@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

@article {pmid41024711,
year = {2025},
author = {Sanchez, A and Zhou, C and Tulaiha, R and Ramirez, F and Wang, L and Zhang, X},
title = {CRISPR Screen Identifies BAP1 as a Deubiquitinase Regulating SPIN4 Stability.},
journal = {Biochemistry},
volume = {64},
number = {20},
pages = {4318-4326},
pmid = {41024711},
issn = {1520-4995},
support = {R35 GM146979/GM/NIGMS NIH HHS/United States ; R35 GM154945/GM/NIGMS NIH HHS/United States ; T32 GM149439/GM/NIGMS NIH HHS/United States ; },
mesh = {*Ubiquitin Thiolesterase/metabolism/genetics ; Humans ; *Tumor Suppressor Proteins/metabolism/genetics ; *CRISPR-Cas Systems ; *Cell Cycle Proteins/metabolism/genetics ; Ubiquitination ; HEK293 Cells ; Protein Stability ; Ubiquitin-Protein Ligases/metabolism ; Proteolysis ; Microtubule-Associated Proteins ; Phosphoproteins ; },
abstract = {Protein homeostasis is tightly controlled by the coordinated actions of E3 ubiquitin ligases and deubiquitinases (DUBs). We previously identified Spindlin-4 (SPIN4), a histone H3K4me3 reader, as a degradation substrate of DCAF16. In this study, we confirmed this degradation pathway using an E3 ligase-focused CRISPR-Cas9 knockout screen. Furthermore, through a DUB-focused CRISPR-Cas9 knockout screen and biochemical analyses, we demonstrated that the deubiquitinase BAP1 interacts with and stabilizes SPIN4 via its deubiquitination activity. Inhibition or loss of BAP1 reduces SPIN4 levels, highlighting its critical role in maintaining SPIN4 homeostasis. Proteomics and interactome analyses further support this regulatory axis. These findings reveal a dynamic balance controlling SPIN4 stability, with potential implications for epigenetic regulation and disease processes.},
}

*Ubiquitin Thiolesterase/metabolism/genetics
Humans
*Tumor Suppressor Proteins/metabolism/genetics
*CRISPR-Cas Systems
*Cell Cycle Proteins/metabolism/genetics
Ubiquitination
HEK293 Cells
Protein Stability
Ubiquitin-Protein Ligases/metabolism
Proteolysis
Microtubule-Associated Proteins
Phosphoproteins

@article {pmid41109516,
year = {2025},
author = {Pandya, K and Kumar, D},
title = {CRISPR/Cas Genome Editing for Neurodegenerative Diseases: Mechanisms, Therapeutic Advances, and Clinical Prospects.},
journal = {Ageing research reviews},
volume = {},
number = {},
pages = {102922},
doi = {10.1016/j.arr.2025.102922},
pmid = {41109516},
issn = {1872-9649},
abstract = {Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD) are major public health challenges. Current treatments are only symptomatic and do not address the underlying pathogenic genetic mechanisms. The development of the CRISPR/Cas genome editing tool has increased possibilities for targeted repair of pathological mutations. CRISPR/Cas9, Cas12, and Cas13 systems enable targeted editing and transcriptome modulation in various preclinical models. CRISPR/Cas9 disruption of mutant APP and Tau genes in AD models has restored normal genetic function. This technique reduces toxic protein aggregation. Results in neurodegenerative phenotype in these models are ameliorated. Correction of CAG nucleotide repeats in HD, and reduction of alpha-synuclein expression in PD. Advancements in engineered Cas variants with enhanced specificity, such as SpCas9-HF1 and prime editors, with innovative delivery strategies including adeno-associated virus (AAVs) and nanoparticle-based systems, have improved genome editing. However, challenges remain, including off-target effects, mosaicism, and delivery across the BBB, and long-term safety. Ethical consideration focuses on somatic versus germline editing, equitable access, and regulatory oversight. While somatic editing shows acceptance in treating neurological disorders. Germline interventions face strict regulations due to potential multigeneration impacts. Collectively, these technologies are the vanguard of precision molecular medicine, advancing from symptom management towards potentially curative gene therapies for neurological disorders.},
}

@article {pmid41109509,
year = {2025},
author = {Gujjar, RS and Sherin, S and Batra, S and Mahto, A and Goswami, SK and Kumar, R and Tiwari, RK and Singh, A and Kumari, M},
title = {Emerging trends in the development of efficient CAS nucleases for meticulous gene editing in plants.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {},
number = {},
pages = {112820},
doi = {10.1016/j.plantsci.2025.112820},
pmid = {41109509},
issn = {1873-2259},
abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease) is a formidable and precise gene-editing system, invented as a natural immune system of bacterial defense. The system relies on a guide RNA/CrRNA (Crispr RNA) sequence, specifically designed to bind to the target nucleic acid, enabling the Cas nuclease to act like a molecular scissor and cleave the target sequence. Recent research has substantially advanced the development, optimization, and application of Cas nucleases, particularly Cas9, Cas12, and their engineered orthologs to enhance the fidelity, flexibility, and proficiency of specific edits at the target site. This review focuses on the latest advancements in the research on Cas nucleases such as Cas9, Cas12a, Cas12b, CasΦ, Cas13, and Cas14, along with their mode of function and the exclusive features of each nuclease. Further, we have elaborated on the emergence of redesigned Cas9 orthologs, such as dCas9 (deactivated Cas9) and nCas9 (Cas9 nickase). The modified Cas9 orthologs have empowered the CRISPR-based editing with sophisticated techniques such as base editing and prime editing, which are capable of providing preferred edits with more precision and specificity. These advanced genome editing approaches have been favorably employed to improve the desirable agronomic traits such as stress tolerance, herbicide resistance, and yield stability in a wide range of crops, including rice, maize, wheat, tomato, and cotton. KEY MESSAGE: Our manuscript offers the recent advancement in the research on Cas nucleases, and their modified orthologs to perform CRISPR-based gene edits with enhanced more precision and specificity.},
}

@article {pmid41108631,
year = {2025},
author = {Harzli, I and Mostafa, K and Mutlu, VA and Shamsi, IH and Kavas, M},
title = {Exploring Novel Microbial Approaches for Enhancing Crop Resilience to Abiotic Stress: Mechanisms and Applications.},
journal = {Physiologia plantarum},
volume = {177},
number = {5},
pages = {e70582},
doi = {10.1111/ppl.70582},
pmid = {41108631},
issn = {1399-3054},
support = {121O463//Tübitak/ ; },
mesh = {*Crops, Agricultural/microbiology/physiology ; *Stress, Physiological ; Mycorrhizae/physiology ; Gene Editing ; Plant Breeding ; },
abstract = {The current state of agriculture heavily relies on chemical fertilizers and pesticides, which can negatively impact plant nutritional quality, plant health, and productivity. Additionally, abiotic stresses pose significant challenges to global agricultural productivity, threatening food security and crop sustainability. Therefore, developing and implementing sustainable alternatives to chemical fertilizers and pesticides is crucial to enhance agricultural productivity and resilience. Recent research highlights the potential of microorganisms, such as plant growth-promoting rhizobacteria (PGPR), mycorrhizal fungi, and endophytes, as sustainable solutions to improve plant resilience under abiotic stress conditions. However, challenges including scalability, ecological impacts, and the need for standardized application methods persist. This review explores novel microbial approaches to improving crop resilience against abiotic stress, focusing on how microorganisms interact with plants to mitigate stress impacts. Key mechanisms include the production of stress-alleviating compounds, enhanced nutrient uptake, and modulation of plant stress response pathways. We also examine advanced strategies in plant breeding, emphasizing CRISPR/Cas-mediated genome editing technologies as powerful tools for elucidating plant-microbe interactions. A thorough understanding of these interactions is essential for effectively applying genome editing to enhance the functional capacities of plants or associated microbes, ultimately improving key agronomic traits. This review provides a comprehensive overview of these innovative microbial approaches and their practical applications in sustainable agriculture, offering insights into future research directions, such as developing novel microbial strains and optimizing field applications.},
}

*Crops, Agricultural/microbiology/physiology
*Stress, Physiological
Mycorrhizae/physiology
Gene Editing
Plant Breeding

@article {pmid41108328,
year = {2025},
author = {Liu, C and Yao, D and Jiang, Y and Huo, C and Zhang, J and Yi, S and Yang, W and Han, Y and Liu, Y and Wang, X},
title = {Electroporation-based CRISPR/Cas9 Gene Editing in Haliotis Discus Hannai.},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {27},
number = {6},
pages = {148},
pmid = {41108328},
issn = {1436-2236},
support = {ZR2023QC107//Natural Science Foundation of Shandong Province/ ; ZR2022QC233//Natural Science Foundation of Shandong Province/ ; 2022LZGC015//Key Research and Development Program of Shandong Province/ ; SDAIT-14-01//Modern Agricultural Industry Technology System of Shandong Province/ ; tstp20240518//Special Funds for the Taishan Scholar Project of Shandong Province/ ; 202203//Special Supporting Funds for Leading Talents above Provincial Level in Yantai City/ ; 2023HD//Aquaculture Seed Industry Promotion Project of Yantai City/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Electroporation/methods ; *Myostatin/genetics ; *Gastropoda/genetics/embryology ; Aquaculture ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Abalone, a marine mollusk with significant economic and ecological value, plays a crucial role in sustainable aquaculture. The development and application of CRISPR-Cas9 gene-editing technology have opened up a new path for improving breeding efficiency. CRISPR/Cas9-mediated gene editing has been achieved in abalones via microinjection. In this study, a gene encoding myostatin MSTN in H. discus hannai; was selected as target for conducting the CRISPR-Cas9 gene editing experiment in combination with an electroporation delivery system. Our results showed that all three sgRNAs effectively targeted and cleaved the target segment, with sgRNA1 and sgRNA2 exhibiting high in vitro activity. After electroporation, the effects of transfection on embryonic development of fertilized eggs were observed and statistically analyzed. 12.7 ± 5.4% of the fertilized eggs were damaged and deformed after electroporation. Twenty-four hours after electroporation, surviving larvae were collected for DNA extraction and sequencing. Two potential mutations within the target region of MSTN were identified by sequencing. These results provide a reference for the improvement and development of CRISPR-mediated gene editing methods in marine mollusks such as abalones.},
}

Animals
*CRISPR-Cas Systems
*Gene Editing/methods
*Electroporation/methods
*Myostatin/genetics
*Gastropoda/genetics/embryology
Aquaculture
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41107544,
year = {2025},
author = {Whyms, C and Zhao, Y and Addo-Yobo, D and He, H and Whittington, AC and Trasanidou, D and Salazar, CRP and Staals, RHJ and Li, H},
title = {The twist-and-squeeze activation of CARF-fused adenosine deaminase by cyclic oligoadenylates.},
journal = {The EMBO journal},
volume = {},
number = {},
pages = {},
pmid = {41107544},
issn = {1460-2075},
support = {R35 GM152081/GM/NIGMS NIH HHS/United States ; },
abstract = {The recently identified CARF (CRISPR-associated Rossman-fold) family of proteins play a critical role in prokaryotic defense, mediating cOA (cyclic oligoadenylate)-stimulated ancillary immune responses in the type III CRISPR-Cas systems. Whereas most previously characterized CARF proteins contain nucleic acids or protein degradation effectors, a subset of the family, including the CARF-fused adenosine deaminase (ADA) (Cad1), has recently been shown to convert ATP to ITP. The enzymatic mechanism and the activation process of Cad1, however, remain incompletely understood. Here we present biochemical and structural analyses of a ring nuclease Cad1, revealing its substrate binding specificity and a sequential activation process by cOAs. Despite an overall structural similarity to canonical ADA enzymes, the ADA domain of Cad1 possesses unique structural features that confer a specificity for ATP. Supported by mutational analysis, our structural work demonstrates an allosteric link between the cOA-binding CARF and the ADA domain through a protein network within the hexameric enzyme assembly. Binding of a cA4 molecule to paired CARF domains induces a twisting of the linked ADA domains around one another, which remodels their active sites and alters interactions with neighboring ADA domains, thereby driving a sequential conformational activation mechanism.},
}

@article {pmid41005541,
year = {2025},
author = {Kristof, A and Karunakaran, K and Ferry, Y and Mizote, P and Allen, C and Briggs, S and Blazeck, J},
title = {A next-generation platform for highly optimized CRISPR-mediated transcriptional repression.},
journal = {Journal of biotechnology},
volume = {408},
number = {},
pages = {192-200},
doi = {10.1016/j.jbiotec.2025.09.012},
pmid = {41005541},
issn = {1873-4863},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; HEK293 Cells ; Methyl-CpG-Binding Protein 2/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Knockdown Techniques ; Protein Engineering/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Repressor Proteins/genetics/metabolism ; Nuclear Localization Signals/genetics ; },
abstract = {CRISPR interference (CRISPRi), the fusion of transcriptional repressor domains to nuclease-inactive Cas9, is a powerful genetic tool enabling site-specific suppression of gene expression. However, its performance remains inconsistent across cell lines, gene targets, or single guide RNAs (sgRNAs) employed. This study described the development process of a novel, highly optimized CRISPRi repressor for mammalian gene regulation through a multi-pronged protein engineering approach: (1) truncating established domains, (2) characterizing candidate domains, (3) creating combinatorial domain fusion libraries, and (4) optimizing NLS configuration. First, by evaluating several truncations of MeCP2, a well-established repressor, we see that the ultra-compact NCoR/SMRT interaction domain (NID) significantly enhances CRISPRi gene knockdown performance, exceeding levels observed with canonical MeCP2 subdomains by an average of ∼40 %. Incorporating this optimized MeCP2 NID truncation with a diverse panel of authenticated repressor domains, we next assemble and screen combinatorial multi-domain libraries, discovering four new repressor fusions. Upon follow-up nuclear localization signal (NLS) configuration analysis, we see that affixing one carboxy-terminal NLS enhances gene knockdown efficiency of the repressors by an average of ∼50 %. Through rigorous validation of NLS-tagged repressor fusions across several cell lines, multiple sgRNA targets, and genome-wide dropout screens, we establish that our strongest system, dCas9-ZIM3-NID-MXD1-NLS, achieves superior gene silencing capabilities over alternative CRISPRi platforms. In addition to developing dCas9-ZIM3-NID-MXD1-NLS, a uniquely potent transcriptional repressor, we envision that the multi-domain engineering approach utilized in this study will be valuable framework enabling future strides in CRISPR platform development.},
}

*CRISPR-Cas Systems/genetics
Humans
HEK293 Cells
Methyl-CpG-Binding Protein 2/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Knockdown Techniques
Protein Engineering/methods
Clustered Regularly Interspaced Short Palindromic Repeats
*Repressor Proteins/genetics/metabolism
Nuclear Localization Signals/genetics

