@article {pmid40966091,
year = {2025},
author = {Wang, Y and Hu, M and Zou, Y and Wang, X},
title = {In vivo CRISPR screening protocol to identify metastasis mediators using iteratively selected mouse models.},
journal = {STAR protocols},
volume = {6},
number = {4},
pages = {104042},
pmid = {40966091},
issn = {2666-1667},
mesh = {Animals ; Mice ; Female ; *CRISPR-Cas Systems/genetics ; Humans ; Disease Models, Animal ; *Neoplasm Metastasis/genetics ; *Ovarian Neoplasms/genetics/pathology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {In vivo CRISPR screens uncover metastasis genes in native contexts, surpassing in vitro model limitations. Here, we present a protocol to identify metastasis-driving genes in ovarian cancer using an in vivo CRISPR screening technique. Key steps include single-guide RNA (sgRNA) library design and validation, lentiviral transduction, establishment of metastatic mouse models, tissue collection, sgRNA amplification for sequencing, bioinformatics-based candidate gene identification, and functional validation. For complete details on the use and execution of this protocol, please refer to Wang et al.[1].},
}

Animals
Mice
Female
*CRISPR-Cas Systems/genetics
Humans
Disease Models, Animal
*Neoplasm Metastasis/genetics
*Ovarian Neoplasms/genetics/pathology
RNA, Guide, CRISPR-Cas Systems/genetics
Cell Line, Tumor
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid40975871,
year = {2025},
author = {Washer, SJ and Navarro-Guerrero, E and Cowley, SA and Ebner, DV and Bassett, AR},
title = {Protocol for pooled FACS-based CRISPR knockout screening in human iPSC-derived microglia.},
journal = {STAR protocols},
volume = {6},
number = {4},
pages = {104111},
pmid = {40975871},
issn = {2666-1667},
mesh = {Humans ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Microglia/cytology/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; *Flow Cytometry/methods ; Lentivirus/genetics ; Phagocytosis/genetics ; },
abstract = {Here, we present a protocol for CRISPR knockout screening in human induced pluripotent stem cell (hiPSC)-derived microglia (iMGL) using lentiviral delivery of CRISPR-Cas9 and co-transduction of VPX virus-like particles (VPX-VLPs). We first describe large-scale production of iMGL from hiPSCs, production of the lentiviral and VPX-VLP libraries, and titration. Next, we describe how to perform a pooled CRISPR screen for phagocytosis including the computational analysis pipeline of CRISPR screening data. For complete details on the use and execution of this protocol, please refer to Perez-Alcantara et al.[1].},
}

Humans
*Induced Pluripotent Stem Cells/cytology/metabolism
*Microglia/cytology/metabolism
*CRISPR-Cas Systems/genetics
*Gene Knockout Techniques/methods
*Flow Cytometry/methods
Lentivirus/genetics
Phagocytosis/genetics

@article {pmid41014566,
year = {2025},
author = {Hazan, JM and Lahoud-Jeries, N and Bester, AC},
title = {Protocol for simplified parallel perturbations using an abridged long non-coding RNA CRISPR library.},
journal = {STAR protocols},
volume = {6},
number = {4},
pages = {104110},
pmid = {41014566},
issn = {2666-1667},
mesh = {*RNA, Long Noncoding/genetics ; *Gene Library ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; HEK293 Cells ; },
abstract = {High-throughput CRISPR interference (CRISPRi) screens are invaluable for discovering novel functional genes, but applying such screens to long non-coding RNAs (lncRNAs) is more challenging. Here, we present a protocol for designing and executing pooled CRISPRi screens targeting lncRNAs using an abridged cell-type-specific dual single-guide RNA (sgRNA) library. We describe steps for library design and synthesis, followed by stable lentiviral transduction. We then provide guidelines for performing multiple parallel perturbations tailored to the research question, followed by gRNA amplification and data analysis.},
}

*RNA, Long Noncoding/genetics
*Gene Library
RNA, Guide, CRISPR-Cas Systems/genetics
Humans
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
HEK293 Cells

@article {pmid41108681,
year = {2025},
author = {Espinoza, A and Ingersoll, S and Ren, X},
title = {Protocol for quantifying phase-separated condensates in living cells using HILO microscopy imaging and genetic engineering.},
journal = {STAR protocols},
volume = {6},
number = {4},
pages = {104151},
pmid = {41108681},
issn = {2666-1667},
mesh = {Humans ; *Genetic Engineering/methods ; CRISPR-Cas Systems/genetics ; *Biomolecular Condensates ; Microscopy, Fluorescence/methods ; *Microscopy/methods ; },
abstract = {Spatial clustering of epigenetic factors through phase separation is an emerging concept in transcriptional regulation and genome organization. Here, we present a protocol to quantify the phase separation capacity of epigenetic factors in living cells via HILO (highly inclined and laminated optical sheet) microscopy. We describe steps for using CRISPR-Cas9 to generate a cell line with two distinct fusion proteins containing fluorophores and either CRE-LoxP- or dTAG-inducible degradation systems. We then detail the procedures for imaging fusion proteins to analyze and quantify phase separation capacity. For complete details on the use and execution of this protocol, please refer to Brown et al.[1].},
}

Humans
*Genetic Engineering/methods
CRISPR-Cas Systems/genetics
*Biomolecular Condensates
Microscopy, Fluorescence/methods
*Microscopy/methods

@article {pmid41201092,
year = {2025},
author = {Wu, Y and Zhong, A and Ramirez, B and Wai, SS and Zhou, T},
title = {Protocol to rapidly verify silent gene reporter in human pluripotent stem cells using CRISPR activation.},
journal = {STAR protocols},
volume = {6},
number = {4},
pages = {104164},
pmid = {41201092},
issn = {2666-1667},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; UM1 HG012654/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *Pluripotent Stem Cells/metabolism/cytology ; *Genes, Reporter/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Transcriptional Activation/genetics ; },
abstract = {Validating human pluripotent stem cell (hPSC) reporters targeting silent genes typically requires inducing gene expression through cell state transitions, which can be time consuming and complex. Here, we present a rapid workflow to verify reporter knockins at unexpressed loci in hPSCs using CRISPR-mediated transcriptional activation (CRISPRa). We detail steps for designing and cloning single-guide RNA (sgRNA), delivery of CRISPRa into reporter cells, and detection of reporter gene. In this protocol, we illustrate this process using KLF17-GFP reporter hPSCs. For complete details on the use and execution of this protocol, please refer to Wu et al.[1].},
}

Humans
*Pluripotent Stem Cells/metabolism/cytology
*Genes, Reporter/genetics
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Transcriptional Activation/genetics

@article {pmid41240344,
year = {2025},
author = {Banerjee, N and Walsh, B and Patel, R and Castelletto, ML and Hallem, EA},
title = {Protocol to generate stable knockout lines in the human-parasitic nematode Strongyloides stercoralis.},
journal = {STAR protocols},
volume = {6},
number = {4},
pages = {104201},
pmid = {41240344},
issn = {2666-1667},
mesh = {Animals ; *Strongyloides stercoralis/genetics ; *Gene Knockout Techniques/methods ; Humans ; CRISPR-Cas Systems/genetics ; Gerbillinae ; },
abstract = {A major limitation to the study of gene function in parasitic nematodes was the inability to make stable mutant lines. Here, we present a protocol for generating stable knockout lines in the human-parasitic nematode Strongyloides stercoralis. We describe steps for generating CRISPR components and microinjecting them into worms. We also detail procedures for identifying potential gene disruptions and propagating mutants by host passage in gerbils to generate stable homozygous knockout lines. This protocol enables studies of gene function in S. stercoralis. For complete details on the use and execution of this protocol, please refer to Banerjee et al.[1].},
}

Animals
*Strongyloides stercoralis/genetics
*Gene Knockout Techniques/methods
Humans
CRISPR-Cas Systems/genetics
Gerbillinae

@article {pmid41277604,
year = {2026},
author = {Xu, M and Yan, L and Zhu, M and Zhan, Z and Chen, H and Wang, D and Zheng, Z and Zhang, Y and Xiong, L and He, Y},
title = {TKC-MC: An Effective Strategy for Generating Heritable Heterozygous Mutations in Essential Genes in Rice.},
journal = {Plant biotechnology journal},
volume = {24},
number = {4},
pages = {2092-2104},
doi = {10.1111/pbi.70472},
pmid = {41277604},
issn = {1467-7652},
support = {2023ZD04074//Biological Breeding-Major Projects/ ; 32200335//National Natural Science Foundation of China/ ; U21A20207//National Natural Science Foundation of China/ ; Y2023QC39//Youth innovation Program of Chinese Academy of Agricultural Sciences/ ; YBXM2504//Nanfan special project of CAAS/ ; YBXM2505//Nanfan special project of CAAS/ ; CARS-01//Earmarked Fund for China Agriculture Research System/ ; },
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Heterozygote ; Plants, Genetically Modified/genetics ; *Mutation/genetics ; *Genes, Essential/genetics ; *Genes, Plant/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR/Cas9 gene-editing technology has been widely used in defining gene functions and crop improvement. However, some genes are essential for plant growth and development. Loss-of-function homozygous mutations in essential genes lead to plant death or sterility. Mutations in essential genes need to be maintained and propagated in heterozygous plants. CRISPR/Cas9 technology is highly efficient in generating homozygous or bi-allelic mutations at T0 generation in rice, making it difficult to generate useful genetic materials for essential genes using traditional gene editing technology. In this study, we designed Transgene-Killer CRISPR (TKC)-mediated mismatch-spacer targeting (TKC-M) to efficiently generate heritable heterozygous mutations in essential genes in rice. Leveraging our earlier transgenic offspring self-elimination TKC platform, TKC-M relied on timely self-elimination of Cas9 and engineered gRNA-target mismatches to enrich heritable heterozygous or mosaic incomplete-edited T0 mutants and heterozygous progeny. We found that the sensitivity of targets to spacer mismatch(es) varies. A single-base mismatch at gRNA positions 11 or 17 yielded abundant heritable heterozygotes in sensitive targets. For insensitive targets, dual mismatches at positions 8 and 15 maximised heritable heterozygotes. Co-transformation of rice with TKC vectors carrying gRNA without mismatches (G1), gRNA with a mismatch at position 11 (M11) and M8 + M15 spacers, termed TKC-M Cocktail (TKC-MC) significantly increased the incomplete-edited mutant ratio compared with using G1 alone. This work establishes a technical foundation for generating mutant libraries that cover every single gene in a plant genome and for in-depth research on essential genes.},
}

