROS production, SYK phosphorylation, and cytokine responses.

RESULTS AND DISCUSSION: Both CD16b[-/-] and CD32a[-/-] HL-60-derived clones maintained neutrophilic differentiation and phagocytic capacity but displayed impaired FcγR-mediated ROS production and SYK phosphorylation, with more pronounced defects in CD16b[-/-] cells. Cytokine production was altered in both lines, with CD16b[-/-] cells producing less IL-6 and IL-1β, and CD32a[-/-] cells producing less TNF-α and IL-10. This model provides new insights into the distinct roles of CD16b and CD32a in neutrophil activation and immune responses.},
}

Humans
*Receptors, IgG/genetics/metabolism/immunology
*Neutrophils/immunology/metabolism
*CRISPR-Cas Systems
*Signal Transduction/immunology
HL-60 Cells
Phagocytosis
GPI-Linked Proteins/genetics
Reactive Oxygen Species/metabolism
Cytokines/metabolism

@article {pmid41110011,
year = {2025},
author = {De La Mora, CL and Havey, MJ and Krysan, PJ},
title = {Efficient production of gene-edited onion (Allium cepa) plants using biolistic delivery of cas9 RNPs and transient expression constructs.},
journal = {Plant cell reports},
volume = {44},
number = {11},
pages = {243},
pmid = {41110011},
issn = {1432-203X},
support = {1844304//Division of Integrative Organismal Systems/ ; },
mesh = {*Onions/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Biolistics/methods ; *CRISPR-Associated Protein 9/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; Plant Proteins/genetics/metabolism ; },
abstract = {Delivery of Cas9/sgRNA RNPs and DNA vectors designed to transiently express morphogenic regulatory genes or an antibiotic resistance gene enabled efficient recovery of gene edited onion lines. We developed a protocol for efficiently producing gene edited onion plants that does not depend on stable transformation. The process makes use of transient gene expression to enrich for gene editing among plants regenerated from immature embryos bombarded with ribonucleoprotein (RNP) complexes composed of CRISPR-associated protein 9 (Cas9) and single guide RNAs (sgRNAs). We used the Allium cepa Downy Mildew Resistant 6 (AcDMR6) gene as the target gene for our studies and produced a total of 47 onion plants with edited AcDMR6 alleles, including 13 homozygous plants, 12 biallelic plants, 7 heterozygous plants, and 15 chimeric plants. The most effective strategy for producing gene edited onion plants involved co-delivering plasmids encoding a hygromycin-resistance protein and plant developmental regulator genes along with the Cas9/sgRNA RNPs followed by transient Hygromycin selection for 48 h. Using this approach, up to 12% of the regenerated onion plants carried edited alleles of AcDMR6. By comparison, no editing was observed among the 146 plants regenerated from explants transfected with the Cas9/sgRNA RNPs alone. The strategy we describe here for using transient gene expression to enrich for gene editing in onion could potentially be extended to other crop species as well.},
}

*Onions/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
*Biolistics/methods
*CRISPR-Associated Protein 9/metabolism/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Ribonucleoproteins/genetics/metabolism
Plant Proteins/genetics/metabolism

@article {pmid41071600,
year = {2025},
author = {Zhang, W and Feng, ZY and Feng, Z and Lian, R and Liu, Z and Zhang, J},
title = {A CRISPR-Customizable Copper-Coordinated DNA Nanoplatform Potentiates Cuproptosis Through Circadian and Metabolic Pathway Manipulation.},
journal = {ACS nano},
volume = {19},
number = {41},
pages = {36701-36717},
doi = {10.1021/acsnano.5c12641},
pmid = {41071600},
issn = {1936-086X},
mesh = {*Copper/chemistry/pharmacology ; Humans ; Animals ; *DNA/chemistry/pharmacology ; *Circadian Rhythm/drug effects ; Mice ; Metabolic Networks and Pathways/drug effects ; Apoptosis/drug effects ; *Antineoplastic Agents/pharmacology/chemistry ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; ARNTL Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Despite the promise of cuproptosis in antitumor therapy, developing strategies to enhance its therapeutic efficacy within the tumor microenvironment remains a challenge. Inspired by the chronotherapy that manipulate circadian rhythms to enhance drug effectiveness, herein we report for a CRISPR-customized copper-DNA nanoplatform (Cu-RNP) that synergistically induces multimodal cell death, including potentiated cuproptosis, by manipulating circadian and metabolic pathways. Cu-RNP integrates coordination-driven self-assembly of Cu[2+]-DNA nanospheres with Cas13d/crRNA ribonucleoproteins targeting BMAL1. Upon cellular internalization, the acidic and reducing endo/lysosomal environment triggers Cu-RNP disassembly, releasing RNP to silence BMAL1 and disrupt circadian oscillations, leading to WEE1 downregulation and p21 upregulation, thereby inducing apoptosis. Simultaneously, liberated Cu[2+] generates cytotoxic hydroxyl radicals for chemodynamic therapy (CDT) and concurrently depletes GSH, promoting mitochondrial copper overload for cuproptosis. Importantly, we demonstrate that silencing BMAL1 disrupts circadian rhythms, inhibits glycolysis, enhances mitochondrial respiration, and redirects metabolic flux to the TCA cycle, thereby amplifying the cell's vulnerability to copper-induced cuproptosis. In vitro and in vivo results demonstrate that Cu-RNP sensitizes cancer cells to cuproptosis and elicit strong antitumor response through the synergistic combination of cuproptosis, CDT, apoptosis, and circadian-metabolic modulation. This study demonstrates a mechanistic link between BMAL1-regulated circadian rhythms and cuproptosis sensitivity, suggesting a potential treatment strategy for multimodal, cuproptosis-potentiating cancer therapies.},
}

*Copper/chemistry/pharmacology
Humans
Animals
*DNA/chemistry/pharmacology
*Circadian Rhythm/drug effects
Mice
Metabolic Networks and Pathways/drug effects
Apoptosis/drug effects
*Antineoplastic Agents/pharmacology/chemistry
Cell Line, Tumor
*Clustered Regularly Interspaced Short Palindromic Repeats
ARNTL Transcription Factors/genetics/metabolism
CRISPR-Cas Systems