@article {pmid40962145,
year = {2025},
author = {Gao, Y and Zhou, Y and Wang, L and Zhang, N and Qin, W and Meng, W and Zhou, C},
title = {Synergistic strategy for high-yield 2,3-butanediol and acetoin production in Bacillus licheniformis MW03 based on metabolic engineering.},
journal = {Journal of biotechnology},
volume = {408},
number = {},
pages = {232-243},
doi = {10.1016/j.jbiotec.2025.09.006},
pmid = {40962145},
issn = {1873-4863},
mesh = {*Acetoin/metabolism/analysis ; *Butylene Glycols/metabolism ; *Bacillus licheniformis/genetics/metabolism ; *Metabolic Engineering/methods ; Bacterial Proteins/genetics/metabolism ; Fermentation ; Alcohol Oxidoreductases/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Gene Editing ; },
abstract = {Bacillus licheniformis is an efficient platform for 2,3-butanediol (2,3-BD) and acetoin production due to its rapid glucose utilization rate and adaptability to industrial fermentation conditions. Here, we isolated the B. licheniformis strain MW03 with high yield of acetoin and 2,3-BD, which carried genetic mutations in acoR and budC, respectively encoding an acetoin dehydrogenase regulator and meso-2,3-BD dehydrogenase. To further confirm the physiological effects on acetoin and 2,3 BD biosynthesis, gene editing was performed using the CRISPR-Cas9 system, followed by phenotypic screening and genotype validation. The knockout of acoR and budC increased the acetoin maximum titer by 21.2 % and 49.2 %, respectively. Moreover, the optical purity of D-(-)-2,3-BD reached 92.7 % following the knockout of budC. Heterologous expression of acoR from B. licheniformis 2709 in both the wild type and acoR knockout mutant strongly inhibited acetoin accumulation compared to native acoR, which emphasized the regulatory role of AcoR in acetoin accumulation. Conversely, complementation of budC restored the synthesis of meso-2,3-BD synthesis, emphasizing its importance in this process. Overexpression of alsD in the acoR mutant increased the 2,3-BD titer by 61.9 % to 121.97 g/L, while the productivity reached 2.03 g/L·h. Finally, co-expression of bdhA and gldA increased 2,3-BD production by 25.6 %. This study elucidated the dual regulatory roles of acoR and budC in acetoin and 2,3-BD metabolism, establishing a "knockout-overexpression" synergic strategy, which offers theoretical support and practical guidance for further strain optimization.},
}

*Acetoin/metabolism/analysis
*Butylene Glycols/metabolism
*Bacillus licheniformis/genetics/metabolism
*Metabolic Engineering/methods
Bacterial Proteins/genetics/metabolism
Fermentation
Alcohol Oxidoreductases/genetics/metabolism
CRISPR-Cas Systems
Gene Knockout Techniques
Gene Editing

@article {pmid41106392,
year = {2025},
author = {Yang, M and Liu, S and Chen, G and Liu, X and Sun, D and Zhang, J and Wang, Y and Chen, S and Tian, R and Hu, Z},
title = {Structural and functional bases of F. rodentium Cas9 provide insights into CRISPR-Cas protein engineering.},
journal = {Cell genomics},
volume = {},
number = {},
pages = {101039},
doi = {10.1016/j.xgen.2025.101039},
pmid = {41106392},
issn = {2666-979X},
abstract = {The Faecalibaculum rodentium (Fr) CRISPR-Cas9 system exhibits enhanced gene-editing precision and efficiency compared to SpCas9, with distinctive advantages in targeting the TATA box in eukaryotic promoters. However, the underlying molecular mechanisms remained unexplored. Here, we present cryo-electron microscopy structures of the FrCas9-single guide RNA (sgRNA)-DNA complex in both the R-loop expansion and pre-catalytic states, shedding light on its specialized recognition of the 5'-NRTA-3' protospacer adjacent motif (PAM) and the unusual overwinding of the sgRNA-DNA heteroduplex. Our investigations into the structure and extensive mutational analyses reveal that the phosphate lock loop plays a pivotal role in finely adjusting FrCas9's off-target sensitivity and catalytic efficiency. Remarkably, targeted residue substitutions in the phosphate lock loop and the PAM-distal region were found to synergistically enhance both the editing precision and efficiency of FrCas9. These findings advance our understanding of Cas9's accuracy and potency mechanisms while providing a molecular foundation for the rational design and development of next-generation CRISPR technologies.},
}

@article {pmid41106357,
year = {2025},
author = {Gehrke, F and Puchta, H},
title = {CRISPR meets AI-based robotics: Advancing sustainable agriculture.},
journal = {Cell},
volume = {188},
number = {21},
pages = {5785-5787},
doi = {10.1016/j.cell.2025.09.011},
pmid = {41106357},
issn = {1097-4172},
mesh = {*Artificial Intelligence ; *Robotics/methods ; Gene Editing/methods ; *Crops, Agricultural/genetics ; *Agriculture/methods ; *CRISPR-Cas Systems ; },
abstract = {In this issue of Cell, Xu and colleagues develop an approach integrating genome editing, artificial intelligence, and robotics to enhance crop improvement. By reconfiguring reproductive traits for automated pollination in crops such as tomatoes and soybeans, their approach accelerates hybrid seed production and yields crops with better stress tolerance, flavor, and resilience, supporting sustainable agriculture and crop diversity.},
}

*Artificial Intelligence
*Robotics/methods
Gene Editing/methods
*Crops, Agricultural/genetics
*Agriculture/methods
*CRISPR-Cas Systems

@article {pmid41104521,
year = {2025},
author = {Braunreiter, K and Kempton, A and Mejia-Guerra, MK and Murray, A and Baine, S and Adegboye, K and Haile, A and Kumar Ahuja, SJ and Fedoce, A and Liu, C and Burch, P and Kabadi, AM},
title = {Characterization of a humanized mouse model of Duchenne muscular dystrophy to support the development of genetic medicines.},
journal = {Disease models & mechanisms},
volume = {18},
number = {10},
pages = {},
doi = {10.1242/dmm.052182},
pmid = {41104521},
issn = {1754-8411},
support = {//Sarepta Therapeutics/ ; },
mesh = {Animals ; *Muscular Dystrophy, Duchenne/therapy/genetics/physiopathology/pathology/blood ; Disease Models, Animal ; Humans ; Dystrophin/metabolism/genetics ; Muscle, Skeletal/pathology/physiopathology ; Mice, Inbred mdx ; *Genetic Therapy ; Mice ; Male ; Diaphragm/pathology ; Biomarkers/blood/metabolism ; Gene Editing ; Muscle Fibers, Skeletal/pathology/metabolism ; CRISPR-Cas Systems/genetics ; Myocardium/pathology/metabolism ; Fibrosis ; },
abstract = {Duchenne muscular dystrophy (DMD) is a rare, progressive neuromuscular disease resulting from DMD variants, leading to loss of functional dystrophin. To evaluate human-targeted genetic medicines for functional dystrophin restoration, humanized genetic models containing the full human locus are required. This study characterized the hDMDΔ52/mdx mouse model previously reported by Pickar-Oliver and colleagues. Genomic characterization confirmed complete DMD duplication with identical exon 52 deletion junctions on both copies. Histological analysis showed increased diaphragm fibrosis and skeletal muscle central nuclei in hDMDΔ52/mdx mice versus hDMD/mdx controls. hDMDΔ52/mdx mice demonstrated reduced tibialis anterior specific force, decreased skeletal muscle fiber diameter, decreased resistance to eccentric contraction-induced damage and cardiac defects. Multiple serum biomarkers of disease were identified. Using a CRISPR/Cas9 gene-editing strategy to restore human functional dystrophin protein expression, detectable dystrophin expression in the heart and skeletal muscle and increased resistance to injury in the tibialis anterior muscle were observed. In summary, hDMDΔ52/mdx mice display multiple physiological and functional deficits associated with DMD pathology, which can be restored by human-targeted therapy, confirming the suitability of this model for developing human-targeted genetic medicines.},
}

Animals
*Muscular Dystrophy, Duchenne/therapy/genetics/physiopathology/pathology/blood
Disease Models, Animal
Humans
Dystrophin/metabolism/genetics
Muscle, Skeletal/pathology/physiopathology
Mice, Inbred mdx
*Genetic Therapy
Mice
Male
Diaphragm/pathology
Biomarkers/blood/metabolism
Gene Editing
Muscle Fibers, Skeletal/pathology/metabolism
CRISPR-Cas Systems/genetics
Myocardium/pathology/metabolism
Fibrosis

@article {pmid41104129,
year = {2025},
author = {Pan, L and Wei, L and Luo, S and Ren, B and Li, M and Liang, L and Li, X and Wei, G},
title = {Klebsiella pneumoniae detection by a light-controlled one-pot RPA-CRISPR/Cas12a method.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1669860},
pmid = {41104129},
issn = {2235-2988},
mesh = {*Klebsiella pneumoniae/isolation & purification/genetics ; *CRISPR-Cas Systems ; Humans ; *Klebsiella Infections/diagnosis/microbiology ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; Ultraviolet Rays ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: Klebsiella pneumoniae (KP) is a significant pathogenic bacterium responsible for severe infections in hospitals. However, existing traditional detection techniques, such as culture and PCR, are relatively inefficient. Therefore, this study aims to establish a rapid and convenient method for detecting KP.

METHODS: This study developed a single-tube detection method combining recombinant polymerase amplification (RPA) and light-controlled CRISPR/Cas12a. RPA primers were designed and screened for the rcsA gene of KP to effectively amplify the target. A light-controlled CRISPR/Cas12a system was created using crRNA modified with a photocleavable group (NPOM). The two systems were integrated into a single tube. Following RPA amplification, UV light-controlled release of crRNA inhibition activates CRISPR-mediated target recognition and Cas12a trans-cleavage, detecting fluorescent signals (FD) in conjunction with UV analysis.

RESULTS: The light-controlled RPA-CRISPR/Cas12a detection platform developed in this study uses a 15 μL reaction system. By optimizing key parameters such as RPA amplification time (20 min), primer concentration (400 nM), UV light activation time (30 s), and crRNA/Cas12a concentration (300 nM), the platform achieves optimal detection efficiency. The platform has a fluorescence detection limit of 4.072×10[2] copies/reaction and can specifically identify KP in seven common clinical strains. Clinical sample validation demonstrated that the method yields results fully consistent with PCR detection (30/30 agreement rate of 100%), showcasing excellent detection performance and clinical application potential.

CONCLUSION: We have successfully developed a light-controlled RPA-CRISPR/Cas12a detection system capable of rapidly and highly sensitively detecting KP. This system demonstrates significant advantages in terms of detection speed (completed in as little as 50 minutes), sensitivity (as low as 4.072×10[2] copies/reaction), and ease of use, providing an efficient and reliable solution for clinical pathogen detection.},
}

*Klebsiella pneumoniae/isolation & purification/genetics
*CRISPR-Cas Systems
Humans
*Klebsiella Infections/diagnosis/microbiology
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
Bacterial Proteins/genetics
Ultraviolet Rays
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41039096,
year = {2025},
author = {Johnston, M and Dissanayake-Perera, S and Collins, JJ and Stevens, MM and Dincer, C},
title = {Convergence of nanotechnology and CRISPR-based diagnostics.},
journal = {Nature nanotechnology},
volume = {20},
number = {10},
pages = {1365-1373},
pmid = {41039096},
issn = {1748-3395},
support = {13GW0493//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/ ; CiET2021\94//Royal Academy of Engineering/ ; },
mesh = {*Nanotechnology/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; },
abstract = {In addition to its broad application in genome engineering and therapeutics, clustered regularly interspaced short palindromic repeats (CRISPR) technology provides field-deployable methods for the highly sensitive and selective detection of nucleic acids. From a diagnostic perspective, CRISPR-based assays hold clear clinical potential for identifying a range of both infectious and non-communicable diseases. In this Perspective we evaluate recent nanotechnologies and nanomaterials that have been engineered to interface with CRISPR systems on a nanoscale level to realize the full potential of this versatile diagnostic tool. We assess biomolecules such as enzymes and oligonucleotides, some of the more commonly used synthetic nanoparticles and detection platforms that integrate nanotechnologies in new and innovative ways. We discuss current trends and look ahead to future challenges and opportunities, including non-nucleic acid target detection, pre-amplification-free detection of nucleic acids, the development of wearable devices and integration with artificial intelligence workflows.},
}

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

@article {pmid40975295,
year = {2026},
author = {Zheng, X and Xu, H and Huang, Y and Liu, X and Zhu, S and Liu, H and Gao, S},
title = {Development of an RT-RAA-CRISPR-Cas12a assay for rapid, sensitive and visual detection of Tilapia Lake Virus (TiLV).},
journal = {Journal of virological methods},
volume = {339},
number = {},
pages = {115266},
doi = {10.1016/j.jviromet.2025.115266},
pmid = {40975295},
issn = {1879-0984},
mesh = {Animals ; *Fish Diseases/virology/diagnosis ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Tilapia/virology ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/genetics ; Aquaculture ; RNA, Viral/genetics ; DNA, Single-Stranded/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {In this study，we developed a new, highly efficient, and sequence-specific method for detecting Tilapia Lake Virus (TiLV) based on the clustered regularly interspaced short palindromic repeats (CRISPR) - CRISPR-associated protein 12a (Cas12a) system. TiLV is a highly contagious virus that has caused significant damage to the global aquaculture industry. Specific primers, CRISPR RNA (crRNA), and single-stranded DNA (ssDNA) reporters were designed to detect TiLV genome segment 3, with the ssDNA reporters modified at the 5' and 3' ends with fluorophore and quencher groups, respectively. The assay showed no cross-reactivity with other bacterial and viral pathogens in fish. The detection limit was 9.10 copies per reaction for recombinant plasmid standards and 91.82 fg/μL for TiLV RNA, demonstrating high sensitivity. The reverse transcription recombinase aided amplification (RT-RAA) coupled CRISPR/Cas12a method showed 100 % concordance with the standard fluorescence method, indicating its accuracy and suitability for clinical testing. This study innovatively combined the RT-RAA technique with the CRISPR/Cas12a reaction system, offering a new diagnostic method for TiLV that is fast, portable, highly specific, and sensitive. This enables on-site rapid screening for TiLV, ensuring aquaculture safety and the secure circulation of aquatic animal products.},
}