*Oryza/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Heterozygote
Plants, Genetically Modified/genetics
*Mutation/genetics
*Genes, Essential/genetics
*Genes, Plant/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41432570,
year = {2026},
author = {Hu, J and Li, X and Gao, Y and Guo, Y and Liu, Y and Wang, C and Xu, G and Du, C and Liu, S and Zhao, Z and Wang, Y and Wu, Y and Dong, X and Li, C and Wan, J},
title = {Cas9-Embedding Hyperactive TadA8e Confers Efficient and Highly Specific A-To-G Base Editing in Rice.},
journal = {Plant biotechnology journal},
volume = {24},
number = {4},
pages = {2576-2591},
doi = {10.1111/pbi.70511},
pmid = {41432570},
issn = {1467-7652},
support = {2023ZD04074//the Biological Breeding-Major Projects/ ; 2023YFD1202900//the National Key Research and Development Program/ ; ZSBBL-KY2023-04//the Zhongshan Biological Breeding Laboratory/ ; NAUSY-ZZ03//the Guidance Foundation of the Sanya Institute of Nanjing Agricultural University/ ; BK20230038//the Jiangsu Province Natural Science Foundation/ ; //the Nanjing U35 program/ ; 2023AB006-02//the Bingtuan Key Science and Technology Program of Xinjiang Province/ ; KYT2024005//the Fundamental Research Funds for the Central Universities/ ; 31872806//the National Natural Science Foundation of China/ ; //Ministry of Education of the People's Republic of China/ ; },
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *CRISPR-Associated Protein 9/metabolism/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; *Adenosine Deaminase/genetics/metabolism ; Plants, Genetically Modified ; Plant Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Adenine base editors (ABEs) produce precise A-to-G conversion in the genomic target sites without causing double-strand breaks. However, the hyperactive adenosine deaminase TadA8e raises safety concerns on genome-wide off-target edits. We engineered 11 chimeric proteins for ABEs (CP-ABEs) by embedding hyperactive TadA8e within Cas9 nickase to minimise the sgRNA-independent off-target effects. Four CP-ABEs exhibited robust on-target activity with minimal sgRNA-independent off-target edits. Then we developed four chimeric high-fidelity ABEs (CH-ABEs) to minimise both sgRNA-dependent and sgRNA-independent off-target effects by employing high-fidelity Cas9 variants. The CH-ABEs achieved reductions of up to 7.0-fold and 79.4-fold in the respective off-target edits, while generating 22.0%-72.4% homozygous and biallelic rice mutants. Whole-genome and whole-transcriptome sequencing (WGS/WTS) confirmed the specificity of CH-ABEs. Incorporating Sniper2L into CH-ABEs further enhanced both specificity and on-target activity. Two PAM-less SpRY variants (SpRY-K2, SpRY-KK) expanded the targeting scope of CP-ABEs and boosted activity by 80.0%. Furthermore, we demonstrated that CP-ABE8e-RY[KK] could discriminate paralogous targets in rice and successfully applied it to create herbicide-resistant rice by precisely installing the OsALS-K591E mutation.},
}

*Oryza/genetics
*Gene Editing/methods
*CRISPR-Associated Protein 9/metabolism/genetics
CRISPR-Cas Systems/genetics
Genome, Plant/genetics
*Adenosine Deaminase/genetics/metabolism
Plants, Genetically Modified
Plant Proteins/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41433155,
year = {2026},
author = {Hemani, D and Grissom, JH and Chi, RJ},
title = {Protocol for marker-free genome editing in Saccharomyces cerevisiae using universal donor templates and multiplexed CRISPR-Cas9.},
journal = {STAR protocols},
volume = {7},
number = {1},
pages = {104280},
pmid = {41433155},
issn = {2666-1667},
mesh = {*Saccharomyces cerevisiae/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genome, Fungal/genetics ; },
abstract = {Here, we present a protocol for marker-free genome editing in Saccharomyces cerevisiae by combining PCR-based selectable marker cassettes with CRISPR-Cas9. We describe steps for generating gene deletions using MX6 markers and excising the markers by introducing a reusable guide RNA (gRNA)-Cas9 plasmid and universal repair templates, allowing multiplex removal in a single step. Final verification by PCR yields marker-free strains that can be iteratively edited using the same selectable markers. For complete details on the use and execution of this protocol, please refer to Grissom et al.[1].},
}

*Saccharomyces cerevisiae/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
*Genome, Fungal/genetics

@article {pmid41575844,
year = {2026},
author = {Bourner, LA and Acken, KA and Long, H and Chung, LA and Roth, KD and Dorsey, FC},
title = {Protocol to study the role of endogenously produced itaconate using CRISPR-Cas9 technology in THP-1 cells.},
journal = {STAR protocols},
volume = {7},
number = {1},
pages = {104304},
pmid = {41575844},
issn = {2666-1667},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Succinates/metabolism/analysis ; THP-1 Cells ; Macrophages/metabolism ; Chromatography, Liquid/methods ; Mass Spectrometry ; Carboxy-Lyases ; },
abstract = {Itaconate mimetics inadequately represent endogenous itaconate, a negative regulator of innate immune-driven pro-inflammatory cytokines. We present a CRISPR-Cas9 protocol to delete a 4-nucleotide region in the immunoresponsive gene 1 (IRG1), ablating ACOD1 (the itaconate-producing enzyme) in THP-1 cells. We describe the functional validation of ACOD1 deletion using immunoblotting, ELISA, and liquid chromatography-mass spectrometry (LC-MS) quantification of itaconate, enabling the study of endogenous itaconate in THP-1 macrophages. For complete details on the use and execution of this protocol, please refer to Bourner et al.[1].},
}

Humans
*CRISPR-Cas Systems/genetics
*Succinates/metabolism/analysis
THP-1 Cells
Macrophages/metabolism
Chromatography, Liquid/methods
Mass Spectrometry
Carboxy-Lyases

@article {pmid41575845,
year = {2026},
author = {Priesmeier, L and Tiburcy, M and Zelarayán, LC},
title = {Protocol for differentiation of vascular smooth muscle cells from human iPSCs and their application in CRISPRa-mediated gene regulation.},
journal = {STAR protocols},
volume = {7},
number = {1},
pages = {104345},
pmid = {41575845},
issn = {2666-1667},
mesh = {Humans ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Cell Differentiation/genetics ; *Muscle, Smooth, Vascular/cytology ; *Myocytes, Smooth Muscle/cytology/metabolism ; *Cell Culture Techniques/methods ; *CRISPR-Cas Systems/genetics ; Cells, Cultured ; Gene Expression Regulation ; },
abstract = {Directed differentiation of human induced pluripotent stem cells (hiPSCs) holds major promise for the development of disease models, drug screening platforms, and regenerative medicine. Here, we provide a step-by-step highly reproducible protocol for differentiating vascular smooth muscle cells (vSMCs) from hiPSCs, including hiPSC culture, hiPSC differentiation, and vSMC passaging under chemically defined conditions. We also detail molecular and functional analysis procedures for hiPSC-derived contractile vSMCs along with endogenous transcriptional activation modulation ready for any downstream application.},
}

Humans
*Induced Pluripotent Stem Cells/cytology/metabolism
*Cell Differentiation/genetics
*Muscle, Smooth, Vascular/cytology
*Myocytes, Smooth Muscle/cytology/metabolism
*Cell Culture Techniques/methods
*CRISPR-Cas Systems/genetics
Cells, Cultured
Gene Expression Regulation

@article {pmid41626796,
year = {2026},
author = {Jones, LE and Kellermeyer, R and Anand, R and Watson, J and Smith, L and Huang, X and Wang, Z and Yan, W and Zhang, H and Mastick, CC and Kidd, T and Mastick, GS},
title = {SLIT2 repellent is cleaved by TLL1 protease and promotes sensory axon fasciculation.},
journal = {Development (Cambridge, England)},
volume = {153},
number = {16},
pages = {},
doi = {10.1242/dev.205124},
pmid = {41626796},
issn = {1477-9129},
support = {P20 GM103650/GM/NIGMS NIH HHS/United States ; P30 GM145646/GM/NIGMS NIH HHS/United States ; P30 GM145646; P20 GM103650/NH/NIH HHS/United States ; RO1 NS114219/NH/NIH HHS/United States ; RO1 NS114219/NH/NIH HHS/United States ; },
mesh = {Animals ; Ganglia, Spinal/metabolism/cytology/embryology ; *Axons/metabolism ; *Nerve Tissue Proteins/metabolism/genetics ; Mice ; *Intercellular Signaling Peptides and Proteins/metabolism/genetics ; Slit Homolog 2 Protein ; Mice, Knockout ; *Sensory Receptor Cells/metabolism ; CRISPR-Cas Systems ; Proteolysis ; *Tolloid-Like Metalloproteinases/metabolism/genetics ; },
abstract = {SLIT2 is a secreted protein that repels axons from the CNS midline. Full-length SLIT2 (SLIT2-FL) is proteolytically cleaved into two fragments, SLIT2-N and SLIT2-C. SLIT2-FL and SLIT2-N have opposing biological effects on cultured dorsal root ganglion (DRG) axons. This study identified SLIT2 cleavage mechanisms and functional significance for DRG axon guidance. The Tolloid-related protease TLL1 cleaved SLIT2 in cultured cells, with TLL1 requiring activation by furin/prohormone convertases. We used CRISPR editing in mice to produce a Slit2ΔTLS allele lacking the TLL1 cleavage site. Slit2ΔTLS embryos retained dorsal repulsion of DRG axons, in contrast to DRG midline invasion in Slit2 knockouts. However, DRG axons in Slit2 knockouts and Slit2ΔTLS mutants showed reduced fasciculation of rootlets and longitudinal DRG projections. In vitro, SLIT2-N promoted fasciculation of DRG axons. These results suggest that proteolytic cleavage generates additional SLIT2 biological functions for organizing DRG central axon projections.},
}