Animals
*Fish Diseases/virology/diagnosis
*CRISPR-Cas Systems
Sensitivity and Specificity
*Tilapia/virology
*Nucleic Acid Amplification Techniques/methods
CRISPR-Associated Proteins/genetics
Aquaculture
RNA, Viral/genetics
DNA, Single-Stranded/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40935117,
year = {2026},
author = {Terada, T and Fujii, S and Yamanishi, N and Kajihara, R and Watanabe, T and Ezaki, R and Horiuchi, H and Matsuzaki, M},
title = {Potential of recombinant avian adeno-associated virus as a viral vector for CRISPR/Cas9 delivery to avian cells.},
journal = {Journal of virological methods},
volume = {339},
number = {},
pages = {115263},
doi = {10.1016/j.jviromet.2025.115263},
pmid = {40935117},
issn = {1879-0984},
mesh = {Animals ; *CRISPR-Cas Systems ; *Genetic Vectors/genetics ; Chickens/genetics ; *Gene Editing/methods ; *Dependovirus/genetics ; Cell Line ; Green Fluorescent Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Fibroblasts/virology ; Staphylococcus aureus/genetics/enzymology ; },
abstract = {While genome editing has been established in chickens, where cultured primordial germ cell (PGC) systems are available, the implementation of genome editing remains a major challenge in many other birds due to the lack of robust PGC culture methods. Therefore, the development of reliable and efficient tools can significantly accelerate precision genome modification in avian species. Here, we evaluated the applicability of recombinant avian adeno-associated virus (rA3V) as a delivery vector for a CRISPR/Cas9 construct in avian cells using Staphylococcus aureus-derived Cas9 (SaCas9) and single-guide RNA (sgRNA). Infection with rA3V particles carrying an EGFP expression cassette (rA3V-EGFP) successfully induced EGFP expression in chicken fibroblasts (DF-1) cells, with approximately 80 % EGFP-positive cells at the maximum multiplicity of infection (MOI = 10,000). In plasmid-based transfection experiments, sgRNAs targeting the chicken tyrosinase locus and SaCas9 exhibited DNA cleavage activity in DF-1 cells. Furthermore, infection with rA3V particles encoding these CRISPR components successfully introduced indel mutations into the tyrosinase gene in DF-1 cells, with a calculated indel frequency of approximately 5.4 % at MOI = 40,000 without drug selection. Although EGFP expression was observed in quail fibrosarcoma cells, the percentage of EGFP-positive cells was much lower than that in DF-1 cells. In addition, in vivo infection with rA3V-EGFP of the chicken blastoderm failed to induce EGFP expression in germline cells, even at the highest applicable viral dose. In summary, rA3V can be used as a genome-editing vector in birds, although further investigation of its infectivity and tropism is necessary to expand its applicability to diverse avian species.},
}

Animals
*CRISPR-Cas Systems
*Genetic Vectors/genetics
Chickens/genetics
*Gene Editing/methods
*Dependovirus/genetics
Cell Line
Green Fluorescent Proteins/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Fibroblasts/virology
Staphylococcus aureus/genetics/enzymology

@article {pmid40915071,
year = {2026},
author = {Li, Z and Jiao, Y and Tang, J and Dong, X and Thomas, R and Xie, B and Li, Y and Jacques, P},
title = {The pH-responsive regulator PlPacC and GATA transcription factor PlAreB are involved in the regulation of the biosynthesis of the antifungal lipopeptaibols leucinostatins in Purpureocillium lilacinum.},
journal = {Microbiological research},
volume = {302},
number = {},
pages = {128324},
doi = {10.1016/j.micres.2025.128324},
pmid = {40915071},
issn = {1618-0623},
mesh = {Hydrogen-Ion Concentration ; *Transcription Factors/genetics/metabolism ; *Antifungal Agents/metabolism ; *Fungal Proteins/genetics/metabolism ; *Hypocreales/metabolism/genetics/growth & development ; Gene Expression Regulation, Fungal ; Nitrogen/metabolism ; Culture Media/chemistry ; CRISPR-Cas Systems ; },
abstract = {The biocontrol fungus Purpureocillium lilacinum PLBJ-1 produces leucinostatins, a class of non-ribosomal peptides (NRPs) with broad-spectrum antimicrobial activities. However, the molecular mechanisms underlying the optimization of culture conditions for leucinostatin production remain unexplored. Previous research showed that PLBJ-1 synthesizes leucinostatins more effectively in hand-made Potato Dextrose Broth (PDB-M) than in commercially available PDB (PDB-C). Elementary analysis of these two media indicated that the difference in leucinostatin yield was correlated with variations in pH dynamics and nitrogen content. Subsequent experiments under different initial pH and nitrogen levels confirmed that an alkaline environment and reduced nitrogen availability could enhance leucinostatin production. To investigate the regulators involved, CRISPR-Cas9-mediated gene disruptions were performed on the pH-responsive transcription factor PlPacC and the nitrogen regulator PlAreB. The disruption of either PlPacC or PlAreB resulted in a marked reduction in biomass and sporulation in P. lilacinum PLBJ-1. Specifically, PlPacC disruption impaired environmental pH regulation and significantly decreased leucinostatin production. In contrast, PlAreB disruption led to an increased leucinostatin yield. Overall, these findings demonstrate that environmental pH and nitrogen availability are the critical factors governing leucinostatin biosynthesis, acting through two key transcriptional regulators, PlPacC and PlAreB. This study lays a molecular foundation for future large-scale optimization of leucinostatin fermentation.},
}

Hydrogen-Ion Concentration
*Transcription Factors/genetics/metabolism
*Antifungal Agents/metabolism
*Fungal Proteins/genetics/metabolism
*Hypocreales/metabolism/genetics/growth & development
Gene Expression Regulation, Fungal
Nitrogen/metabolism
Culture Media/chemistry
CRISPR-Cas Systems

@article {pmid40789509,
year = {2025},
author = {Tsai, HY and Chen, MH and Yun, J and Lai, LA and Valentine, JF and Bronner, MP and Brentnall, TA and Pan, S and Chen, R},
title = {Restricting metabolic plasticity enhances stress adaptation through the modulation of PDH and HIF1A in TRAP1-depleted colon cancer.},
journal = {Cancer letters},
volume = {632},
number = {},
pages = {217977},
doi = {10.1016/j.canlet.2025.217977},
pmid = {40789509},
issn = {1872-7980},
support = {P30 DK056338/DK/NIDDK NIH HHS/United States ; R01 CA211892/CA/NCI NIH HHS/United States ; R01 CA276173/CA/NCI NIH HHS/United States ; },
mesh = {*Colonic Neoplasms/metabolism/genetics/pathology ; Humans ; *Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics ; Glycolysis ; Glucose Transporter Type 1/genetics/metabolism ; Pyruvate Dehydrogenase Acetyl-Transferring Kinase ; Cell Line, Tumor ; Oxidative Phosphorylation/drug effects ; HCT116 Cells ; Reactive Oxygen Species/metabolism ; Gene Expression Regulation, Neoplastic ; Monocarboxylic Acid Transporters/genetics/metabolism ; Adaptation, Physiological ; CRISPR-Cas Systems ; HSP90 Heat-Shock Proteins ; Symporters ; },
abstract = {Metabolic plasticity allows cancer cells to survive under adverse conditions. To investigate the role of mitochondrial chaperone tumor necrosis factor receptor-associated protein 1 (TRAP1) in this process, we used CRISPR/Cas9 mediated genetic deletion to knock out (KO) TRAP1 in colon cancer cells. Depletion of TRAP1 triggered a series of events: induced metabolic reprogramming, increased glycolytic flux, downregulation of mitochondrial complex I, and elevated ROS generation. TRAP1-deficient cells showed tolerance to Oxidative Phosphorylation (OXPHOS) inhibitors and exhibited a higher extracellular acidification rate (ECAR). Additionally, TRAP1 depletion activated hypoxia response elements (HREs) and upregulated HIF1A target genes such as GLUT1 and MCT1. Furthermore, pyruvate dehydrogenase kinases 1 (PDK1) was upregulated in KO cells, leading to the inactivation of the tricarboxylic acid (TCA) cycle enzyme, pyruvate dehydrogenase (PDH). This metabolic shift towards glycolytic metabolism resulted in increased glycolytic metabolism, elevated lactic acid production, and higher glucose consumption, making TRAP1-depleted cancer cells more dependent on this altered metabolism for survival. Treatment with DCA, a PDK inhibitor, restored PDH activity, exacerbated oxidative stress, and increased cell death in KO cells. Our study here sheds light on how TRAP1 depletion affects metabolic plasticity, driving colon cancer cells to adapt to metabolic and oxidative stress. These findings highlight TRAP1 as a promising therapeutic target for manipulating metabolic plasticity and overcoming drug resistance in cancer therapy.},
}

*Colonic Neoplasms/metabolism/genetics/pathology
Humans
*Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics
Glycolysis
Glucose Transporter Type 1/genetics/metabolism
Pyruvate Dehydrogenase Acetyl-Transferring Kinase
Cell Line, Tumor
Oxidative Phosphorylation/drug effects
HCT116 Cells
Reactive Oxygen Species/metabolism
Gene Expression Regulation, Neoplastic
Monocarboxylic Acid Transporters/genetics/metabolism
Adaptation, Physiological
CRISPR-Cas Systems
HSP90 Heat-Shock Proteins
Symporters

@article {pmid41103153,
year = {2025},
author = {Chen, L and Yun, M and Chen, B and Xie, S and Liu, W and Wang, M and Yan, J and Cai, J and Yang, S and Peng, Q and Xie, D and Lin, Y and Jiang, B},
title = {Loss of CsCLV2 function causes dwarfism and determinates growth in cucumber.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {1},
pages = {e70525},
doi = {10.1111/tpj.70525},
pmid = {41103153},
issn = {1365-313X},
support = {32202477//National Natural Science Foundation of China/ ; },
mesh = {*Cucumis sativus/genetics/growth & development/metabolism ; *Plant Proteins/genetics/metabolism/physiology ; *Dwarfism/genetics ; Phenotype ; Meristem/growth & development/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Cucumber (Cucumis sativus L.) is a globally important vegetable crop. Ideal plant architecture optimizes spatial utilization, enhances economic coefficient, and facilitates mechanized cultivation. In this study, we identified a dwarf mutant, csdw3, exhibiting reduced plant height, shortened internodes, and fewer internodes. Genetic analysis showed that this dwarf phenotype is controlled by a single recessive gene. Fine-mapping localized the causal locus to an 80 kb region on chromosome 1, where we discovered a 102 bp deletion in CsCLV2, a gene encoding a leucine-rich repeat receptor-like protein homologous to Arabidopsis CLAVATA2. CRISPR-Cas9-generated loss-of-function mutants recapitulated the dwarf phenotype, confirming the role of CsCLV2 in plant height regulation. Histological examination revealed that CsCLV2 disruption causes premature termination of shoot apical meristem (SAM) development, reducing both internode number and length. Protein interaction assays further demonstrated that CsCLV2 associates with receptor-like kinase CsCIK1 (CLAVATA3 INSENSITIVE RECEPTOR KINASES 1), indicating their cooperative function in the CLV-WUS signaling pathway to maintain meristem activity. Our findings uncover a regulator of plant height in cucumber and provide valuable genetic resources for breeding ideotypes optimized for yield and cultivation efficiency.},
}

*Cucumis sativus/genetics/growth & development/metabolism
*Plant Proteins/genetics/metabolism/physiology
*Dwarfism/genetics
Phenotype
Meristem/growth & development/genetics
Gene Expression Regulation, Plant
CRISPR-Cas Systems

@article {pmid41102733,
year = {2025},
author = {Marchenko, N and Nesbitt, NM and Alexandrova, E and Reisz, JA and D'Alessandro, A and Suh, J and Uryasev, S and Pennacchia, L and Bahou, WF},
title = {Biliverdin reductase B as a new target in breast cancer.},
journal = {Breast cancer research : BCR},
volume = {27},
number = {1},
pages = {179},
pmid = {41102733},
issn = {1465-542X},
support = {NCI CA284999/CA/NCI NIH HHS/United States ; P30CA046934/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Breast Neoplasms/genetics/pathology/metabolism/drug therapy ; Female ; Animals ; Cell Line, Tumor ; Mice ; *Oxidoreductases Acting on CH-CH Group Donors/genetics/metabolism/antagonists & inhibitors ; Cell Proliferation ; Oxidation-Reduction ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Enhanced metabolic and mitochondrial activity inherent in actively proliferating cancer cells is associated with intracellular redox imbalance that impacts cellular viability. To restore redox homeostasis cancer cells evolve to activate redox protective mechanisms. This differential activation of redox defense pathways compared to normal cells provides a therapeutic window for novel targeted therapies in cancer. Although heme metabolism emerges as a crucial regulator of redox homeostasis and iron metabolism in cancer cells with frequent alteration in breast cancer, it remains largely unexplored, and no targeted translational approaches have been developed. Heme-regulated redox homeostasis is coordinately maintained through biosynthetic and degradation pathways. As a byproduct of TCA cycle, cytotoxic heme is initially derivatized by heme oxygenases and progressively metabolized to the potent antioxidant bilirubin by two non-redundant biliverdin reductases, BLVRA and BLVRB. BLVRB overexpression has been observed in breast cancers, although its function in breast cancer pathogenesis remains unknown.

METHODS: CRISPR/Cas9 deletion of BLVRB in multiple breast cancer cell lines demonstrated its profound effect on intracellular redox state and cell proliferation in vitro and in xenograft models. Integrated proteomic, metabolomic, and lipidomic studies identified and validated BLVRB-mediated adaptive metabolic responses required for breast cancer cell cytoprotection.

RESULTS: We have established BLVRB as a requisite component of the pro-survival redox defense mechanism in breast cancer cells. Targeted deletion of BLVRB induces reductive stress, leading to alterations in endoplasmic reticulum proteostasis and lipid composition. These defects impact plasma membrane functionality and endosomal recycling of multiple oncogenic receptors, such as HER2 and transferrin receptors.

CONCLUSIONS: These data collectively identify BLVRB as a novel metabolic target in breast cancer, distinct from other redox-regulating pathways. This study, along with our recent progress in developing novel specific BLVRB inhibitors, offers a unique translational opportunity for targeted therapies in personalized breast cancer medicine.},
}

Humans
*Breast Neoplasms/genetics/pathology/metabolism/drug therapy
Female
Animals
Cell Line, Tumor
Mice
*Oxidoreductases Acting on CH-CH Group Donors/genetics/metabolism/antagonists & inhibitors
Cell Proliferation
Oxidation-Reduction
Xenograft Model Antitumor Assays
CRISPR-Cas Systems

@article {pmid41102523,
year = {2025},
author = {Savage, N and Danis, E and Chokshi, CR and Custers, S and Shaikh, MV and Miletic, P and Venugopal, C and Brown, KR and Vibhakar, R and Moffat, J and Singh, SK},
title = {CRISPR screen reveals SOX2 as a critical regulator of CD133 and cellular stress response in glioblastoma.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {36228},
pmid = {41102523},
issn = {2045-2322},
support = {1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; },
mesh = {*AC133 Antigen/metabolism/genetics ; *SOXB1 Transcription Factors/genetics/metabolism ; *Glioblastoma/genetics/metabolism/pathology ; Humans ; *CRISPR-Cas Systems ; Neoplastic Stem Cells/metabolism/pathology ; Cell Line, Tumor ; *Brain Neoplasms/genetics/metabolism/pathology ; Gene Expression Regulation, Neoplastic ; *Stress, Physiological ; Animals ; Mice ; },
abstract = {Glioblastoma (GBM) remains a formidable challenge in clinical settings due to limited treatments available. The surface protein CD133 marks glioblastoma stem cells (GSCs), cells capable of overcoming therapeutic pressures and correlate with more aggressiveness tumor phenotypes. In this study, we employed a CRISPR-Cas9 functional screen to deconvolute CD133 dynamics in tumors. This led us to establish that SOX2 is a key player in controlling the PROM1 gene, which in turn influences how cells react to stress factors, including those induced by chemoradiation treatment. The discoveries in this study shed light on the complex web of mechanisms that control the survival and resistance of GSCs, offering promising new avenues for targeting and potentially overcoming therapy resistance.},
}

*AC133 Antigen/metabolism/genetics
*SOXB1 Transcription Factors/genetics/metabolism
*Glioblastoma/genetics/metabolism/pathology
Humans
*CRISPR-Cas Systems
Neoplastic Stem Cells/metabolism/pathology
Cell Line, Tumor
*Brain Neoplasms/genetics/metabolism/pathology
Gene Expression Regulation, Neoplastic
*Stress, Physiological
Animals
Mice

@article {pmid41101942,
year = {2025},
author = {Lim, SL and Chin, CH and Chiou, YJ and Hsu, MT and Chiang, PW and Chen, HJ and Tu, YC and Tzou, WS and Tang, SL},
title = {Unveiling Unusual Ecofunctional Traits of Endozoicomonas Species Through Comprehensive Comparative Genomics.},
journal = {Environmental microbiology},
volume = {27},
number = {10},
pages = {e70191},
doi = {10.1111/1462-2920.70191},
pmid = {41101942},
issn = {1462-2920},
support = {AS-IA-109-L05//Academia Sinica/ ; },
mesh = {*Genome, Bacterial ; Genomics ; Phylogeny ; Prophages/genetics ; Bacterial Proteins/genetics ; *Gammaproteobacteria/genetics ; Quorum Sensing ; },
abstract = {Endozoicomonas is an omnipresent marine bacterial genus, associated with various marine organisms, that contributes to host health, nutrient cycling and disease resistance. Nonetheless, its genomic features remain poorly characterised due to a paucity of high-quality genomes. In this study, we sequenced 5 novel Endozoicomonas strains and re-sequenced 1 known strain to improve genomic resolution. By integrating these 6 high-quality genomes with 31 qualified published genomes, our pan-genomic analysis revealed variation in genetic traits among clades. Notably, Endozoicomonas lacks quorum-sensing capabilities, suggesting resistance to quorum quenching mechanisms. It also lacks the capacity to synthesise and transport vitamin B12, indicating that it does not supply this nutrient to holobionts. Remarkably, Endozoicomonas genomes encode 92 identified giant proteins (15-65 kbp). These proteins cluster into three major groups associated with antimicrobial peptide synthesis, exotoxin production and cell adhesion. Additionally, we found that Endozoicomonas has acquired prophages from diverse sources via infection or other types of gene transfer. Notably, CRISPR-Cas sequences suggest evolutionary trajectories independent of both prophage acquisition and phylogenetic lineage, implying potential geographic influences or environmental pressures. This study provides new insights into the genomic diversity of Endozoicomonas and its genetic adaptations to diverse hosts.},
}

*Genome, Bacterial
Genomics
Phylogeny
Prophages/genetics
Bacterial Proteins/genetics
*Gammaproteobacteria/genetics
Quorum Sensing

@article {pmid41100915,
year = {2025},
author = {Kim, I and Suh, JY},
title = {Old and new tactics of CRISPR-centric competition between bacteria and bacteriophages.},
journal = {Current opinion in structural biology},
volume = {95},
number = {},
pages = {103168},
doi = {10.1016/j.sbi.2025.103168},
pmid = {41100915},
issn = {1879-033X},
abstract = {The CRISPR-Cas system provides adaptive immunity for prokaryotes against mobile genetic elements (MGEs) such as bacteriophages and plasmids. As a countermeasure, MGEs have evolved various anti-CRISPR (Acr) mechanisms that neutralize the CRISPR-mediated immunity. Canonical Acr proteins block target binding of Cas proteins in a stoichiometric or enzymatic manner. New findings reveal that Acr also disintegrates functional Cas complexes, induces promiscuous target binding, and mimics Cas proteins and crRNA with defective mutations. Here, we summarize a broad repertoire of structural and functional mechanisms underlying CRISPR-centric competition, highlighting recent discoveries of molecular machinery that modulates CRISPR function.},
}

@article {pmid41100703,
year = {2025},
author = {Liao, X and Li, Y and Wu, Y and Wen, L and Jing, M and Chen, B and Li, X and Shang, X},
title = {TEMC-Cas: Accurate Cas Protein Classification via Combined Contrastive Learning and Protein Language Models.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00631},
pmid = {41100703},
issn = {2161-5063},
abstract = {The accurate classification of Cas proteins is crucial for understanding CRISPR-Cas systems and developing genome-editing tools. Here, we present TEMC-Cas, a deep learning framework for accurate classification of Cas proteins that combines a finely tuned ESM protein language model with contrastive learning. Unlike traditional methods that rely on sequence similarity (e.g., BLAST, HMMs) or structural prediction, TEMC-Cas leverages evolutionary-scale modeling to capture distant homology while employing contrastive learning to distinguish closely related subtypes. The framework incorporates LoRA for efficient parameter adaptation and addresses class imbalance through weighted loss functions. TEMC-Cas achieves superior performance in classifying the Cas1-Cas13 families and 17 Cas12 subtypes, demonstrating particular strength in identifying remote homology. This approach provides a robust tool for the discovery of the CRISPR system and expands the toolbox for genome engineering applications. TEMC-Cas is now freely accessible at https://github.com/Xingyu-Liao/TEMC-Cas.},
}

@article {pmid41099996,
year = {2025},
author = {Shafi, Z and Shahid, M and Ilyas, T and Singh, K},
title = {Next-generation perspectives on microbially synthesized siderophores: molecular engineering, multi-omics insights, and applications for smart climate-resilient crops.},
journal = {World journal of microbiology & biotechnology},
volume = {41},
number = {10},
pages = {393},
pmid = {41099996},
issn = {1573-0972},
mesh = {*Siderophores/biosynthesis/genetics/metabolism ; *Crops, Agricultural/growth & development/microbiology ; Iron/metabolism ; *Bacteria/metabolism/genetics ; Metabolomics ; Gene Editing ; Genomics ; Proteomics ; CRISPR-Cas Systems ; Multiomics ; },
abstract = {Siderophores, low-molecular-weight iron-chelating compounds synthesized by microbes, play a crucial role in iron (Fe) acquisition under Fe-limited conditions. In recent years, their significance in sustainable agriculture has gained increasing attention due to their multifaceted roles in plant growth promotion, stress alleviation, and disease suppression. This review presents next-generation insights into the biosynthesis, regulation, and applications of microbial siderophores, with a focus on advanced molecular and omics-based approaches. Innovations in synthetic biology and CRISPR/Cas-mediated genome editing have enabled precise manipulation of siderophore biosynthetic gene clusters, enhancing their production and functionality. Multi-omics platforms-genomics, transcriptomics, proteomics, and metabolomics-have revealed complex regulatory networks, unveiling cryptic pathways and inter-microbial variability in siderophore synthesis. Furthermore, the use of siderophore-producing plant growth-promoting rhizobacteria (PGPR) has shown promise in improving nutrient uptake, inducing systemic resistance, and mitigating abiotic stresses in crops. The integration of nano-formulations and encapsulation technologies has enhanced the stability and field efficacy of siderophore-based bioinoculants. This review also explores emerging strategies for developing microbial consortia and smart delivery systems to meet the challenges of climate-resilient agriculture. By bridging molecular insights with field-level applications, this article underscores the potential of siderophores as eco-friendly tools for next-generation sustainable farming practices.},
}

*Siderophores/biosynthesis/genetics/metabolism
*Crops, Agricultural/growth & development/microbiology
Iron/metabolism
*Bacteria/metabolism/genetics
Metabolomics
Gene Editing
Genomics
Proteomics
CRISPR-Cas Systems
Multiomics

@article {pmid41097718,
year = {2025},
author = {Lye, SH and Polycarp, N and Durojaye, TJ and Tollefsbol, TO},
title = {Functional Heterogeneity and Context-Dependent Roles of LncRNAs in Breast Cancer.},
journal = {Cancers},
volume = {17},
number = {19},
pages = {},
pmid = {41097718},
issn = {2072-6694},
support = {R01CA178441/CA/NCI NIH HHS/United States ; },
abstract = {As with other non-coding RNAs (ncRNAs), the aberrant expression of long non-coding RNAs (lncRNAs) can be associated with different forms of cancers, including breast cancer (BC). Various lncRNAs may either promote or suppress cell proliferation, metastasis, and other related cancer signaling pathways by interacting with other cellular machinery, thus affecting the expression of BC-related genes. However, lncRNAs are characterized by features that are unlike protein-coding genes, which pose unique challenges when it comes to their study and utility. They are highly diverse and may display contradictory functions depending on factors like the BC subtype, isoform diversity, epigenetic regulation, subcellular localization, interactions with various molecular partners, and the tumor microenvironment (TME), which contributes to the intratumoral heterogeneity and phenotypic plasticity. While lncRNAs have potential clinical utility, their functional heterogeneity coupled with a current paucity of knowledge of their functions present challenges for clinical translation. Strategies to address this heterogeneity include improving classification systems, employing CRISPR/Cas tools for functional studies, utilizing single-cell and spatial sequencing technologies, and prioritizing robust targets for therapeutic development. A comprehensive understanding of the lncRNA functional heterogeneity and context-dependent behavior is crucial for advancing BC research and precision medicine. This review discusses the sources of lncRNA heterogeneity, their implications in BC biology, and approaches to resolve knowledge gaps in order to harness lncRNAs for clinical applications.},
}

@article {pmid41027589,
year = {2025},
author = {Jiang, W and Georgiadis, I and Fumagalli, T and Wang, S and Vasileiou, C and Dahlin, J and Borodina, I},
title = {In Vivo DNA Assembly in Yarrowia lipolytica.},
journal = {ACS synthetic biology},
volume = {14},
number = {10},
pages = {4116-4121},
doi = {10.1021/acssynbio.5c00296},
pmid = {41027589},
issn = {2161-5063},
mesh = {*Yarrowia/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Rad52 DNA Repair and Recombination Protein/genetics/metabolism ; Saccharomyces cerevisiae/genetics ; Homologous Recombination/genetics ; *DNA/genetics/metabolism ; Gene Editing/methods ; Green Fluorescent Proteins/genetics/metabolism ; },
abstract = {The oleaginous yeast Yarrowia lipolytica is an important platform organism for biotechnology applications. In this study, we established an in vivo DNA assembly system leveraging CRISPR-Cas9 for efficient genomic integration of multiple DNA fragments into the genome of Y. lipolytica. Using the green fluorescent protein mNeonGreen as a model, we demonstrated 53% correct assembly of three DNA fragments with homology arms as short as 50 bp. The system was further validated by constructing 2-3 step biosynthetic pathways for pigments betaxanthin and betanin. To improve the homologous recombination efficiency of Y. lipolytica, we expressed S. cerevisiae RAD52 (ScRAD52) or a Cas9-hBrex27 fusion protein. While ScRAD52 expression impaired growth, the cas9-hBrex27 fusion enhanced integration efficiency, particularly for multifragment pathway assemblies. The in vivo assembly method simplifies pathway construction and gene overexpression in Y. lipolytica.},
}

*Yarrowia/genetics/metabolism
CRISPR-Cas Systems/genetics
Rad52 DNA Repair and Recombination Protein/genetics/metabolism
Saccharomyces cerevisiae/genetics
Homologous Recombination/genetics
*DNA/genetics/metabolism
Gene Editing/methods
Green Fluorescent Proteins/genetics/metabolism

@article {pmid41021780,
year = {2025},
author = {Golla, SA and Abo-Hashesh, M and Gupta, D and Liu, Y and Mahadevan, R},
title = {Model-Based Optimization of a qCRISPRi Circuit for Dynamic Control of Metabolic Pathways.},
journal = {ACS synthetic biology},
volume = {14},
number = {10},
pages = {3890-3898},
doi = {10.1021/acssynbio.5c00095},
pmid = {41021780},
issn = {2161-5063},
mesh = {Escherichia coli/genetics/metabolism ; *Metabolic Engineering/methods ; *Metabolic Networks and Pathways/genetics ; Quorum Sensing/genetics ; Repressor Proteins/genetics/metabolism ; Trans-Activators/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic/genetics ; Gene Expression Regulation, Bacterial ; },
abstract = {Metabolic engineering enables sustainable chemical production but often imposes metabolic burdens that reduce cellular viability and productivity. Dynamic control strategies, such as quorum sensing (QS)-based circuits, can mitigate these effects by autonomously regulating gene expression in response to cell density. In this study, we investigated a QS-regulated CRISPR interference (qCRISPRi) circuit for the dynamic control of metabolic pathways, focusing on the role of leaky expression and regulator stringency. Using a combination of mathematical modeling and experiments, we evaluated how promoter leakiness and LuxR stringency influence key switching characteristics including maximum gene expression, switching density, fold repression, and transition time. Our results show that high leaky expression of dCas9 reduces switching density and represses GFP prematurely, whereas a high-stringency LuxR variant enhances switching precision by reducing leakiness and enabling sharper transitions. These model predictions were validated experimentally in E. coli, confirming that LuxR stringency improves dynamic circuit performance. Together, this work provides a quantitative framework for optimizing QS-based regulatory systems and offers generalizable design insights for implementing dynamic control in metabolic engineering.},
}