Animals
Ganglia, Spinal/metabolism/cytology/embryology
*Axons/metabolism
*Nerve Tissue Proteins/metabolism/genetics
Mice
*Intercellular Signaling Peptides and Proteins/metabolism/genetics
Slit Homolog 2 Protein
Mice, Knockout
*Sensory Receptor Cells/metabolism
CRISPR-Cas Systems
Proteolysis
*Tolloid-Like Metalloproteinases/metabolism/genetics

@article {pmid41649928,
year = {2026},
author = {Yoon, DE and Yang, J and Jeong, TY and Park, J and Lee, H and Seong, JK and Kim, K},
title = {Protocol for non-invasive delivery of CRISPR RNPs via virus-like particles for mouse model generation.},
journal = {STAR protocols},
volume = {7},
number = {1},
pages = {104352},
pmid = {41649928},
issn = {2666-1667},
mesh = {Animals ; Mice ; *Gene Editing/methods ; *Ribonucleoproteins/genetics ; *CRISPR-Cas Systems/genetics ; Female ; Models, Animal ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {CRISPR-virus-like particle (VLP)-induced targeted mutagenesis (CRISPR-VIM) enables genome editing in mouse embryos through non-invasive delivery of CRISPR ribonucleoproteins (RNPs) via VLPs, eliminating the need for physical manipulation and specialized expertise. We detail protocols for VLP production, titration, and treatment for diverse genome edits. This protocol is compatible with zygotes and in vitro fertilization (IVF)-derived embryos via simple co-culture, facilitating high-efficiency and heritable mutations with minimized off-target effects, independent of specialized equipment and conducive to reduced animal use. For complete details on the use and execution of this protocol, please refer to Jeong et al.[1].},
}

Animals
Mice
*Gene Editing/methods
*Ribonucleoproteins/genetics
*CRISPR-Cas Systems/genetics
Female
Models, Animal
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41665608,
year = {2026},
author = {Hanson, E and Gold, R and Lee, DH and Yigit, MV},
title = {Cas12a-Programmed Modular CRISPR Cascade Reaction on Paper Supports for Dual-Mode Detection of Pathogenic Genomes.},
journal = {ACS sensors},
volume = {11},
number = {3},
pages = {2774-2783},
doi = {10.1021/acssensors.5c04848},
pmid = {41665608},
issn = {2379-3694},
mesh = {*Paper ; *CRISPR-Cas Systems/genetics ; *Listeria monocytogenes/genetics/isolation & purification ; *Biosensing Techniques/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Campylobacter jejuni/genetics/isolation & purification ; *Endodeoxyribonucleases/genetics/metabolism ; *Genome, Bacterial ; *Bacterial Proteins/genetics ; Escherichia coli/genetics/isolation & purification ; Metal Nanoparticles/chemistry ; Silver/chemistry ; DNA, Bacterial/genetics ; },
abstract = {We developed an optical biosensing platform using cost-efficient and scalable paper support for the detection and identification of three major bacterial pathogens using fluorescent DNA-templated silver nanoclusters (FNPs) and an innovative CRISPR-Cas12a cascade reaction. The sensors were fabricated as ∼5 mm letter-shaped paper cutouts, with each letter representing a specific pathogen: "C" for Campylobacter jejuni, "E" for Shiga toxin-producing Escherichia coli, and "L" for Listeria monocytogenes. Detection was initially achieved via an ON-to-OFF mechanism, wherein target recognition by Cas12a led to FNP degradation and fluorescence loss using target strands identified from the conserved genomic regions from each pathogen. This platform successfully detected individual and multiple targets in all possible seven combinations. To enhance diagnostic clarity, we developed a two-step CRISPR-Cas12a cascade reaction enabling an ON signal output when the target is present, a more intuitive and desirable reporting format. In this design, the first Cas12a reaction detects the target and cleaves an activator strand, preventing activation of a second Cas12a reaction that would otherwise degrade FNPs. Consequently, fluorescence is retained in the presence of the target (ON-retention) and lost in its absence, providing a clear ON signal when the target is detected, and an OFF signal when it is not. Finally, we demonstrated both ON-to-OFF and ON-retention detection modes using the whole Listeria genome amplified by isothermal recombinase polymerase amplification, with reliable detection of as few as 40 full genomic copies using fluorescent images on paper substrates. This work represents a significant advancement in Cas12a-based biosensing, uniquely demonstrating multistep biochemical reactions directly on paper support, and offers a promising platform for low-cost, scalable pathogen detection in resource-limited settings.},
}

*Paper
*CRISPR-Cas Systems/genetics
*Listeria monocytogenes/genetics/isolation & purification
*Biosensing Techniques/methods
*CRISPR-Associated Proteins/genetics/metabolism
Campylobacter jejuni/genetics/isolation & purification
*Endodeoxyribonucleases/genetics/metabolism
*Genome, Bacterial
*Bacterial Proteins/genetics
Escherichia coli/genetics/isolation & purification
Metal Nanoparticles/chemistry
Silver/chemistry
DNA, Bacterial/genetics

@article {pmid41686171,
year = {2026},
author = {Su, M and Lv, MM and Pan, MX and Zha, CJ and Nie, YG and Ying, ZM},
title = {From Self-Processing to Responsive Assembly Enabling an Autocatalytic Cas13a Circuit for Enhanced Biosensing.},
journal = {ACS sensors},
volume = {11},
number = {3},
pages = {2697-2706},
doi = {10.1021/acssensors.5c04673},
pmid = {41686171},
issn = {2379-3694},
mesh = {*Biosensing Techniques/methods ; Humans ; CRISPR-Cas Systems ; Survivin/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; MicroRNAs/analysis/genetics ; DNA, Viral/analysis/genetics ; RNA, Messenger/analysis/genetics ; DNA-Directed RNA Polymerases ; Viral Proteins ; },
abstract = {Despite the success of non-classical crRNA designs in Cas12a-based biosensing, application to Cas13a systems faces fundamental challenges. Our research discovers that site-specific splitting within the crRNA seed region enables effective activation of Cas13a trans-cleavage. Here, we developed an autocatalytic Cas13a circuit that transitions from self-processing to responsive assembly for enhanced biosensing (PRA-Cas13a). The system employs engineered pre-crRNA as a molecular switch, which undergoes self-processing upon target binding to assemble an active Cas13a complex and activate its trans-cleavage activity. By integrating a dual-UUU site DNA switch template and a T7 RNA polymerase-mediated signal amplification module, a "processing-assembly-amplification" cycle is constructed to enhance the detection signal. Through validation using various targets including miRNA, mRNA, and viral DNA, the PRA-Cas13a system not only achieves an attomolar (aM) level detection limit but also enables visual field detection within 10 min using a lateral flow test strip. Analysis of single-base mutations demonstrated that its sensitivity is significantly superior to conventional CRISPR-based methods. Moreover, the system successfully enabled accurate detection of survivin mRNA in different cell lines and HPV16 in clinical cervical swab samples, showing strong concordance with qPCR gold standard methods. The PRA-Cas13a strategy leverages intrinsic self-processing assembly and autocatalytic signal amplification, addresses the critical issue of off-target cleavage inherent in conventional Cas13a systems while expanding the range of applicable targets, and demonstrates high specificity and point-of-care testing potential in cancer and viral diagnostics.},
}

*Biosensing Techniques/methods
Humans
CRISPR-Cas Systems
Survivin/genetics
*CRISPR-Associated Proteins/genetics/metabolism
MicroRNAs/analysis/genetics
DNA, Viral/analysis/genetics
RNA, Messenger/analysis/genetics
DNA-Directed RNA Polymerases
Viral Proteins

@article {pmid41699288,
year = {2026},
author = {Hu, C and Tai, H and Zhu, R and Shu, Z and Guo, G and Ma, D and Zuo, S and Tang, L and Zeng, Z},
title = {CRISPR-Cas9 screening identifies ATOX1-driven cisplatin resistance mechanisms in liver cancer and evaluates targeted inhibitor efficacy.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41699288},
issn = {2399-3642},
mesh = {*Cisplatin/pharmacology ; Humans ; *Drug Resistance, Neoplasm/genetics ; *Liver Neoplasms/drug therapy/genetics/metabolism/pathology ; *CRISPR-Cas Systems ; *Antineoplastic Agents/pharmacology ; Animals ; Cell Line, Tumor ; Mice ; *Copper Transport Proteins/genetics/antagonists & inhibitors/metabolism ; Female ; Gene Expression Regulation, Neoplastic/drug effects ; Male ; Receptor, Notch1/genetics/metabolism ; },
abstract = {Liver cancer treatment with cisplatin is often hindered by drug resistance. This study aimed to identify key genes associated with cisplatin resistance in liver cancer and develop targeted inhibitors. Using genome-wide CRISPR-Cas9 screening, ATOX1 was identified as a critical gene for cisplatin resistance. ATOX1 was highly expressed in liver cancer tissues and associated with poor prognosis. Knockdown of ATOX1 in liver cancer cells enhanced cisplatin sensitivity in vitro and in vivo. Molecular dynamics simulation and virtual screening identified compound 8 as a potent ATOX1 inhibitor with high affinity (Kd = 12.5 μM) and exhibited synergistic effects with cisplatin on liver cancer cell growth. Mechanistically, compound 8 inhibits the activity of ATOX1, leading to intracellular copper accumulation. The elevated copper levels subsequently promote increased DNA methylation at the NOTCH1 promoter, resulting in suppression of the NOTCH1/HES1 signaling pathway and enhancing the sensitivity of liver cancer cells to cisplatin. In conclusion, ATOX1 is crucial for cisplatin resistance in liver cancer and linked to poor prognosis. Targeting ATOX1 with compound 8 may be a novel therapeutic strategy for overcoming cisplatin resistance.},
}