Escherichia coli/genetics/metabolism
*Metabolic Engineering/methods
*Metabolic Networks and Pathways/genetics
Quorum Sensing/genetics
Repressor Proteins/genetics/metabolism
Trans-Activators/genetics/metabolism
CRISPR-Cas Systems/genetics
Promoter Regions, Genetic/genetics
Gene Expression Regulation, Bacterial

@article {pmid40992138,
year = {2026},
author = {Xiao, G and Shi, H and Lin, Q and Li, S and He, J and Zhang, G},
title = {A rapid CRISPR-Cas12a/T7EI integrated workflow for high-throughput screening of homozygous mutant cell lines.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {267},
number = {},
pages = {117152},
doi = {10.1016/j.jpba.2025.117152},
pmid = {40992138},
issn = {1873-264X},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *High-Throughput Screening Assays/methods ; Gene Editing/methods ; *Mutation/genetics ; Homozygote ; Workflow ; THP-1 Cells ; Cell Line ; },
abstract = {Efficient screening for homozygous mutant cell lines, particularly those resulting from low-efficiency CRISPR-Cas9 editing, remains challenging. Here, we developed HomoSelect-CT, an integrated workflow combining CRISPR-Cas12a nucleic acid detection with T7 Endonuclease I (T7EI) genotyping, designed to streamline the screening process for homozygous mutant cell lines. This method requires no specialized instrumentation, enhancing accessibility and efficiency. We validated HomoSelect-CT by successfully identifying homozygous mutants in CRISPR-Cas9-edited THP-1 cells, which was confirmed by Sanger sequencing and Western blot (WB). These findings demonstrate that HomoSelect-CT is a robust and efficient alternative for the rapid isolation of genome-edited cell lines. The entire screening workflow, from monoclonal cultures to confirmed homozygous mutants, is completed in under 4 h, requiring only standard PCR equipment and routine reagents. Thus, HomoSelect-CT represents a significant advancement in CRISPR screening methodology, offering remarkable simplicity and enabling high-throughput screening that is particularly suitable for mutants arising from low-efficiency editing events.},
}

Humans
*CRISPR-Cas Systems/genetics
*High-Throughput Screening Assays/methods
Gene Editing/methods
*Mutation/genetics
Homozygote
Workflow
THP-1 Cells
Cell Line

@article {pmid40829585,
year = {2025},
author = {Xu, C and Niu, X and Sun, H and Yan, H and Tang, W and Ke, A},
title = {Conversion of IscB and Cas9 into RNA-guided RNA editors.},
journal = {Cell},
volume = {188},
number = {21},
pages = {5847-5861.e11},
doi = {10.1016/j.cell.2025.07.032},
pmid = {40829585},
issn = {1097-4172},
mesh = {Humans ; *RNA Editing ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/metabolism/genetics ; HEK293 Cells ; Adenosine Deaminase/metabolism/genetics ; RNA, Messenger/metabolism/genetics ; Gene Editing/methods ; RNA-Binding Proteins/metabolism/genetics ; DNA, Single-Stranded/metabolism ; Trans-Splicing ; },
abstract = {RNA-guided RNA editing represents an attractive alternative to DNA editing. However, the prevailing tool, CRISPR-Cas13, has collateral RNA cleavage activity that causes undesirable cytotoxicity in human cells. Here, we report an ultracompact RNA-editing platform engineered from IscB, which has comparable or higher activity than Cas13 but without cytotoxicity concerns. We show that IscB, the evolutionary ancestor of Cas9, has an intrinsic affinity for complementary single-stranded (ss)DNA and RNA. This activity becomes dominant when its double-stranded DNA binding activity is switched off through the deletion of its target-adjacent motif domain. The resulting R-IscB is comparable to or better than Cas13, can efficiently alter splicing outcomes in human cells, and can further mediate trans-splicing to correct any mutation at the mRNA level. R-IscB also drives efficient A-to-I editing on mRNA when fused to adenosine deaminase acting on RNA 2 (ADAR2) and mediates cleavage-based mRNA knockdown upon HNH engineering. Finally, we show that the same approach converts some Cas9s to RNA-targeting tools.},
}

Humans
*RNA Editing
*RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/metabolism/genetics
HEK293 Cells
Adenosine Deaminase/metabolism/genetics
RNA, Messenger/metabolism/genetics
Gene Editing/methods
RNA-Binding Proteins/metabolism/genetics
DNA, Single-Stranded/metabolism
Trans-Splicing

@article {pmid40774253,
year = {2025},
author = {Zhou, Z and Zhu, S and Hong, Y and Jin, G and Ma, R and Lin, F and Zhang, Y and Lee, HY and Liu, N},
title = {Composite transposons with bivalent histone marks function as RNA-dependent enhancers in cell fate regulation.},
journal = {Cell},
volume = {188},
number = {21},
pages = {5878-5894.e18},
doi = {10.1016/j.cell.2025.07.014},
pmid = {40774253},
issn = {1097-4172},
mesh = {Humans ; *DNA Transposable Elements/genetics ; Erythropoiesis/genetics ; *Histone Code ; CRISPR-Cas Systems ; *Enhancer Elements, Genetic ; *Histones/metabolism ; Animals ; *RNA/metabolism ; Myelopoiesis/genetics ; },
abstract = {Discrete genomic units can recombine into composite transposons that transcribe and transpose as single units, but their regulation and function are not fully understood. We report that composite transposons harbor bivalent histone marks, with activating and repressive marks in distinct regions. Genome-wide CRISPR-Cas9 screening, using a reporter driven by the hominid-specific composite transposon SVA (SINE [short interspersed nuclear element]-VNTR [variable number of tandem repeats]-Alu) in human cells, identified diverse genes that modify bivalent histone marks to regulate SVA transcription. SVA transcripts are critical for SVA's cis-regulatory function in selectively contacting and activating long-range gene expression. Remarkably, a subset of bivalent SVAs is activated during erythropoiesis to boost multiple erythroid gene expression, and knocking down these SVAs leads to deficient erythropoiesis. The RNA-dependent cis-regulatory function of SVA activates genes for myelopoiesis and can contribute to aging-associated myeloid-biased hematopoiesis. These results reveal that the cis-regulatory functions of composite transposons are bivalently regulated to control cell fate transitions in development and aging.},
}

Humans
*DNA Transposable Elements/genetics
Erythropoiesis/genetics
*Histone Code
CRISPR-Cas Systems
*Enhancer Elements, Genetic
*Histones/metabolism
Animals
*RNA/metabolism
Myelopoiesis/genetics

@article {pmid40769155,
year = {2025},
author = {Park, JC and Uhm, H and Kim, YW and Oh, YE and Lee, JH and Yang, J and Kim, K and Bae, S},
title = {AI-generated MLH1 small binder improves prime editing efficiency.},
journal = {Cell},
volume = {188},
number = {21},
pages = {5831-5846.e21},
doi = {10.1016/j.cell.2025.07.010},
pmid = {40769155},
issn = {1097-4172},
mesh = {Humans ; *Gene Editing/methods ; Animals ; *MutL Protein Homolog 1/metabolism/genetics/chemistry ; HeLa Cells ; Mice ; Mismatch Repair Endonuclease PMS2/metabolism ; CRISPR-Cas Systems ; DNA Mismatch Repair ; Protein Binding ; RNA, Guide, CRISPR-Cas Systems/metabolism ; },
abstract = {The prime editing (PE) system consists of a Cas9 nickase fused to a reverse transcriptase, which introduces precise edits into the target genomic region guided by a PE guide RNA. However, PE efficiency is limited by mismatch repair. To overcome this limitation, transient expression of a dominant-negative MLH1 (MLH1dn) has been used to inhibit key components of mismatch repair. Here, we designed a de novo MLH1 small binder (MLH1-SB) that binds to the dimeric interface of MLH1 and PMS2 using RFdiffusion and AlphaFold 3. The compact size of MLH1-SB enabled its integration into existing PE architectures via 2A systems, creating a PE-SB platform. The PE7-SB2 system significantly improved PE efficiency, achieving an 18.8-fold increase over PEmax and a 2.5-fold increase over PE7 in HeLa cells, as well as a 3.4-fold increase over PE7 in mice. This study highlights the potential of generative AI in advancing genome editing technology.},
}

Humans
*Gene Editing/methods
Animals
*MutL Protein Homolog 1/metabolism/genetics/chemistry
HeLa Cells
Mice
Mismatch Repair Endonuclease PMS2/metabolism
CRISPR-Cas Systems
DNA Mismatch Repair
Protein Binding
RNA, Guide, CRISPR-Cas Systems/metabolism

@article {pmid40706591,
year = {2025},
author = {Marzook, NB and Song, OR and Baumgärtel, L and Bernitz, N and Mkandawire, TT and Watson, LC and Nunes, V and Warchal, S and MacRae, JI and Howell, M and Sateriale, A},
title = {The essential host genome for Cryptosporidium survival exposes metabolic dependencies that can be leveraged for treatment.},
journal = {Cell},
volume = {188},
number = {21},
pages = {5947-5961.e15},
doi = {10.1016/j.cell.2025.07.001},
pmid = {40706591},
issn = {1097-4172},
mesh = {Animals ; *Cryptosporidiosis/parasitology/drug therapy/genetics/metabolism ; Humans ; *Cryptosporidium/genetics/metabolism/drug effects ; Glutathione/metabolism ; CRISPR-Cas Systems ; Mice ; Epithelial Cells/metabolism/parasitology ; Farnesyl-Diphosphate Farnesyltransferase/antagonists & inhibitors/metabolism ; Cholesterol/biosynthesis ; Host-Parasite Interactions/genetics ; },
abstract = {Cryptosporidium is a leading cause of diarrheal disease, yet little is known regarding the infection cell biology of this intracellular intestinal parasite. To this end, we implemented an arrayed genome-wide CRISPR-Cas9 knockout screen to microscopically analyze multiple phenotypic features of a Cryptosporidium infection following individual host gene ablation. We discovered parasite survival within the host epithelial cell hinges on squalene, an intermediate metabolite in the host cholesterol biosynthesis pathway. A buildup of squalene within intestinal epithelial cells creates a reducing environment, making more reduced glutathione available for parasite uptake. Remarkably, the Cryptosporidium parasite has lost the ability to synthesize glutathione and has become dependent on this host import. This dependency can be leveraged for treatment with the abandoned drug lapaquistat, an inhibitor of host squalene synthase that shifts the redox environment, blocking Cryptosporidium growth in vitro and in vivo.},
}

Animals
*Cryptosporidiosis/parasitology/drug therapy/genetics/metabolism
Humans
*Cryptosporidium/genetics/metabolism/drug effects
Glutathione/metabolism
CRISPR-Cas Systems
Mice
Epithelial Cells/metabolism/parasitology
Farnesyl-Diphosphate Farnesyltransferase/antagonists & inhibitors/metabolism
Cholesterol/biosynthesis
Host-Parasite Interactions/genetics

@article {pmid40685751,
year = {2025},
author = {Qin, W and Lin, SJ and Zhang, Y and Huang, K and Petree, C and Boyd, K and Varshney, P and Varshney, GK},
title = {Rationally Designed TadA-Derived Cytosine Editors Enable Context-Independent Zebrafish Genome Editing.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {39},
pages = {e09800},
doi = {10.1002/advs.202509800},
pmid = {40685751},
issn = {2198-3844},
support = {R24 OD034438/OD/NIH HHS/United States ; R24OD034438/RI/ORIP NIH HHS/United States ; /NH/NIH HHS/United States ; R24OD034438/RI/ORIP NIH HHS/United States ; },
mesh = {Animals ; *Zebrafish/genetics ; *Gene Editing/methods ; *Cytosine/metabolism ; CRISPR-Cas Systems/genetics ; Genome/genetics ; },
abstract = {CRISPR base editors are crucial for precise genome manipulation. Existing APOBEC-based cytosine base editors (CBEs), while powerful, exhibit indels and sequence context limitations, and editing CC and GC motifs is challenging and inefficient. To address these challenges, existing tRNA adenine deaminase (TadA)-derived CBEs are evaluated in zebrafish, and a series of zTadCBE variants is developed that demonstrate high editing efficiency, minimized off-target effects, and an expanded targeting range compared to existing tools. The approach integrates beneficial mutations from TadA-based adenine base editors (ABEs) with SpRYCas9n-enhanced protospacer-adjacent motif (PAM) compatibility. The expanded window zTadCBE variants enable the targeting of cytosines at a broader range of nucleotide positions relative to the PAM sequence, further enhancing the versatility of this tool. Using zTadCBEs, four zebrafish disease models affecting the auditory, nervous, metabolic, and muscular systems are generated directly in the F0 generation-models that cannot be efficiently produced using earlier CBE tools. Together, zTadCBE variants provide a robust and flexible toolkit for efficient and precise C-to-T base editing in zebrafish, facilitating rapid in vivo functional assessment of genetic variants.},
}

Animals
*Zebrafish/genetics
*Gene Editing/methods
*Cytosine/metabolism
CRISPR-Cas Systems/genetics
Genome/genetics

@article {pmid40683408,
year = {2025},
author = {Zhou, J and Zhou, C and Jiang, G and Wang, Y and Pan, L and Jia, X and Tu, X and Li, W and Wang, C},
title = {Engineering an Escherichia coli with performance-enhanced switch utilizing CRISPR-Cas9 system as living quorum quencher for biofilm formation inhibition.},
journal = {Environmental research},
volume = {285},
number = {Pt 2},
pages = {122383},
doi = {10.1016/j.envres.2025.122383},
pmid = {40683408},
issn = {1096-0953},
mesh = {*Biofilms/growth & development ; *Escherichia coli/genetics/physiology ; *Quorum Sensing/genetics ; *CRISPR-Cas Systems ; Genetic Engineering ; },
abstract = {Quorum quenching (QQ) of signal molecules plays a critical role in disrupting bacterial communication, thereby suppressing biofilm formation. However, the wild-type QQ bacteria lacks the regulatory capacity to modulate gene expression levels. In this study, the QQ gene aiiO and reporter gene GFP were chromosomally integrated into Escherichia coli BW25113 using the clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) system. The performance-enhanced switch in the engineering bacteria (EB) allowed it to express aiiO weakly without the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG) and express aiiO strongly with IPTG, and 1.00 mM IPTG induction enhanced EB's QQ activity by 2.34-fold. In activated sludge systems, the inoculation of EB reduced biofilm formation by 18.56 % versus controls after 168 h, with the performance-enhanced switch enhancing inhibition to 24.72 %. EB reduced biofilm thickness by 22.96 %, total microbial biomass by 57.68 %, and significantly decreased extracellular polymeric substances secretion and adhesion strength of the biofilm (maximum reductions: 29.88 % and 34.31 %, respectively) across all sampling points versus controls. 1.00 mM IPTG addition sustainedly intensified these biofilm-inhibitory effects by EB, demonstrating the genetic switch's persistent functionality under environmentally relevant conditions. Furthermore, the genetically modified strain exhibited minimal environmental impact according to standardized assessments. Therefore, this study successfully constructed an implementable strategy for engineering bacteria-mediated biofilm control, with demonstrated applicability in complex environmental systems.},
}

*Biofilms/growth & development
*Escherichia coli/genetics/physiology
*Quorum Sensing/genetics
*CRISPR-Cas Systems
Genetic Engineering

@article {pmid41097540,
year = {2025},
author = {Chen, C and Wu, T and Liu, J and Gao, J},
title = {Threat and Control of tet(X)-Mediated Tigecycline-Resistant Acinetobacter sp. Bacteria.},
journal = {Foods (Basel, Switzerland)},
volume = {14},
number = {19},
pages = {},
doi = {10.3390/foods14193374},
pmid = {41097540},
issn = {2304-8158},
support = {32402890//National Natural Science Foundation of China/ ; 2023M732993//China Postdoctoral Science Foundation/ ; },
abstract = {Tigecycline is regarded as one of the last-resort antibiotics against multidrug-resistant (MDR) Acinetobacter sp. bacteria. Recently, the tigecycline-resistant Acinetobacter sp. isolates mediated by tet(X) genes have emerged as a class of global pathogens for humans and food-producing animals. However, the genetic diversities and treatment options were not systematically discussed in the era of One Health. In this review, we provide a detailed illustration of the evolution route, distribution characteristics, horizontal transmission, and rapid detection of tet(X) genes in diverse Acinetobacter species. We also detail the application of chemical drugs, plant extracts, phages, antimicrobial peptides (AMPs), and CRISPR-Cas technologies for controlling tet(X)-positive Acinetobacter sp. pathogens. Despite excellent activities, the antibacterial spectrum and application safety need further evaluation and resolution. It is noted that deep learning is a promising approach to identify more potent antimicrobial compounds.},
}

@article {pmid41097079,
year = {2025},
author = {Wu, KC and Chang, YH and Chiang, RY and Ding, DC},
title = {CAP-LAMP2b-Modified Stem Cells' Extracellular Vesicles Hybrid with CRISPR-Cas9 Targeting ADAMTS4 to Reverse IL-1β-Induced Aggrecan Loss in Chondrocytes.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199812},
pmid = {41097079},
issn = {1422-0067},
support = {TCRD 113-065//Hualien Tzu Chi Hospital/ ; },
mesh = {*Extracellular Vesicles/metabolism ; Humans ; *Chondrocytes/metabolism/cytology ; *ADAMTS4 Protein/genetics/metabolism ; *CRISPR-Cas Systems ; *Mesenchymal Stem Cells/metabolism/cytology ; *Aggrecans/metabolism ; *Interleukin-1beta/metabolism ; Cell Differentiation ; Cells, Cultured ; Gene Editing ; },
abstract = {Extracellular vesicles (EVs) from mesenchymal stem cells hold therapeutic promise for inflammatory and degenerative diseases; however, limited delivery and targeting capabilities hinder their clinical use. In this study, we sought to enhance the anti-inflammatory and chondroprotective effects of EVs through CAP-LAMP2b (chondrocyte affinity peptide fused to an EV membrane protein) engineering and ADAMTS4 gene editing hybrid vesicle formation. Human umbilical cord MSCs (hUCMSCs) were characterized via morphology, immunophenotyping, and trilineage differentiation. EVs from control and CAP-LAMP2b-transfected hUCMSCs were fused with liposomes carrying CRISPR-Cas9 ADAMTS4 gRNA. DiI-labeled EV uptake was assessed via fluorescence imaging. CAP-LAMP2b was expressed in hUCMSCs and their EVs. EVs exhibited the expected size (~120 nm), morphology, and exosomal markers (CD9, CD63, CD81, HSP70). CAP-modified hybrid EVs significantly enhanced chondrocyte uptake compared to control EVs and liposomes. IL-1β increased ADAMTS4 expression, whereas CAP-LAMP2b-ADAMTS4 EVs, particularly clone SG3, reversed these effects by reducing ADAMTS4 and restoring aggrecan. Western blotting confirmed suppressed ADAMTS4 and elevated aggrecan protein. CAP-LAMP2b-ADAMTS4 EVs, therefore, showed superior uptake and therapeutic efficacy in inflamed chondrocytes, attenuating inflammatory gene expression and preserving matrix integrity. These results support engineered EVs as a promising cell-free approach for cartilage repair and osteoarthritis treatment.},
}

*Extracellular Vesicles/metabolism
Humans
*Chondrocytes/metabolism/cytology
*ADAMTS4 Protein/genetics/metabolism
*CRISPR-Cas Systems
*Mesenchymal Stem Cells/metabolism/cytology
*Aggrecans/metabolism
*Interleukin-1beta/metabolism
Cell Differentiation
Cells, Cultured
Gene Editing

@article {pmid41097026,
year = {2025},
author = {Jeong, SK and Park, JR and Kim, EG and Kim, KM},
title = {Development of Resistance to Damping-Off in Rice, Oryza sativa L., Using CRISPR/Cas9.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199761},
pmid = {41097026},
issn = {1422-0067},
mesh = {*Oryza/genetics/microbiology/growth & development ; *CRISPR-Cas Systems ; *Disease Resistance/genetics ; *Plant Diseases/genetics/microbiology ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; Rhizoctonia/pathogenicity ; Plant Proteins/genetics ; },
abstract = {Damping-off disease hinders rice seedling growth and reduces yield. Current control methods, such as seed or soil sterilization, rely on chemicals that cause environmental pollution and promote pathogen resistance. As a sustainable alternative, we targeted the damping-off resistance-related gene OsDGTq1 using CRISPR/Cas9. Field experiments first verified OsDGTq1's significance in resistance. The CRISPR/Cas9 system, delivered via Agrobacterium-mediated transformation, was used to edit OsDGTq1 in rice cultivar Ilmi. Lesions from major damping-off pathogens, Rhizoctonia solani and Pythium graminicola, were observed on G0 plants. All 37 regenerated plants contained T-DNA insertions. Among them, edits generated by sgRNA1-1, sgRNA1-2, and sgRNA1-3 resulted in the insertion of two thymine bases as target mutations. Edited lines were assigned names and evaluated for agronomic traits, seed-setting rates, and pathogen responses. Several lines with edited target genes showed distinct disease responses and altered gene expression compared to Ilmi, likely due to CRISPR/Cas9-induced sequence changes. Further studies in subsequent generations are needed to confirm the stability of these edits and their association with resistance. These results confirm that genome editing of OsDGTq1 alters resistance to damping-off. The approach demonstrates that gene-editing technology can accelerate rice breeding, offering an environmentally friendly strategy to develop resistant varieties. Such varieties can reduce chemical inputs, prevent pollution, and minimize seedling loss, ultimately enhancing food self-sufficiency and stabilizing rice supply.},
}

*Oryza/genetics/microbiology/growth & development
*CRISPR-Cas Systems
*Disease Resistance/genetics
*Plant Diseases/genetics/microbiology
Gene Editing/methods
Plants, Genetically Modified/genetics
Rhizoctonia/pathogenicity
Plant Proteins/genetics

@article {pmid41097019,
year = {2025},
author = {Kapitonova, MA and Shabalina, AV and Dedkov, VG and Dolgova, AS},
title = {CRISPR-Cas12a-Based Isothermal Detection of Mammarenavirus machupoense Virus: Optimization and Evaluation of Multiplex Capability.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199754},
pmid = {41097019},
issn = {1422-0067},
support = {Ensuring chemical and biological safety of the Russian Federation//state program/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *Endodeoxyribonucleases/genetics ; *CRISPR-Associated Proteins/genetics ; *Molecular Diagnostic Techniques/methods ; *Arenaviridae/genetics/isolation & purification ; Animals ; Bacterial Proteins ; },
abstract = {Bolivian hemorrhagic fever (BHF) is a zoonotic disease caused by Mammarenavirus machupoense (MACV) featuring severe neurological and hemorrhagic symptoms and a high mortality rate. BHF is usually diagnosed by serological tests or real-time polymerase chain reaction (RT-PCR); these methods are often inaccessible in endemic regions due to a lack of laboratory infrastructure, creating a demand for sensitive and rapid equipment-free alternatives. Here, we present an isothermal method for MACV nucleic acid detection based on the Cas12a-based DETECTR system combined with recombinase polymerase amplification (RPA) in a single tube: the RT-RPA/DETECTR assay. We demonstrate the possibility of using more than one primer set for the simultaneous detection of MACV genetic variants containing multiple point mutations. The method was optimized and tested using specially developed virus-like armored particles containing the target sequence. The multiplex RT-RPA/DETECTR method achieved a limit of detection of approximately 5 × 10[4] copies/ mL (80 aM) of armored particles. The method was validated using clinical samples spiked with virus-like particles. The assay proved to be selective and reliable in detecting certain nucleotide substitutions simultaneously.},
}

*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
Humans
*Endodeoxyribonucleases/genetics
*CRISPR-Associated Proteins/genetics
*Molecular Diagnostic Techniques/methods
*Arenaviridae/genetics/isolation & purification
Animals
Bacterial Proteins

@article {pmid41096922,
year = {2025},
author = {Xu, J and Pan, M and Zhu, Y and Wang, P and Jiang, L and Xu, D and Wang, X and Chen, L and Guo, W and Yang, H and Cao, D},
title = {CRISPR/Cas9-Mediated Targeted Mutagenesis of GmAS1/2 Genes Alters Leaf Shape in Soybean.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199657},
pmid = {41096922},
issn = {1422-0067},
support = {YBXM2529//National Nanfan Research Institute of CAAS/ ; },
mesh = {*Glycine max/genetics/anatomy & histology/growth & development ; *CRISPR-Cas Systems ; *Plant Leaves/genetics/anatomy & histology/growth & development ; Gene Expression Regulation, Plant ; Phenotype ; *Plant Proteins/genetics/metabolism ; *Mutagenesis ; *Genes, Plant ; Gene Editing ; Transcription Factors/genetics ; Gene Expression Profiling ; Mutation ; },
abstract = {ASYMMETRIC LEAVES1 (AS1) and AS2 play essential roles in regulating leaf development in plants. However, their functional roles in soybean remain poorly understood. Here, we identified two members of the soybean AS1 gene family, GmAS1a and GmAS1c, which exhibit high expression levels in stem and leaf tissues. Using the CRISPR/Cas9 system, we targeted four GmAS1 and three GmAS2 genes, generating mutant lines with distinct leaf development phenotypes, including wrinkling (refers to fine lines and creases on the leaf surface, like aged skin texture), curling (describes the inward or outward rolling of leaf edges, deviating from the typical flat shape), and narrow. We found that functional redundancy exists among the four GmAS1 genes in soybean. GmAS1 and GmAS2 cooperatively regulate leaf curling, leaf crinkling phenotypes, and leaf width in soybean, with functional redundancy also observed between these two genes. Transcriptome sequencing analysis of w3 mutant (as1b as1c as1d as2a as2b as2c) identified 1801 differentially expressed genes (DEGs), including 192 transcription factors (TFs). Gene ontology enrichment analysis revealed significant enrichment of DEGs in pathways associated with plant hormone biosynthesis and signal transduction. A detailed examination of the DEGs showed several genes involved in the development of leaf lateral organs, such as KNOX (SHOOT MERISTEMLESS (STM), KNAT1, KNAT2, and KNAT6), LOB (LBD25, LBD30), and ARP5, were down-regulated in w3/WT (wild-type) comparison. CRISPR/Cas9-mediated targeted mutagenesis of the GmAS1/2 genes significantly impairs leaf development and polarity establishment in soybean, providing valuable germplasm resources and a theoretical framework for future studies on leaf morphogenesis.},
}

*Glycine max/genetics/anatomy & histology/growth & development
*CRISPR-Cas Systems
*Plant Leaves/genetics/anatomy & histology/growth & development
Gene Expression Regulation, Plant
Phenotype
*Plant Proteins/genetics/metabolism
*Mutagenesis
*Genes, Plant
Gene Editing
Transcription Factors/genetics
Gene Expression Profiling
Mutation

@article {pmid41096782,
year = {2025},
author = {Huang, C and Liu, M and Kok, J},
title = {Chromosomal and Plasmid-Based CRISPRi Platforms for Conditional Gene Silencing in Lactococcus lactis.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199516},
pmid = {41096782},
issn = {1422-0067},
support = {201706350298//China Scholarship Council/ ; 201505990303//China Scholarship Council/ ; },
mesh = {*Lactococcus lactis/genetics ; *Plasmids/genetics ; *CRISPR-Cas Systems ; *Gene Silencing ; Streptococcus pyogenes/genetics ; Bacterial Proteins/genetics ; Gene Expression Regulation, Bacterial ; *Chromosomes, Bacterial/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; Promoter Regions, Genetic ; },
abstract = {Inducible CRISPR interference (CRISPRi) systems were established in Lactococcus lactis using both plasmid and chromosomal approaches. Expression of nuclease-deficient Cas9 (dCas9) from Streptococcus pyogenes was placed under the control of the nisin-inducible promoter PnisA, while sgRNAs were transcribed from the constitutive Pusp45 promoter. To monitor expression, dCas9 was fused with superfolder GFP. Plasmid-based constructs successfully repressed a luciferase reporter gene and silenced the gene of the major autolysin, AcmA, leading to the expected morphological phenotype. However, plasmid systems showed leaky expression, producing mutant phenotypes even without induction. Chromosomal integration of dCas9 reduced its expression level by approximately 20-fold compared with plasmid-based expression, thereby preventing leaky activity and ensuring tight regulation. This chromosome-based (cbCRISPRi) platform enabled controlled repression of the essential gene ybeY, which resulted in severe growth defects. Restoration of wild-type phenotypes was achieved by introducing a synonymous codon substitution in the sgRNA target region. Transcriptome analysis of ybeY-silenced cells revealed downregulation of ribosomal protein genes and widespread effects on membrane-associated proteins, ATP synthase subunits, and various transporters. These inducible CRISPRi platforms provide robust and tunable tools for functional genomics in L. lactis, particularly for studying essential genes that cannot be deleted.},
}

*Lactococcus lactis/genetics
*Plasmids/genetics
*CRISPR-Cas Systems
*Gene Silencing
Streptococcus pyogenes/genetics
Bacterial Proteins/genetics
Gene Expression Regulation, Bacterial
*Chromosomes, Bacterial/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing
Promoter Regions, Genetic