*Cisplatin/pharmacology
Humans
*Drug Resistance, Neoplasm/genetics
*Liver Neoplasms/drug therapy/genetics/metabolism/pathology
*CRISPR-Cas Systems
*Antineoplastic Agents/pharmacology
Animals
Cell Line, Tumor
Mice
*Copper Transport Proteins/genetics/antagonists & inhibitors/metabolism
Female
Gene Expression Regulation, Neoplastic/drug effects
Male
Receptor, Notch1/genetics/metabolism

@article {pmid41702943,
year = {2026},
author = {de Menezes, MN and Chen, AXY and Kulkarni, N and Sampurno, S and Saw, NYL and Yap, KM and Pérez-Núñez, I and Roth, S and Deguit, CDT and Haugen, B and Ramsbottom, KM and Munoz, I and Beavis, PA and Parish, IA},
title = {High efficiency CRISPR knock-in demonstrates that TCF1 is insufficient to reverse T cell exhaustion.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41702943},
issn = {2041-1723},
support = {MCRF21019//Victorian Cancer Agency (VCA)/ ; MCRF20011//Victorian Cancer Agency (VCA)/ ; Lloyd J. Old STAR Grant CRI5578//Cancer Research Institute (CRI)/ ; },
mesh = {Animals ; *Hepatocyte Nuclear Factor 1-alpha/genetics/metabolism ; *Gene Knock-In Techniques/methods ; Mice ; *CD8-Positive T-Lymphocytes/immunology/metabolism/cytology ; Cell Differentiation ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; *T Cell Transcription Factor 1/genetics/metabolism ; Mice, Transgenic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; T-Cell Exhaustion ; },
abstract = {CD8[+] T cell exhaustion is a regulatory state triggered by chronic antigen stimulation in both cancer and persistent infection. The less differentiated stem-like sub-populations of exhausted T cells have been heavily studied given their importance to the efficacy of current immunotherapies. While the transcription factor TCF1 is both necessary and sufficient for formation and maintenance of these stem-like populations, it remains unclear whether TCF1 can actively de-differentiate more terminally exhausted subsets back into a stem-like state. To address this question, here we utilize and optimize a high efficiency CRISPR knock-in methodology, compatible with mouse in vivo exhaustion models, to engineer T cells that either constitutively over-express TCF1, or conditionally over-express TCF1 following differentiation of the cells into a CX3CR1[+] intermediate-exhausted state. Strikingly, we find that only constitutive, and not conditional, TCF1 over-expression can increase the size of the stem-like T cell pool. Thus, while TCF1 can slow stem-like T cell differentiation, it is insufficient to revert more differentiated cells back into a stem-like state.},
}

Animals
*Hepatocyte Nuclear Factor 1-alpha/genetics/metabolism
*Gene Knock-In Techniques/methods
Mice
*CD8-Positive T-Lymphocytes/immunology/metabolism/cytology
Cell Differentiation
Mice, Inbred C57BL
CRISPR-Cas Systems
*T Cell Transcription Factor 1/genetics/metabolism
Mice, Transgenic
*Clustered Regularly Interspaced Short Palindromic Repeats
T-Cell Exhaustion

@article {pmid41715004,
year = {2026},
author = {Metz, P and Alves-Vasconcelos, S and Wallbank, R and Riepsaame, J and Brown, S and Hassan, AB},
title = {Variation in guide RNA library representation results in gene effect score bias in genome-wide CRISPR screens.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {},
pmid = {41715004},
issn = {1471-2164},
support = {OxPOS//F. Hoffmann-La Roche/ ; GEO//John Fell Fund, University of Oxford/ ; MPNST//Grenfell Shaw Charity/ ; Oxford Clinical Fellowships/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; *Gene Library ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Human ; },
abstract = {Genome wide CRISPR-based perturbation screens are powerful discovery tools enabling the identification of novel gene dependencies through either gain or loss of function. While genome wide guide RNA (gRNA) libraries have advantages when using enAsCas12a, such as multiplex single gRNAs per gene, they may be subject to similar confounding factors that can affect the interpretation of large genome-wide datasets. Here, we examine the impact of these variables in over twenty enAsCas12a multiple gRNA based perturbation screens performed using Humagne C, Humagne D and Inzolia libraries in human cells, as well as external datasets containing Cas9-based CRISPR library screens, including from DepMap. We demonstrate that the choice of CRISPR library is often the most significant factor that influences genetic perturbation results, outweighing other variables such as either target cell lines or culture media conditions. A potential contributor to this effect is gRNA representation within a given CRISPR library, where lower gRNA representation can lead to variable and more pronounced gene effect scores using either log fold change or Chronos analysis. These effects may be mitigated by using either multiple gRNA constructs per gene, by optimisation of CRISPR library production processes or by targeting with multiple independent gRNA libraries. Importantly, we also propose strategies for addressing gRNA representation bias during CRISPR screen hit prioritisation. CRISPR library gRNA representation dependent bias remains a major challenge in the interpretation of gene essentiality in perturbation screens.},
}

Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems
*Gene Library
*Clustered Regularly Interspaced Short Palindromic Repeats
Genome, Human

@article {pmid41715150,
year = {2026},
author = {Zhao, Y and Li, X and Du, Y},
title = {AI-driven CRISPR screening: optimizing gene editing through automation and intelligent decision support.},
journal = {Journal of translational medicine},
volume = {24},
number = {1},
pages = {},
pmid = {41715150},
issn = {1479-5876},
support = {No. Z04J2024E107-B-12//Bethune Charitable Foundation/ ; },
mesh = {Humans ; *Artificial Intelligence ; *Gene Editing ; *Automation ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Decision Support Techniques ; *CRISPR-Cas Systems/genetics ; Deep Learning ; *Genetic Testing/methods ; },
abstract = {BACKGROUND: CRISPR-based genetic screening has become a central methodology in functional genomics, enabling systematic interrogation of gene function, genetic interactions and context-dependent vulnerabilities at scale. However, the rapid expansion of screening modalities-including multi-condition designs, combinatorial perturbations, in vivo applications and single-cell readouts-has exposed fundamental limitations of heuristic-driven experimental design and post hoc statistical analysis.

MAIN BODY: This Review synthesizes how artificial intelligence is reshaping CRISPR screening by introducing predictive, adaptive and system-level intelligence across the experimental lifecycle. We organize recent advances into two tightly coupled modules. First, machine learning and deep learning (ML/DL) methods optimize experimental design by learning context-dependent perturbation behavior, anticipating confounding effects and enabling iterative, information-efficient screening strategies. Second, large language model-agent (LLM-agent) systems complement these advances by externalizing scientific reasoning, integrating biological knowledge at scale and coordinating analysis and decision-making in human-in-the-loop workflows.

CONCLUSIONS: Together, ML/DL and LLM-agent approaches reframe CRISPR screening from a static analytical pipeline into an intelligent experimental system, with important implications for robustness, scalability and biological discovery.},
}

Humans
*Artificial Intelligence
*Gene Editing
*Automation
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Decision Support Techniques
*CRISPR-Cas Systems/genetics
Deep Learning
*Genetic Testing/methods

@article {pmid41718462,
year = {2026},
author = {Ren, Y and Wu, R and Yang, X and Yao, B and Wang, Y},
title = {Membrane-Confined CRISPR-Cas12a Activation via Split Activator for Wash-Free Detection of Tumor-Specific Extracellular Vesicles.},
journal = {ACS sensors},
volume = {11},
number = {3},
pages = {2356-2365},
doi = {10.1021/acssensors.5c04166},
pmid = {41718462},
issn = {2379-3694},
mesh = {*Extracellular Vesicles/metabolism/chemistry ; *CRISPR-Cas Systems ; Humans ; Aptamers, Nucleotide/chemistry/metabolism ; *Biosensing Techniques/methods ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism ; *Neoplasms/diagnosis ; },
abstract = {Current CRISPR-Cas12a biosensing systems for protein analysis rely on the release of a blocked activator from an aptamer through a competitive mechanism. However, the design of the complementary length between the activator and the aptamer involves a critical trade-off: excessively long sequences impede aptamer-target binding, whereas overly short sequences fail to activate Cas12a effectively. To overcome this limitation, we split the full-length Cas12a activator into two short single-stranded DNA fragments (S1 and S2; 10 nt each). S1 was specifically sequestered within the hairpin structure of an allosteric aptamer. The binding of the target protein to the aptamer triggered a conformational change, exposing S1 and thereby enabling its assembly with S2 to activate Cas12a. The strategy successfully preserved the binding affinity of the aptamer without compromising the efficiency of Cas12a activation. Furthermore, we anchored the Cas12a substrate to the membrane surface of extracellular vesicles (EVs) via hydrophobic interactions, localizing the cleavage reaction to the EVs interface and thereby significantly enhancing local catalytic efficiency. Finally, the strategy provided highly sensitive detection of tumor-specific EVs, not only accurately distinguishing cancer patients from healthy individuals (77.5% accuracy for early-stage and 100% for advanced-stage) but also holding potential for monitoring dynamic postoperative changes. Overall, our study provided a wash-free, one-pot detection platform for EVs analysis and established a new paradigm for CRISPR-based clinical diagnostics.},
}

*Extracellular Vesicles/metabolism/chemistry
*CRISPR-Cas Systems
Humans
Aptamers, Nucleotide/chemistry/metabolism
*Biosensing Techniques/methods
*CRISPR-Associated Proteins/metabolism
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism
*Neoplasms/diagnosis