@article {pmid41096720,
year = {2025},
author = {Peng, H and Li, J and Sun, K and Tang, H and Huang, W and Li, X and Wang, S and Ding, K and Han, Z and Li, Z and Xu, L and Wang, K},
title = {Advances and Applications of Plant Base Editing Technologies.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199452},
pmid = {41096720},
issn = {1422-0067},
support = {32472182//National Natural Science Foundation of China/ ; 2024ZD0407704//Biological Breeding-National Science and Technology Major Project/ ; 2022BBF02039//Science and Technology Department of Ningxia in China/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Crops, Agricultural/genetics ; Genome, Plant ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; },
abstract = {Base editing represents a major breakthrough in the field of genome editing in recent years. By fusing deaminases with the CRISPR/Cas system, it enables precise single-base modifications of DNA. This review systematically summarizes the development of base editing technologies, including cytosine base editors (CBEs), adenine base editors (ABEs), and glycosylase base editors (GBEs), with a particular focus on their applications in crop improvement as well as future trends and prospects. We highlight advances in the creation of novel germplasm with enhanced stress resistance and desirable agronomic traits through base editing in rice, wheat, maize, potato, and other crops, particularly for improving herbicide resistance, disease resistance, and grain quality. Furthermore, we analyze factors that influence base editing efficiency, noting that challenges remain, such as PAM sequence constraints, limited base conversion types, off-target effects, narrow editing windows, and efficiency variation. Future efforts should aim to optimize deaminase activity, expand PAM compatibility, and develop versatile tools to facilitate the broad application of base editing in molecular breeding. This review provides a timely reference for researchers and breeders, offering theoretical guidance and practical insights into harnessing base editing for crop genetic improvement.},
}

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

@article {pmid41096693,
year = {2025},
author = {He, J and Shi, N and Yao, H and Li, J and Wang, Y and Zhang, J},
title = {Genome Editing in the Chicken: From PGC-Mediated Germline Transmission to Advanced Applications.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199426},
pmid = {41096693},
issn = {1422-0067},
support = {2023ZD04053//the Biological Breeding-National Science and Technology Major Project/ ; 2025YFHZ0042//the Natural Science Foundation of Sichuan Province/ ; SCU2025D003//the fundamental research funds for the central universitie/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Chickens/genetics ; *Germ Cells/metabolism ; CRISPR-Cas Systems ; },
abstract = {Avian genome editing has historically lagged behind mammalian research. This disparity is primarily due to a unique reproductive biology that precludes standard techniques like pronuclear injection. A pivotal breakthrough, however, came from the development of efficient in vitro culture systems for primordial germ cells (PGCs). This has established the chicken as a tractable and powerful model for genetic engineering. Our review chronicles the technological evolution this has enabled, from early untargeted methods to the precision of modern CRISPR-based systems. We then analyze the broad applications of these tools, which are now used to engineer disease resistance, enhance agricultural traits, and develop novel platforms such as surrogate hosts and oviduct bioreactors. Collectively, these advances have established PGC-based genome editing as a robust and versatile platform. Looking forward, emerging precision editors and the expansion of these techniques to other avian species are poised to drive the next wave of innovation in poultry science and biotechnology.},
}

Animals
*Gene Editing/methods
*Chickens/genetics
*Germ Cells/metabolism
CRISPR-Cas Systems

@article {pmid41096689,
year = {2025},
author = {Haldrup, SB and McClements, ME and Cehajic-Kapetanovic, J and Corydon, TJ and MacLaren, RE},
title = {Gene Therapy Strategies for the Treatment of Bestrophinopathies.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199421},
pmid = {41096689},
issn = {1422-0067},
support = {NA//Aarhus University/ ; NA//Fight for Sight Denmark/ ; NA//Synoptik Fonden/ ; NA//Maskinfabrikant Jochum Jensen og hustru Mette Marie Jensen, f. Poulsens Mindelegat (fond)/ ; NA//A.P. Møller Foundation/ ; NA//Mette Warburgs Fond/ ; NA//APT Holding/ ; 00038189//VELUX Foundation/ ; 24OC0088426//Novo Nordisk Foundation/ ; NA//NIHR Oxford Biomedical Research Centre/ ; 304408/Z/23/Z//Wellcome Discovery Award/ ; },
mesh = {Humans ; *Genetic Therapy/methods ; *Bestrophins/genetics/metabolism ; *Retinal Diseases/therapy/genetics ; *Eye Diseases, Hereditary/therapy/genetics ; Gene Editing/methods ; Mutation ; Animals ; CRISPR-Cas Systems ; Chloride Channels/genetics ; },
abstract = {The BEST1 gene encodes a transmembrane protein in the retinal pigment epithelium (RPE) in the eye, that functions as a calcium-dependent chloride channel (CaCC). Pathogenic variants in BEST1 are the underlying cause for bestrophinopathies, a group of inherited retinal disorders that vary in their pattern of inheritance, clinical appearance, and underlying molecular disease mechanisms. Currently, there are no treatments available for any of the bestrophinopathies, and gene therapy represents an attractive strategy due to the accessibility of the eye and slow disease progression. While gene augmentation may be effective for a subset of bestrophinopathies, others require allele-specific silencing or correction of the disease-causing variant to reconstitute expression of the BEST1 protein. This review aims to give an overview of the clinical diversity of bestrophinopathies and proposes the molecular disease mechanism of the pathogenic BEST1 variant as an important parameter for the choice of treatment strategy. Furthermore, we discuss the potential of different mutation-specific and mutation-independent CRISPR/Cas9-based gene editing strategies as a future treatment approach for bestrophinopathies.},
}

Humans
*Genetic Therapy/methods
*Bestrophins/genetics/metabolism
*Retinal Diseases/therapy/genetics
*Eye Diseases, Hereditary/therapy/genetics
Gene Editing/methods
Mutation
Animals
CRISPR-Cas Systems
Chloride Channels/genetics

@article {pmid41096677,
year = {2025},
author = {Șerban, M and Toader, C and Covache-Busuioc, RA},
title = {CRISPR and Artificial Intelligence in Neuroregeneration: Closed-Loop Strategies for Precision Medicine, Spinal Cord Repair, and Adaptive Neuro-Oncology.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199409},
pmid = {41096677},
issn = {1422-0067},
mesh = {Humans ; *Artificial Intelligence ; *Precision Medicine/methods ; Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; *Spinal Cord Regeneration ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Nerve Regeneration/genetics ; },
abstract = {Repairing the central nervous system (CNS) remains one of the most difficult obstacles to overcome in translational neurosciences. This is due to intrinsic growth inhibitors, extracellular matrix issues, the glial scar-form barrier, chronic neuroinflammation, and epigenetic silencing. The purpose of this review is to bring together findings from recent developments in genome editing and computational approaches, which center around the possible convergence of clustered regularly interspaced short palindromic repeats (CRISPR) platforms and artificial intelligence (AI), towards precision neuroregeneration. We wished to outline possible ways in which CRISPR-based systems, including but not limited to Cas9 and Cas12 nucleases, RNA-targeting Cas13, base and prime editors, and transcriptional regulators such as CRISPRa/i, can be applied to potentially reactivate axon-growth programs, alter inhibitory extracellular signaling, reprogram or lineage transform glia to functional neurons, and block oncogenic pathways in glioblastoma. In addition, we wanted to highlight how AI approaches, such as single-cell multi-omics, radiogenomic prediction, development of digital twins, and design of adaptive clinical trials, will increasingly be positioned to act as system-level architects that allow translation of complex datasets into predictive and actionable therapeutic approaches. We examine convergence consumers in spinal cord injury and adaptive neuro-oncology and discuss expanse consumers in ischemic stroke, Alzheimer's disease, Parkinson's disease, and rare neurogenetic syndromes. Finally, we discuss the ethical and regulatory landscape around beyond off-target editing and genomic stability of CRISPR, algorithmic bias, explainability, and equitable access to advanced neurotherapies. Our intent was not to provide a comprehensive inventory of possibilities but rather to provide a conceptual tool where CRISPR acts as a molecular manipulator and AI as a computational integrator, converging to create pathways towards precision neuroregeneration, personalized medicine, and adaptive neurotherapeutics that are ethically sound.},
}

Humans
*Artificial Intelligence
*Precision Medicine/methods
Gene Editing/methods
*CRISPR-Cas Systems
Animals
*Spinal Cord Regeneration
*Clustered Regularly Interspaced Short Palindromic Repeats
*Nerve Regeneration/genetics

@article {pmid41096563,
year = {2025},
author = {Simoni, S and Fambrini, M and Pugliesi, C and Rogo, U},
title = {Genome Editing by Grafting.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199294},
pmid = {41096563},
issn = {1422-0067},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Genome, Plant ; *Plants, Genetically Modified/genetics ; },
abstract = {Grafting is the process of joining parts of two plants, allowing the exchange of molecules such as small RNAs (including microRNAs and small interfering RNAs), messenger RNAs, and proteins between the rootstock and the scion. Genome editing by grafting exploits RNAs, such as tRNA-like sequences (TLS motifs), to deliver the components (RNA) of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system from transgenic rootstock to wild-type scion. The complex Cas9 protein and sgRNA-TLS produced in the scion perform the desired modification without the integration of foreign DNA in the plant genome, resulting in heritable transgene-free genome editing. In this review, we examine the current state of the art of this innovation and how it helps address regulatory problems, improves crop recovery and selection, exceeds the usage of viral vectors, and may reduce potential off-target effects. We also discuss the promise of genome editing by grafting for plants recalcitrant to in vitro culture and for agamic-propagated species that must maintain heterozygosity for plant productivity, fruit quality, and adaptation. Furthermore, we explore the limitations of this technique, including variable efficiency, graft incompatibility among genotypes, and challenges in large-scale application, while highlighting its considerable potential for further improvement and future broader applications for crop breeding.},
}

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

@article {pmid41093884,
year = {2025},
author = {Moon, J and Zhang, J and Guan, X and Yang, R and Guo, C and Schalper, KT and Avery, L and Banach, D and LaSala, R and Warrier, R and Liu, C},
title = {CRISPR anti-tag-mediated room-temperature RNA detection using CRISPR/Cas13a.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9142},
pmid = {41093884},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Hepacivirus/genetics/isolation & purification ; *RNA, Viral/genetics/blood/analysis ; Temperature ; *CRISPR-Associated Proteins/metabolism/genetics ; HIV Infections/virology/diagnosis/blood ; Hepatitis C/diagnosis/virology ; HIV/genetics/isolation & purification ; HIV-1/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The CRISPR/Cas13a enzyme serves as a powerful tool for RNA detection due to its RNA-targeting capabilities. However, simple and highly sensitive detection using Cas13a faces challenges, such as the need for pre-amplification and elevated reaction temperatures. In this study, we investigate the allosteric regulation mechanism of Cas13a activation by target RNAs with various structures containing the CRISPR anti-tag sequence. We discover that the target RNA secondary structure and anti-tag sequences inhibit the trans-cleavage reaction of Cas13a. By designing and introducing a specific CRISPR anti-tag hairpin, we develop CRISPR Anti-tag Mediated Room-temperature RNA Detection (CARRD) using a single CRISPR/Cas13a enzyme. This method enables one-step cascade signal amplification for RNA detection without the need for pre-amplification. We apply the CARRD method to detect human immunodeficiency virus (HIV) and hepatitis C virus (HCV), achieving a detection sensitivity of 10 aM. Furthermore, we validate its clinical feasibility by detecting HIV clinical plasma samples, demonstrating a simple, affordable, and efficient approach for viral RNA detection. Due to its simplicity, sensitivity, and flexible reaction temperature, the CARRD method is expected to have broad applicability, paving the way for the development of field-deployable diagnostic tools.},
}

*CRISPR-Cas Systems/genetics
Humans
Hepacivirus/genetics/isolation & purification
*RNA, Viral/genetics/blood/analysis
Temperature
*CRISPR-Associated Proteins/metabolism/genetics
HIV Infections/virology/diagnosis/blood
Hepatitis C/diagnosis/virology
HIV/genetics/isolation & purification
HIV-1/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41093851,
year = {2025},
author = {Zhou, SK and Luo, JT and Chen, YF and Lu, ZD and Jian, QH and Jiang, SQ and Li, J and Zhang, XQ and Tan, XY and Yang, XZ and Xu, CF and Wang, J},
title = {Muscle-specific gene editing therapy via mammalian fusogen-directed virus-like particles.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9145},
pmid = {41093851},
issn = {2041-1723},
support = {32430059//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32471434//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32271442//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Muscle, Skeletal/metabolism ; Mice ; *Muscular Dystrophy, Duchenne/therapy/genetics ; CRISPR-Cas Systems ; Dystrophin/genetics/metabolism ; *Genetic Therapy/methods ; Humans ; Disease Models, Animal ; CRISPR-Associated Protein 9/genetics/metabolism ; Male ; Ribonucleoproteins/genetics/metabolism ; Exons ; Mice, Inbred C57BL ; },
abstract = {Muscle genetic defects can lead to impaired movement, respiratory failure, and other severe symptoms. The development of curative therapies is challenging due to the need for the delivery of gene-editing tools into skeletal muscle cells throughout the body. Here, we use muscular fusogens (Myomaker and Myomerger) to engineer muscle-specific virus-like particles (MuVLPs) for the systemic delivery of gene-editing tools. We demonstrate that MuVLPs can be loaded with diverse payloads, including EGFP, Cre and Cas9/sgRNA ribonucleoproteins (Cas9 RNPs), and can be delivered into skeletal muscle cells via targeted membrane fusion. Systemic administration of MuVLPs carrying Cas9 RNPs enables skeletal muscle-specific gene editing, which excised the exon containing a premature terminator codon mutation in a mouse model for Duchenne muscular dystrophy (DMD). This treatment restores dystrophin expression in various skeletal muscle tissues, including the diaphragm, quadriceps, tibialis anterior, gastrocnemius, and triceps. As a result, the treated mice exhibit a significantly increased capacity for exercise and endurance. This study established a platform for precise gene editing in skeletal muscle tissues.},
}

Animals
*Gene Editing/methods
*Muscle, Skeletal/metabolism
Mice
*Muscular Dystrophy, Duchenne/therapy/genetics
CRISPR-Cas Systems
Dystrophin/genetics/metabolism
*Genetic Therapy/methods
Humans
Disease Models, Animal
CRISPR-Associated Protein 9/genetics/metabolism
Male
Ribonucleoproteins/genetics/metabolism
Exons
Mice, Inbred C57BL

@article {pmid41093849,
year = {2025},
author = {Shang, M and Li, Y and Cao, Q and Ren, J and Zeng, Y and Wang, J and Gonzalez, RVL and Zhang, X},
title = {A motif preferred adenine base editor with minimal bystander and off-targets editing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9153},
pmid = {41093849},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; Animals ; Humans ; Mice ; *Adenine/metabolism/chemistry ; *Nucleotide Motifs/genetics ; HEK293 Cells ; CRISPR-Cas Systems ; },
abstract = {47% of hereditable diseases are caused by single C•G-to-T•A base conversions, which means efficient A-to-G base editing tools (ABEs) have great potential for the treatment of these diseases. However, the existing efficient ABEs, while catalyzing targeted A-to-G conversion, cause high A or C bystander editing and off-target events, which poses safety concerns for their clinical applications. To overcome this shortcoming, we have developed ABE8e-YA (ABE8e with TadA-8e A48E) for efficient and accurate editing of As in YA motifs with YAY > YAR (Y = T or C, R = A or G) hierarchy through structure-oriented rational design. Compared with ABE3.1, which is currently the only ABE version with a YAC motif preference, ABE8e-YA exhibits an average A-to-G editing efficiency improvement of an up to 3.1-fold increase in the indicated YA motif while maintaining reduced bystander C editing and minimized DNA or RNA off-targets. Additionally, we demonstrate that ABE8e-YA efficiently and precisely corrects pathogenic mutations in human cells, suggesting its high suitability for addressing 9.3% of pathogenic point mutations, higher than that of ABE8e and ABE9. Moreover, by using ABE8e-YA, we efficiently and precisely generate hypocholesterolemia and tail-loss mouse models mimicking human-associated disease, as well as performed in vivo mouse proprotein convertase subtilisin/kexin type 9 (Pcsk9) base editing for hypercholesterolemia gene therapy. Together these data indicate its great potential in broad applications for disease modeling and gene therapy.},
}

*Gene Editing/methods
Animals
Humans
Mice
*Adenine/metabolism/chemistry
*Nucleotide Motifs/genetics
HEK293 Cells
CRISPR-Cas Systems

@article {pmid41093834,
year = {2025},
author = {Shibue, K and Kahraman, S and Castillo-Quan, JI and De Jesus, DF and Hu, J and Morita, H and Blackwell, TK and Yi, P and Kulkarni, RN},
title = {Genome-wide CRISPR Screen Identifies Sec31A as a Key Regulator of Alpha Cell Survival.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9159},
pmid = {41093834},
issn = {2041-1723},
support = {DK067536//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R35GM122610//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01AG054215//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; JP22K16404//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; },
mesh = {Animals ; Humans ; Mice ; Caenorhabditis elegans/metabolism/genetics ; *Vesicular Transport Proteins/genetics/metabolism ; *Glucagon-Secreting Cells/metabolism/cytology ; Cell Survival/genetics ; Endoplasmic Reticulum Stress/genetics ; CRISPR-Cas Systems ; Receptor, Insulin/metabolism ; Endoplasmic Reticulum/metabolism ; Signal Transduction ; Insulin/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Insulin-Secreting Cells/metabolism ; Glucagon/metabolism ; },
abstract = {Glucagon, secreted by pancreatic alpha cells, is essential for maintaining normal blood glucose levels. In type 1 and advanced type 2 diabetes, alpha cells often fail to respond to low glucose, yet the mechanisms underlying their stress resistance remain unclear. To investigate this, we performed a genome-wide CRISPR screen and identify Sec31A, a gene involved in transporting proteins from the endoplasmic reticulum (ER), as a key regulator of alpha cell survival under stress. We show that loss of Sec31A enhances survival in stressed mouse alpha cells and in C. elegans. In human islets, SEC31A expression increases in alpha cells under inflammatory stress, and this upregulation is reversed by reducing ER stress. Functional studies in lab-grown human islet clusters reveal distinct responses in alpha versus beta cells following Sec31A suppression. We also find that Sec31A interacts with the insulin receptor, suggesting a link between stress adaptation and insulin signaling in alpha cells.},
}

Animals
Humans
Mice
Caenorhabditis elegans/metabolism/genetics
*Vesicular Transport Proteins/genetics/metabolism
*Glucagon-Secreting Cells/metabolism/cytology
Cell Survival/genetics
Endoplasmic Reticulum Stress/genetics
CRISPR-Cas Systems
Receptor, Insulin/metabolism
Endoplasmic Reticulum/metabolism
Signal Transduction
Insulin/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats
Insulin-Secreting Cells/metabolism
Glucagon/metabolism

@article {pmid41093525,
year = {2025},
author = {Wei, Z and Luo, H and Huang, D and Wei, P and Zhang, J and Wei, J and Tang, Q and Huang, L and Zhang, K and Liao, X},
title = {Structure-specific electrochemiluminescent biosensor for FEN1 detection via dumbbell probe-mediated transcription and CRISPR/Cas13a-induced G-quadruplexes cleavage.},
journal = {Analytica chimica acta},
volume = {1377},
number = {},
pages = {344662},
doi = {10.1016/j.aca.2025.344662},
pmid = {41093525},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *G-Quadruplexes ; *Flap Endonucleases/analysis/blood/metabolism ; *Electrochemical Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Luminescent Measurements/methods ; *DNA Probes/chemistry/genetics ; Limit of Detection ; Transcription, Genetic ; *CRISPR-Associated Proteins/metabolism ; },
abstract = {Flap endonuclease 1 (FEN1) is crucial for DNA replication, repair, and telomere maintenance. Its dysregulation is linked to various cancers and diseases. Accurate detection of FEN1 is essential for early diagnosis and therapeutic monitoring. Thus, a novel electrochemiluminescent (ECL) biosensor has been developed for the structure-specific and detection of FEN1. The strategy integrates dumbbell DNA probe-mediated transcription and CRISPR/Cas13a-induced trans-cleavage of RNA G-quadruplexes. In the presence of FEN1, the 5'-flap structure of the probe was selectively cleaved and subsequently ligated by T4 DNA ligase to form a closed circular template. This enabled T7 RNA polymerase to transcribe crRNA-encoded RNA strands, which activated Cas13a to cleave surface-tethered G-quadruplexes/hemin complexes on a Ru(II)/Ti3C2/AuNPs-modified electrode, thereby restoring the quenched ECL signal. The biosensor exhibited an ultralow detection limit of 4.82 × 10[-9] U μL[-1] and a wide dynamic range (1 × 10[-8] to 1 × 10[-5] U μL[-1]), along with excellent specificity and stability. Successful application in human serum validated its reliability for complex biological samples. This work presents a powerful platform for sensitive FEN1 monitoring, holding potential for clinical diagnostics and enzymatic analysis.},
}

*Biosensing Techniques/methods
*G-Quadruplexes
*Flap Endonucleases/analysis/blood/metabolism
*Electrochemical Techniques/methods
Humans
*CRISPR-Cas Systems
*Luminescent Measurements/methods
*DNA Probes/chemistry/genetics
Limit of Detection
Transcription, Genetic
*CRISPR-Associated Proteins/metabolism

@article {pmid41093508,
year = {2025},
author = {Wei, Z and Huang, D and Luo, H and Zhang, J and Wei, J and Tang, Q and Huang, L and Zhang, K and Liao, X},
title = {A multi-level signal conversion architecture for enzyme sensing: Integrating MXene nanoplatforms with CRISPR-driven electrochemiluminescence.},
journal = {Analytica chimica acta},
volume = {1377},
number = {},
pages = {344636},
doi = {10.1016/j.aca.2025.344636},
pmid = {41093508},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *Luminescent Measurements/methods ; *CRISPR-Cas Systems ; Humans ; *Flap Endonucleases/analysis/metabolism ; Nanocomposites/chemistry ; Gold/chemistry ; Limit of Detection ; DNA/chemistry ; Nitrites ; Titanium ; Transition Elements ; },
abstract = {Precise and ultrasensitive detection of flap endonuclease 1 (FEN1), a key DNA repair enzyme implicated in cancer diagnostics, remains challenging due to its subtle structural cleavage activity. Herein, we present a cascade-amplified electrochemiluminescence (ECL) biosensor based on a Ti3C2 MXene-supported Ru (bpy)3[2+]/Au nanocomposite integrated with a CRISPR-Cas13a system and DNA walker circuitry. Upon specific recognition and cleavage of a 5'-flap substrate by FEN1, a nicked DNA product is circularized and transcribed via T7 RNA polymerase, yielding RNA activators that trigger Cas13a-mediated collateral cleavage. This event releases a blocked DNA walker, which reorganizes Fc-labeled DNA on the electrode surface and restores the ECL signal suppressed by resonance energy transfer. The system achieves a detection limit as low as 1.48 fU/mL and exhibits a dynamic range spanning five orders of magnitude. Compared to fluorescence-based CRISPR detection systems, the ECL-based platform offers low background, high signal-to-noise ratios, and operational simplicity using standard electrochemical instrumentation, supporting practical deployment in clinical diagnostics. Furthermore, the platform demonstrates high selectivity against other nucleases and proteins, along with excellent performance in spiked human serum samples. This work presents a robust and modular strategy for accurate enzyme activity profiling with promising applications in early-stage disease diagnostics.},
}

*Biosensing Techniques/methods
*Electrochemical Techniques/methods
*Luminescent Measurements/methods
*CRISPR-Cas Systems
Humans
*Flap Endonucleases/analysis/metabolism
Nanocomposites/chemistry
Gold/chemistry
Limit of Detection
DNA/chemistry
Nitrites
Titanium
Transition Elements

@article {pmid41092254,
year = {2025},
author = {Yin, K and Tsai, CJ},
title = {Turbo-charging crop improvement: harnessing multiplex editing for polygenic trait engineering and beyond.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {1},
pages = {e70527},
pmid = {41092254},
issn = {1365-313X},
support = {//Georgia Research Alliance/ ; DE-SC0023166//U.S. Department of Energy/ ; DE-SC0023338//U.S. Department of Energy/ ; ERKP886//U.S. Department of Energy/ ; },
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; *Multifactorial Inheritance/genetics ; Plants, Genetically Modified/genetics ; *Genetic Engineering/methods ; },
abstract = {Multiplex CRISPR editing has emerged as a transformative platform for plant genome engineering, enabling the simultaneous targeting of multiple genes, regulatory elements, or chromosomal regions. This approach is effective for dissecting gene family functions, addressing genetic redundancy, engineering polygenic traits, and accelerating trait stacking and de novo domestication. Its applications now extend beyond standard gene knockouts to include epigenetic and transcriptional regulation, chromosomal engineering, and transgene-free editing. These capabilities are advancing crop improvement not only in annual species but also in more complex systems such as polyploids, undomesticated wild relatives, and species with long generation times. At the same time, multiplex editing presents technical challenges, including complex construct design and the need for robust, scalable mutation detection. We discuss current toolkits and recent innovations in vector architecture, such as promoter and scaffold engineering, that streamline workflows and enhance editing efficiency. High-throughput sequencing technologies, including long-read platforms, are improving the resolution of complex editing outcomes such as structural rearrangements-often missed by standard genotyping-when targeting repetitive or tandemly spaced loci. To fully realize the potential of multiplex genome engineering, there is growing demand for user-friendly, synthetic biology-compatible, and scalable computational workflows for gRNA design, construct assembly, and mutation analysis. Experimentally validated inducible or tissue-specific promoters are also highly desirable for achieving spatiotemporal control. As these tools continue to evolve, multiplex CRISPR editing is poised to become a foundational technology of next-generation crop improvement to address challenges in agriculture, sustainability, and climate resilience.},
}

*Gene Editing/methods
*Crops, Agricultural/genetics
CRISPR-Cas Systems/genetics
Genome, Plant/genetics
*Multifactorial Inheritance/genetics
Plants, Genetically Modified/genetics
*Genetic Engineering/methods

@article {pmid41091748,
year = {2025},
author = {Yew, WN and Dean, CJ and Chan, DKH},
title = {STAG2 mutations in the normal colon induce upregulation of oncogenic pathways in neighbouring wildtype cells.},
journal = {PloS one},
volume = {20},
number = {10},
pages = {pone.0332499.exml},
pmid = {41091748},
issn = {1932-6203},
mesh = {Humans ; Organoids/metabolism ; *Colon/metabolism/pathology/cytology ; *Mutation ; Up-Regulation ; Signal Transduction/genetics ; CRISPR-Cas Systems ; Coculture Techniques ; Gene Editing ; Proto-Oncogene Proteins p21(ras)/metabolism/genetics ; *Carcinogenesis/genetics ; *Colorectal Neoplasms/genetics/pathology ; Tumor Necrosis Factor-alpha/metabolism ; },
abstract = {While driver mutations in the normal colon have been described, characterizing the role and function of these driver mutations in relation to colorectal oncogenesis remains incomplete. Here, we investigated the role of STAG2 mutants in the normal colon using patient-derived wildtype organoids. Using CRISPR-Cas9 gene editing, we generated STAG2 mutants, and co-cultured these mutants with wildtype organoids, mimicking the presence of such STAG2 mutants in the normal colon. We sought to determine the transcriptional impact of co-culture using scRNAseq. Surprisingly, we uncovered a possible cell-cell interaction between STAG2 mutants and wildtype organoids, in which wildtype organoids in co-culture with STAG2 mutants upregulated known oncogenic pathways. This included the upregulation of TNFα-signaling, as well as KRAS-signaling in wildtype organoids. These results suggested that STAG2 mutant cells exert a pro-oncogenic effect in a cell interactive manner, instead of via a cell autonomous approach. In conclusion, our findings demonstrate a novel mechanism of colorectal oncogenesis which can support further investigation.},
}

Humans
Organoids/metabolism
*Colon/metabolism/pathology/cytology
*Mutation
Up-Regulation
Signal Transduction/genetics
CRISPR-Cas Systems
Coculture Techniques
Gene Editing
Proto-Oncogene Proteins p21(ras)/metabolism/genetics
*Carcinogenesis/genetics
*Colorectal Neoplasms/genetics/pathology
Tumor Necrosis Factor-alpha/metabolism

@article {pmid41091245,
year = {2025},
author = {Yang, S and Jiao, X and Liu, J and Liu, Y and Wang, M and Li, S and Qiao, J},
title = {CRISPR-Cas opens a new era of antimicrobial therapy as a powerful gene editing tool.},
journal = {World journal of microbiology & biotechnology},
volume = {41},
number = {10},
pages = {388},
pmid = {41091245},
issn = {1573-0972},
support = {X2024739//College Students' Innovation and Entrepreneurship Training Program of Shandong Second Medical University/ ; ZR2020MH305//Natural Science Foundation of Shandong Province, China/ ; },
}