@article {pmid41736562,
year = {2026},
author = {Purvis, IJ and Ochoa Olmos, OE and Park, KU and Kaufman, ML and Henry, CM and Schaaf, C and Clise, OJ and Tesdahl, CD and Haas, A and Brzezinski, JA},
title = {A robust cis-regulatory network ensures Otx2 expression during retinal development.},
journal = {Development (Cambridge, England)},
volume = {153},
number = {6},
pages = {},
doi = {10.1242/dev.204881},
pmid = {41736562},
issn = {1477-9129},
support = {R01-EY024272/NH/NIH HHS/United States ; //Research to Prevent Blindness/ ; R01 EY024272/EY/NEI NIH HHS/United States ; F31-EY034794/NH/NIH HHS/United States ; F31 EY034794/EY/NEI NIH HHS/United States ; R01-EY024272/NH/NIH HHS/United States ; F31EY034794/NH/NIH HHS/United States ; //Gates Institute/ ; //Anschutz Medical Campus, University of Colorado/ ; //University of Colorado/ ; },
mesh = {*Otx Transcription Factors/genetics/metabolism ; Animals ; *Retina/metabolism/embryology ; *Gene Expression Regulation, Developmental ; Enhancer Elements, Genetic/genetics ; Mice ; *Gene Regulatory Networks/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {The transcription factor Otx2 is essential for photoreceptor and bipolar cell formation during retinal development. Otx2 expression is complex and underlies multiple cell fate decisions during development. To understand how Otx2 expression is regulated, we explored the activity and function of three of its enhancers (DHS2, DHS4 and DHS15). Enhancer reporter assays and lineage tracing show that DHS4 initiates Otx2 expression while DHS2 and DHS15 maintain expression in photoreceptors. Matched CRISPR/Cas9 and CRISPR interference systems were used to mutate or epigenetically silence enhancers, respectively. CRISPR reduced OTX2 expression acutely, but failed to significantly alter cell fate choice over the long term. In contrast, CRISPR interference of these enhancers caused permanent OTX2 loss and corresponding cell fate changes. While these data suggest that each enhancer is needed for normal Otx2 expression, it also highlights that the enhancers can interact and substitute for each other during development. This cis-regulatory element flexibility likely promotes Otx2 expression robustness. Such robustness may enable complex genes, like Otx2, to resist environmental stressors and regulatory disruptions to promote reproducible developmental outcomes.},
}

*Otx Transcription Factors/genetics/metabolism
Animals
*Retina/metabolism/embryology
*Gene Expression Regulation, Developmental
Enhancer Elements, Genetic/genetics
Mice
*Gene Regulatory Networks/genetics
CRISPR-Cas Systems/genetics

@article {pmid41758644,
year = {2026},
author = {Adami, A and Garza, R and Dorazehi, F and Douse, CH and Jakobsson, J},
title = {Protocol for efficient CRISPRi-mediated silencing of retrotransposons in human pluripotent stem cells.},
journal = {STAR protocols},
volume = {7},
number = {1},
pages = {104398},
pmid = {41758644},
issn = {2666-1667},
mesh = {Humans ; *Gene Silencing ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Retroelements/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Pluripotent Stem Cells/metabolism ; },
abstract = {Here, we present a workflow for transcriptional silencing of transposable elements (TEs) in human induced pluripotent stem cells (hiPSCs). We describe steps for designing guide RNAs (gRNAs) to target TE families or unique TE loci. We also detail procedures for validating the efficiency and specificity of large-scale CRISPRi-based silencing using a multiome approach combining bulk RNA sequencing, CUT&RUN epigenetic profiling, and proteomics. This framework optimizes the performance and interpretation of in vitro functional studies based on transcriptional manipulation of TEs in hiPSC models. For complete details on the use and execution of this protocol, please refer to Adami et al.[1].},
}

Humans
*Gene Silencing
*Induced Pluripotent Stem Cells/metabolism/cytology
*Retroelements/genetics
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Pluripotent Stem Cells/metabolism

@article {pmid41800529,
year = {2026},
author = {Yan, J and Wang, S and Xiong, S and Luo, X and Li, Y and Deng, X},
title = {Cas12a Trans-Cleavage of Hairpins Triggers a CHA Cascade for an Ultrasensitive SERS Aptasensor.},
journal = {ACS sensors},
volume = {11},
number = {3},
pages = {2828-2839},
doi = {10.1021/acssensors.6c00041},
pmid = {41800529},
issn = {2379-3694},
mesh = {*Spectrum Analysis, Raman/methods ; *Aptamers, Nucleotide/chemistry/metabolism ; *Biosensing Techniques/methods ; Limit of Detection ; DNA/chemistry ; *CRISPR-Associated Proteins/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/chemistry ; CRISPR-Cas Systems ; Humans ; Gold/chemistry ; Bacterial Proteins ; },
abstract = {Ultrasensitive detection of non-nucleic acid biomarkers using CRISPR/Cas12a remains a major challenge due to the lack of intrinsic signal amplification. Moreover, linear DNA reporters fail to maintain efficient downstream signal amplification after trans-cleavage, while pre-amplification procedures often lead to nonspecific signals, thereby compromising assay accuracy, particularly in complex biological matrices. Here, a highly SERS aptasensor is developed by harnessing CRISPR/Cas12a-driven trans-cleavage of hairpin substrates to trigger catalytic hairpin assembly (Cas12a-CHA), achieving robust cascade signal amplification. Target recognition is converted into customizable DNA triggers that precisely activate Cas12a, while a thymine-rich DNA/RNA reporter with dT5 motifs facilitates high enhances trans-cleavage efficiency, sustaining continuous CHA cycles. The integration of AuNF@4-MBA@Ag@H2 SERS nanotags that generate abundant plasmonic hotspots, the system provides significantly enhanced Raman readout. Benefiting from synergistic molecular amplification and nanostructure engineering, the aptasensor achieves an ultralow detection limit of 1.97 × 10[-17] g/mL, nearly 20,000-fold higher sensitivity than traditional sandwich assays, along with a broad dynamic range and high specificity. Furthermore, it exhibits excellent uniformity, reproducibility, stability, and recovery in spiked serum samples, using FGF2 used as a representative biomarker to validate its performance, highlighting great potential for clinical diagnostics and real-world applications.},
}

*Spectrum Analysis, Raman/methods
*Aptamers, Nucleotide/chemistry/metabolism
*Biosensing Techniques/methods
Limit of Detection
DNA/chemistry
*CRISPR-Associated Proteins/metabolism/chemistry
*Endodeoxyribonucleases/metabolism/chemistry
CRISPR-Cas Systems
Humans
Gold/chemistry
Bacterial Proteins

@article {pmid41805294,
year = {2026},
author = {Anfang, M and Yahya, RH and Caldararu, O and Ben Yaakov, S and Landau, U and Berman, A and Hu, Y and Belew, ZM and Crocoll, C and Xu, D and Nour-Eldin, HH and Mayrose, I and Shani, E},
title = {Targeting redundant gene families: A multiplexed, tissue-specific CRISPR toolbox for Arabidopsis genetic screens.},
journal = {Cell reports},
volume = {45},
number = {3},
pages = {117055},
pmid = {41805294},
issn = {2211-1247},
mesh = {*Arabidopsis/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Genetic Testing/methods ; *Multigene Family ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Organ Specificity/genetics ; Genome, Plant ; },
abstract = {Genome-scale targeted CRISPR libraries for forward genetic screens in plants are powerful tools for functional analysis, but they suffer from limited spatial control, single sgRNA design, and poor handling of genetic redundancy. We develop multiplexed CRISPR libraries in which each construct contains two sgRNAs that simultaneously target multiple members of a gene family. The libraries can also function at the cell-type-specific and tissue levels. A double-barcoding strategy enables efficient tracking and identification of sgRNA combinations at the plant level without individually sequencing each line. Using this platform, we generate over 1,000 Arabidopsis lines that express sgRNAs targeting 707 transporter genes across 114 gene families involved in nutrient uptake. The multiplexed design increases gene coverage and editing efficiency, underscoring its improved targeting capability to reveal hidden phenotypes. This toolbox provides a scalable resource for multi-targeted genome editing and spatially precise forward genetic screens in plants.},
}

*Arabidopsis/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Genetic Testing/methods
*Multigene Family
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Organ Specificity/genetics
Genome, Plant

@article {pmid41816845,
year = {2026},
author = {Chen, WD and Liu, L and Cheng, L},
title = {Nitroreductase-Responsive Oligomeric crRNAs for Enzyme-Triggered Regulation of CRISPR Activity.},
journal = {The Journal of organic chemistry},
volume = {91},
number = {12},
pages = {4494-4501},
doi = {10.1021/acs.joc.5c02287},
pmid = {41816845},
issn = {1520-6904},
mesh = {*Nitroreductases/metabolism/chemistry ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism ; },
abstract = {Hypoxic tumors overexpress nitroreductase (NTR), providing an endogenous trigger for selective biomolecular activation. Here, we describe the synthesis of NTR-responsive clustered regularly interspaced short palindromic repeats (CRISPR) guide RNAs via the site-specific incorporation of a p-nitrobenzyl (p-NB) phosphoramidite at the 5' terminus of crRNAs. Click-mediated oligomerization into trimeric and tetrameric constructs effectively suppressed Cas nuclease activity. Enzymatic reduction by NTR induced linker cleavage, releasing active crRNAs and restoring DNA cleavage in vitro, establishing a strategy for enzyme-regulated CRISPR control.},
}

*Nitroreductases/metabolism/chemistry
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
*RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism

@article {pmid41837829,
year = {2026},
author = {Bao, J and Ju, X and Zhang, H and Tang, D and Yan, J and Cui, C and Gao, SS},
title = {Synergistic CRISPR-Cas9 Host Engineering and Enzyme Evolution for Enantioselective Synthesis of a Vibegron Pyrrolidine Intermediate.},
journal = {Organic letters},
volume = {28},
number = {12},
pages = {3695-3700},
doi = {10.1021/acs.orglett.6c00065},
pmid = {41837829},
issn = {1523-7052},
mesh = {Stereoisomerism ; *Pyrrolidines/chemistry/chemical synthesis/metabolism ; Molecular Structure ; *CRISPR-Cas Systems ; Biocatalysis ; *Oxidoreductases/metabolism/chemistry ; Directed Molecular Evolution ; },
abstract = {The stereoselective synthesis of chiral pyrrolidine motifs is essential to vibegron production but remains challenging using conventional chemical routes. Here we report an imine reductase (IRED) catalyzed asymmetric imine reduction to access a key vibegron intermediate. Directed evolution afforded a highly efficient variant delivering 94% conversion and >99% d.e. Combined enzyme and host engineering enabled clean whole cell catalysis, establishing a robust and scalable biocatalytic platform.},
}

Stereoisomerism
*Pyrrolidines/chemistry/chemical synthesis/metabolism
Molecular Structure
*CRISPR-Cas Systems
Biocatalysis
*Oxidoreductases/metabolism/chemistry
Directed Molecular Evolution

@article {pmid41865271,
year = {2026},
author = {Kahr, J and Diaz-Peregrino, R and Sandalcioglu, IE and John, P and Mawrin, C},
title = {RagC and Map4K3 deficiency in high-grade gliomas drives proliferation and modulates mTORC1-dependent cellular functions.},
journal = {Journal of neuropathology and experimental neurology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jnen/nlag010},
pmid = {41865271},
issn = {1554-6578},
abstract = {Cellular growth and homeostasis via amino acid-responsive pathways are mediated by the mTOR signaling pathway. Rag GTPases and Map4K3 modify mTOR signaling as amino acid sensors. Altered mTOR signaling in relation to amino acid sensors might represent factors that modify proliferation and treatment responses in astrocytic tumors. To investigate this hypothesis, RagC and Map4K3 expression was studied in human gliomas, glioma cells (U87MG/U138MG), and nonglial cells (MCF-7, IOMM-Lee). RagC and Map4K3 knockout in glioma cells was generated using CRISPR-Cas and shRNA. High-grade astrocytomas had significantly reduced immunoreactivity for RagC and Map4K3 compared to low-grade astrocytomas. RagC- and Map4K3-deficient glioma cells had significantly increased proliferation and showed altered morphology and motility. Induced amino acid deficiency (leucine deprivation) reduced proliferation in Map4K3- but not in RagC-deficient cells. mTOR signaling in RagC- and Map4K3-deficient U87 cells was altered with increased phosphorylation of p70S6K and increased expression of RagD and transcription factor EB. In this context, uncoupled, exaggerated autophagy occurred in Map4K3-deficient U87 cells. In contrast, RagC-deficient U87 cells showed increased senescence but no autophagy induction. These data show that losses of RagC and Map4K3 in malignant gliomas have proliferation-inducing effects and differentially modulate key mTOR signaling-dependent cellular mechanisms.},
}

@article {pmid41865648,
year = {2026},
author = {Marková, K},
title = {Affinity-based nanostructured platforms for the selective pretreatment, enrichment and detection of miRNA biomarkers.},
journal = {Journal of chromatography. B, Analytical technologies in the biomedical and life sciences},
volume = {1276},
number = {},
pages = {125017},
doi = {10.1016/j.jchromb.2026.125017},
pmid = {41865648},
issn = {1873-376X},
abstract = {MicroRNAs (miRNAs) are clinically relevant liquid-biopsy biomarkers, yet their reliable quantification is still limited by low abundance, matrix complexity, and frequent association with protective carriers such as Argonaute-2 and exosomes. This review focuses on affinity-based nanostructured platforms as superior tools for the selective pretreatment and enrichment of miRNAs, bridging the gap between raw clinical samples and high-performance analysis. We summarize key nanomaterial architectures, including functional nanoparticles, electrospun nanofibers, 2D nanomaterial platforms, and hybrid nanocomposites, together with surface functionalization strategies that enable the liberation of miRNAs from protein complexes and their subsequent sequence-specific capture. Emphasis is placed on how these enrichment workflows address target accessibility in protein-rich biofluids and mitigate matrix-induced interference. Beyond sample preparation, we evaluate the integration of these platforms with advanced detection modalities, including Surface-Enhanced Raman Spectroscopy (SERS) using inverse molecular sentinel (iMS) nanoprobes, enzyme-free hybridization chain reactions (HCR) and CRISPR/Cas-based assays. These innovative strategies circumvent the limitations of enzymatic amplification, offering high sensitivity and specificity. Finally, we address challenges in automation and standardization, highlighting the need for integrated enrichment-to-detection workflows that accelerate the translation of nanomaterial innovation and next-generation, point-of-care miRNA diagnostics.},
}

@article {pmid41867779,
year = {2026},
author = {Wester, M and Lim, J and Van, AB and Koprowski, K and Valera, E and Bashir, R},
title = {Kinetic Modeling of Target-Amplification-Free CRISPR-Cas-Based Autocatalysis Reactions.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.03.03.709462},
pmid = {41867779},
issn = {2692-8205},
abstract = {CRISPR-Cas-based diagnostics utilize the Cas enzyme's trans-cleavage activity to generate signal and have become popular platforms for sensitive nucleic acid detection. Recently, autocatalytic systems have been demonstrated to improve the time to response and sensitivity in some cases. However, mechanistic description of these assays is limited and optimization relies on simple trial-and-error. In this work, we present the first comprehensive kinetic model that integrates all major biochemical processes involved in these assays, including cleavage reactions, nucleic acid equilibrium kinetics, inhibition of trans-cleavage by single-stranded DNA, and degradation of single-stranded reaction components. We discuss the biochemical foundations and implementation of the ordinary differential equation model, which is built for adaptation to different reaction schemes. We use the full model to investigate the role of nucleic acid stability in assay performance for a typical nucleic acid design and show that our model demonstrates inhibition effects consistent with experimental data. We describe the reaction behavior, derive a simplified analytical model and compare its performance to the full analytical model. Finally, we demonstrate tools developed for rapid in silico optimization to guide the rational design of future target-amplification-free CRISPR-Cas-based autocatalysis assays.},
}

@article {pmid41869310,
year = {2026},
author = {Petri, K and Ferrari, S and Cianciotti, BC},
title = {Editorial: Safety and efficacy of CRISPR/Cas-based genome editing tools: applications and considerations in cell and gene therapy.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1809809},
doi = {10.3389/fimmu.2026.1809809},
pmid = {41869310},
issn = {1664-3224},
}

@article {pmid41870078,
year = {2026},
author = {Bayurova, E and Kostyushev, D and Tikhonov, A and Chulanov, V and Gordeychuk, I},
title = {Broad-acting antivirals: the pursuit of pan-viral therapeutics in the era of pandemics.},
journal = {Journal of virology},
volume = {},
number = {},
pages = {e0007726},
doi = {10.1128/jvi.00077-26},
pmid = {41870078},
issn = {1098-5514},
abstract = {The ever-present threat of new viral epidemics makes the scientific community relentlessly work on the development of universal methods of antiviral therapy. The development of broad-spectrum antivirals (BSAs) focuses either on substances acting directly on viral proteins (direct-acting antivirals [DAA]) or on substances directed at the cell's own proteins (host-targeting antivirals [HTA]). Decades of development have led to the market entry of a number of DAAs with a wide range of antiviral activities; however, their clinical approval has been obtained for individual infections. HTAs have a number of advantages over DAAs, such as a wider range of antiviral activities and a high genetic barrier to viral resistance, which is undoubtedly important when preparing for a battle with an unknown pathogen. The COVID-19 pandemic has allowed for multiple clinical trials for repurposed HTAs, previously licensed for the treatment of other diseases, including cancer. Despite the enormous work done, the arsenal of BSAs capable of protecting against future pandemics caused by pathogen X is very limited. In this review, we described data on the most studied DAAs and HTAs, effective against at least two unrelated viral pathogens, focusing on those that have been studied in late preclinical and clinical trials. In the end, we highlighted alternative new approaches such as CRISPR-Cas therapy.},
}

@article {pmid41870756,
year = {2026},
author = {Gowtham, K and Shanmugaraj, B and Thangavel, LS and Srinivasan, A and Malla, A},
title = {Advancing the frontier of plant-based therapeutics: critical innovations in molecular farming and bioprocess Integration.},
journal = {Biotechnology letters},
volume = {48},
number = {2},
pages = {},
pmid = {41870756},
issn = {1573-6776},
support = {PSGCAS/IRSG/2024-2025/Biotechnology/004//PSG/ ; },
mesh = {*Molecular Farming/methods ; Gene Editing ; CRISPR-Cas Systems ; *Plants, Genetically Modified/genetics/metabolism ; *Plants/genetics/metabolism ; Synthetic Biology ; Biotechnology/methods ; },
abstract = {Plant molecular farming (PMF) has emerged as a promising strategy for producing biopharmaceuticals and high-value biomolecules in plant systems. In this review, we present a comprehensive synthesis of current methodologies while introducing novel approaches to genetic transformation, protein expression, glycan engineering, and downstream processing. We offer in-depth analyses of recent advancements such as CRISPR/Cas9-mediated pathway editing, synthetic biology frameworks for optimizing protein yield and quality, and integrated bioprocessing solutions that enhance purification efficiency. Further, detailed case studies are discussed to illustrate actionable strategies, and future research directions are proposed to bridge current gaps. By focusing on transformative techniques and critical problem-solving perspectives, this review aims to guide researchers toward more effective and scalable PMF applications.},
}

*Molecular Farming/methods
Gene Editing
CRISPR-Cas Systems
*Plants, Genetically Modified/genetics/metabolism
*Plants/genetics/metabolism
Synthetic Biology
Biotechnology/methods

@article {pmid41871065,
year = {2026},
author = {Huang, Y and Liang, W and Huang, M and Deng, Y and Huang, Z and Ai, C and Tan, W and Jiang, L},
title = {Application of CRISPR/Cas13a system on the rapid detection of Salmonella spp.},
journal = {PLoS neglected tropical diseases},
volume = {20},
number = {3},
pages = {e0014150},
doi = {10.1371/journal.pntd.0014150},
pmid = {41871065},
issn = {1935-2735},
mesh = {Humans ; *CRISPR-Cas Systems ; *Salmonella/genetics/isolation & purification ; Sensitivity and Specificity ; *Salmonella Infections/diagnosis/microbiology ; Prospective Studies ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Molecular Diagnostic Techniques/methods ; Diarrhea/microbiology/diagnosis ; Female ; Male ; Adult ; Nucleic Acid Amplification Techniques/methods ; Middle Aged ; },
abstract = {BACKGROUND: Salmonella spp. infections can manifest in various clinical symptoms, from asymptomatic carriage to gastroenteritis, and even severe sepsis. Given the rapid progression of the disease and its potential to cause severe outcomes or trigger cluster outbreaks, making the detection of Salmonella spp. critically important. Although broth enrichment culture is considered the gold standard, it is time-consuming and involves multiple steps, making it difficult to meet urgent diagnostic needs. Hence, prompt and precise detection of Salmonella spp. is crucial not only for early diagnosis and effective treatment, but also for preventing transmission, controlling outbreaks, and screening asymptomatic Salmonella carrier.

METHODS: This study developed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) -SE assay that integrated the advantages of the recombinase polymerase amplification (RPA) and CRISPR/Cas13a system for detecting Salmonella spp. The clinical performance of CRISPR/Cas13a-SE assay was evaluated by a cohort of 94 inpatients with diarrhea and three prospective studies.

RESULTS: The CRISPR/Cas13a-SE assay can be completed within 60 minutes, and its limit of detection was 100 fg/μL. Compared to the broth enrichment culture, the CRISPR/Cas13a-SE assay demonstrated a sensitivity of 87.5% and a specificity of 98.8% in a cohort of 94 inpatients with diarrhea. In our prospective studies involved three distinct cohorts: 1,662 food handlers, 211 outpatients with diarrhea, and 154 inpatients with Gram-negative bacteremia. Compared with broth enrichment culture, CRISPR/Cas13a-SE assay had a high concordance rate of 98.79% (1,642/1,662), 99.52% (210/211), and 100.00% (154/154) respectively.

CONCLUSIONS: We demonstrated that the CRISPR/Cas13a-SE system showed excellent detection performance for infectious diarrhea caused by Salmonella spp. The combined use of CRISPR/Cas13a-SE with the blood culture method enhances the rapid diagnosis of invasive salmonellosis, which is crucial for early target-based therapy. Additionally, screening of asymptomatic Salmonella carrier will be benefit for disease prevention and control.},
}

Humans
*CRISPR-Cas Systems
*Salmonella/genetics/isolation & purification
Sensitivity and Specificity
*Salmonella Infections/diagnosis/microbiology
Prospective Studies
Clustered Regularly Interspaced Short Palindromic Repeats
*Molecular Diagnostic Techniques/methods
Diarrhea/microbiology/diagnosis
Female
Male
Adult
Nucleic Acid Amplification Techniques/methods
Middle Aged

@article {pmid41872458,
year = {2026},
author = {Lkhagvadorj, K and Okamura, E and Taki, T and Suzuki, H and Kuno, A and Itoh, Y and Mizuno, S and Woltjen, K and Ema, M},
title = {Optimizing CRISPR precision in mouse embryos via microhomology-mediated end joining-dominant targeting.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41872458},
issn = {2399-3642},
support = {25K02195//Ministry of Education, Culture, Sports, Science and Technology (MEXT)/ ; 24K18045//Ministry of Education, Culture, Sports, Science and Technology (MEXT)/ ; JP223fa627008//Japan Agency for Medical Research and Development (AMED)/ ; },
mesh = {Animals ; Mice ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *DNA End-Joining Repair ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mouse Embryonic Stem Cells/metabolism ; *Embryo, Mammalian/metabolism ; Female ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Fibroblast Growth Factor 10/genetics ; },
abstract = {CRISPR/Cas9 technology enables efficient gene editing in mice, but its reliance on non-homologous end joining often leads to unpredictable and mosaic mutations in founder (F0) animals. Here, we present a hybrid genome editing strategy that combines in silico prediction software with in vitro validation using mouse embryonic stem cells (mESCs). Although the software was trained on mESC datasets, actual editing outcomes in mESCs more accurately reflected mutation patterns observed in blastocysts and post-implantation embryos. Using this information to develop an integrated pipeline, we pre-selected guide RNAs (gRNAs) predicted to promote microhomology-mediated end joining (MMEJ)-dominant repair and validated them in mESCs prior to embryo injection. Applied to the Tyr and Fgf10 genes, this approach enabled efficient generation of F0 mice with highly uniform genotypes. Our strategy enhances the predictability and reproducibility of CRISPR-based genome editing in mice and may help reduce animal usage in gene editing studies.},
}

Animals
Mice
*Gene Editing/methods
*CRISPR-Cas Systems
*DNA End-Joining Repair
RNA, Guide, CRISPR-Cas Systems/genetics
Mouse Embryonic Stem Cells/metabolism
*Embryo, Mammalian/metabolism
Female
*Clustered Regularly Interspaced Short Palindromic Repeats
Fibroblast Growth Factor 10/genetics

@article {pmid41872465,
year = {2026},
author = {Ahmed, MF and Sarkar, MMH and Mehzabin, K and Hossain, MI and Bhim, M and Chowdhury, SF and Naser, SR and Mumtaz, T and Faruk, MO},
title = {Genomic convergence of multidrug resistance, virulence-associated loci, and phage defense systems in Klebsiella pneumoniae from pharmaceutical wastewater in Bangladesh.},
journal = {Scientific reports},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41598-026-45102-2},
pmid = {41872465},
issn = {2045-2322},
abstract = {Klebsiella pneumoniae strains that combine multidrug resistance and enhanced virulence pose a growing global public health threat. Understanding the genetic basis of these high-risk lineages is critical for surveillance and mitigation. We isolated K. pneumoniae JU-BAEC-01 from treated effluent of antibiotic-manufacturing pharmaceutical facilities in Bangladesh and performed whole-genome sequencing with comparative genomic analyses to characterize its phylogeny, resistome, virulence-associated loci, mobile genetic elements, and predicted antiviral defense systems. JU-BAEC-01 belongs to a phylogenetically distinct lineage, serotype O3b: KL150 with resistance to nearly all clinically relevant antibiotic classes except carbapenems and colistin, mediated by an extensive acquired resistome, including tmexCD3-toprJ3 (tigecycline), armA, aac(6')-Ib-cr, qnrB4, oqxAB, blaDHA-1, blaSHV-182, and blaTEM-1B, mostly carried on conjugative IncC, IncFIB, IncHI1B, and IncR plasmids. Classical hypervirulence markers are present: complete aerobactin (iucABCD-iutA) and salmochelin (iroBCDEN) clusters, rmpA2, type 1 and type 3 fimbriae, T6SS, and pgaABCD. Notably, the strain encodes one of the most elaborate anti-phage defense arsenals reported in Klebsiella to date, comprising functional Type I-E, III-A, and IV-A CRISPR-Cas systems, multiple restriction-modification systems, BREX Type I, abortive infection systems (AbiE, AbiU), and additional novel defenses that coexist with phage-derived anti-CRISPR (AcrIE9) and anti-restriction (ArdA) proteins. K. pneumoniae JU-BAEC-01 is a "perfect storm" pathogen that combines multi-drug resistance (MDR), hypervirulence, and a multilayered, highly developed defense against bacteriophages. Together, these findings highlight the environmental emergence of a genetically distinct, multidrug-resistant K. pneumoniae with substantial virulence potential and complex phage-host interaction capacity, underscoring the need for genomic surveillance of pharmaceutical wastewater systems.},
}

@article {pmid41873079,
year = {2026},
author = {Luan, M and Liu, S and Yang, Q and Zhang, Y and Wang, M and Liu, R and Niu, G},
title = {[Metabolic engineering of Streptomyces for production of hyaluronic acid].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {3},
pages = {1242-1260},
doi = {10.13345/j.cjb.250734},
pmid = {41873079},
issn = {1872-2075},
support = {2023YFD1700700//the National Key Research and Development Program of China/ ; },
mesh = {*Metabolic Engineering/methods ; *Streptomyces/metabolism/genetics ; *Hyaluronic Acid/biosynthesis ; Streptomyces coelicolor/metabolism/genetics ; Hyaluronan Synthases ; },
abstract = {Hyaluronic acid (HA) is a glycosaminoglycan with significant biological activities, which render it widely applicable in the cosmetics and pharmaceutical industries. The development of safe and efficient chassis cells to enhance HA synthesis efficiency has thus emerged as a key factor in HA production. Our study aims to construct a high-performance HA biosynthesis system using Streptomyces as the chassis cell, thereby providing technical support for the efficient microbial production of HA. Thus, our study focused on the metabolic engineering of Streptomyces for strengthening the HA synthesis pathway and then optimized the culture conditions for efficient HA synthesis. First, the HA-synthesizing capabilities of four hyaluronate synthases from different sources were evaluated in two host strains: Streptomyces coelicolor M1146 and Streptomyces albus J1074. The results indicated that the hyaluronate synthases derived from Streptococcus pyogenes exhibited the strongest HA synthesis capability. Notably, the HA yield in S. albus J1074 was higher. Building on this finding, S. albus J1074 was selected as the starting strain to construct a chassis strain tailored for HA synthesis: key genes in the competitive metabolic pathway of HA synthesis were knocked out, while the expression levels of genes associated with the bypass pathway were down-regulated. Furthermore, different combinations of key genes involved in the HA precursor synthesis pathway were designed, and their expression levels were enhanced via a constitutive strong promoter. The recombinant strain obtained therefrom achieved a maximum HA yield of 2.62 g/L. Finally, the synthetic capacity of this high-yield engineered strain was further unleased through the optimization of culture conditions, leading to a final HA yield of 4.63 g/L. The recombinant strain constructed in this study not only lays a foundation for the development of engineered Streptomyces but also provides an excellent chassis strain for the microbial production of HA and other related bioproducts.},
}

*Metabolic Engineering/methods
*Streptomyces/metabolism/genetics
*Hyaluronic Acid/biosynthesis
Streptomyces coelicolor/metabolism/genetics
Hyaluronan Synthases

@article {pmid41873757,
year = {2026},
author = {Yu, Z and Bao, Y and Alcalá-Lalinde, A and Andrade Dos Ramos, Z and Koroglu, M and Berkhout, B and Herrera-Carrillo, E},
title = {Elucidating the kinetics of CRISPR-SaCas9 action to obtain effective HIV DNA excision with two gRNAs.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41873757},
issn = {1362-4962},
support = {1R01AI145045IH//NIH RO1/ ; DGP_EMEC_2023_00154//Junta de Andalucía/ ; PID2024-162385OB-I00//Spanish State Research Agency/ ; },
mesh = {*HIV-1/genetics ; *CRISPR-Cas Systems ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Gene Editing/methods ; Kinetics ; *DNA, Viral/genetics/metabolism ; Proviruses/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics ; HEK293 Cells ; Genome, Viral ; HIV Infections/virology ; },
abstract = {The persistence of integrated human immunodeficiency virus (HIV) proviral DNA poses a major barrier to viral eradication, as the viral reservoir enables rapid rebound upon treatment interruption, despite effective virus inhibition. CRISPR-Cas-based editing strategies, especially those using double-site cleavage, show promise in excising proviral DNA, yet the rate and determinants of excision efficiency remain poorly understood. In this study, we systematically evaluated both single- and dual-SaCas9/gRNA approaches for HIV-1 inactivation. Sequence analysis revealed that SaCas9 can eliminate all wild-type HIV-1 genomes with a single gRNA, unlike other CRISPR-Cas systems. Dual-gRNA strategies improved antiviral efficacy, with the Gag3 + Pol5 combination achieving 97% excision efficiency. Kinetic analysis showed that excision efficiency correlates with the kinetic compatibility of paired gRNAs. Pairs of gRNAs with fast and similar kinetics achieved the highest excision efficiency. In contrast, the Gag3 + Env4 pair exhibited discordant kinetic characteristics (fast and slow), resulting in the failure to induce excision as the cut DNA will be repaired before the second cut is realized. Consequently, no excision but regular editing occurred at the two target sites. These findings provide a mechanistic framework for optimizing CRISPR-Cas-mediated excision, highlighting the critical role of both antiviral activity and kinetic synergy in guiding gRNA selection.},
}

*HIV-1/genetics
*CRISPR-Cas Systems
Humans
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Gene Editing/methods
Kinetics
*DNA, Viral/genetics/metabolism
Proviruses/genetics
*CRISPR-Associated Protein 9/metabolism/genetics
HEK293 Cells
Genome, Viral
HIV Infections/virology

@article {pmid39508585,
year = {2024},
author = {Androsiuk, L and Maane, S and Tal, S},
title = {CRISPR spacers acquired from plasmids primarily target backbone genes, making them valuable for predicting potential hosts and host range.},
journal = {Microbiology spectrum},
volume = {12},
number = {12},
pages = {e0010424},
pmid = {39508585},
issn = {2165-0497},
support = {3-17700//Ministry of Health, State of Israel (Ministry of Health)/ ; //BGU | Kreitman School of Advanced Graduate Studies, Ben-Gurion University of the Negev (Kreitman school of Advanced Graduate Studies)/ ; },
mesh = {*Plasmids/genetics ; *Bacteria/genetics/classification ; *Host Specificity/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Metagenomics/methods ; Gene Transfer, Horizontal ; CRISPR-Cas Systems ; },
abstract = {In recent years, there has been a surge in metagenomic studies focused on identifying plasmids in environmental samples. Although these studies have unearthed numerous novel plasmids, enriching our understanding of their environmental roles, a significant gap remains: the scarcity of information regarding the bacterial hosts of these newly discovered plasmids. Furthermore, even when plasmids are identified within bacterial isolates, the reported host is typically limited to the original isolate, with no insights into alternative hosts or the plasmid's potential host range. Given that plasmids depend on hosts for their existence, investigating plasmids without the knowledge of potential hosts offers only a partial perspective. This study introduces a method for identifying potential hosts and host ranges for plasmids through alignment with CRISPR spacers. To validate the method, we compared the PLSDB plasmids database with the CRISPR spacers database, yielding host predictions for 46% of the plasmids. When compared with reported hosts, our predictions achieved 84% concordance at the family level and 99% concordance at the phylum level. Moreover, the method frequently identified multiple potential hosts for a plasmid, thereby enabling predictions of alternative hosts and the host range. Notably, we found that CRISPR spacers predominantly target plasmid backbone genes while sparing functional genes, such as those linked to antibiotic resistance, aligning with our hypothesis that CRISPR spacers are acquired from plasmid-specific regions rather than insertion elements from diverse sources. Finally, we illustrate the network of connections among different bacterial taxa through plasmids, revealing potential pathways for horizontal gene transfer.IMPORTANCEPlasmids are notorious for their role in distributing antibiotic resistance genes, but they may also carry and distribute other environmentally important genes. Since plasmids are not free-living entities and rely on host bacteria for survival and propagation, predicting their hosts is essential. This study presents a method for predicting potential hosts for plasmids and offers insights into the potential paths for spreading functional genes between different bacteria. Understanding plasmid-host relationships is crucial for comprehending the ecological and clinical impact of plasmids and implications for various biological processes.},
}

*Plasmids/genetics
*Bacteria/genetics/classification
*Host Specificity/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Metagenomics/methods
Gene Transfer, Horizontal
CRISPR-Cas Systems

@article {pmid41191525,
year = {2026},
author = {Wang, K and Wang, J and Feng, R and Dudnyk, K and Kim, YJ and Lim, JYS and Lee, M and Zhang, Y and Gao, X and Cheng, Y and Orkin, SH and Zhou, J and Weiss, MJ and Yue, F and Xu, J},
title = {Silencing of BCL11A by disrupting enhancer-dependent epigenetic insulation.},
journal = {Blood},
volume = {147},
number = {13},
pages = {1470-1484},
doi = {10.1182/blood.2025030211},
pmid = {41191525},
issn = {1528-0020},
mesh = {Humans ; *Enhancer Elements, Genetic ; *Epigenesis, Genetic ; *Gene Silencing ; CRISPR-Cas Systems ; Erythroid Cells/metabolism ; Repressor Proteins ; *Nuclear Proteins/genetics/metabolism ; *Carrier Proteins/genetics/metabolism ; Fetal Hemoglobin/genetics/metabolism ; Chromatin/genetics/metabolism ; },
abstract = {The transcription factor BCL11A is a genetically and clinically validated regulator of the fetal-to-adult hemoglobin switch in human erythroid cells. CRISPR editing of an intronic enhancer within the BCL11A gene reactivates fetal hemoglobin (HbF) in adult erythroid cells, serving as the first CRISPR-based therapy for β-hemoglobinopathies. However, the molecular basis for the remarkable efficacy of CRISPR-mediated enhancer ablation remains elusive. Here, we describe a new genome architecture, an enhancer-dependent chromatin rosette, that is essential for epigenetic insulation and the developmentally regulated, hematopoietic lineage-specific expression of BCL11A. CRISPR-mediated disruption of the BCL11A erythroid enhancer impairs transcription of enhancer-driven RNAs and NIPBL-dependent cohesin loading, leading to the destabilization of the rosette structure, loss of chromatin insulation, and epigenetic silencing of BCL11A. Moreover, targeted depletion of enhancer RNAs using antisense oligonucleotides silences BCL11A by disrupting epigenetic insulation, causing HbF reactivation in adult erythroid cells. These findings uncover an essential role for enhancer-driven epigenetic insulation in transcriptional control, presenting a new strategy for the therapeutic targeting of BCL11A.},
}

Humans
*Enhancer Elements, Genetic
*Epigenesis, Genetic
*Gene Silencing
CRISPR-Cas Systems
Erythroid Cells/metabolism
Repressor Proteins
*Nuclear Proteins/genetics/metabolism
*Carrier Proteins/genetics/metabolism
Fetal Hemoglobin/genetics/metabolism
Chromatin/genetics/metabolism

@article {pmid41717698,
year = {2026},
author = {Tadokoro, T and Li, H and Gan, P and Xu, Z and Tan, W and Alzhanov, D and Sánchez-Ortiz, E and McAnally, JR and Guo, L and Xu, L and Ruan, P and Liu, N and Olson, EN},
title = {Ablation of PKCα Phosphorylation by CRISPR-Cas9 Base Editing Rescues Heart Failure.},
journal = {Circulation research},
volume = {138},
number = {7},
pages = {e326738},
pmid = {41717698},
issn = {1524-4571},
support = {P01 HL160488/HL/NHLBI NIH HHS/United States ; P50 HD087351/HD/NICHD NIH HHS/United States ; R01 HL157281/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *Protein Kinase C-alpha/genetics/metabolism ; *Heart Failure/genetics/therapy/enzymology/physiopathology/pathology/metabolism ; Phosphorylation ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Mice ; Humans ; Myocytes, Cardiac/metabolism/enzymology ; Mice, Inbred C57BL ; Male ; Disease Models, Animal ; Induced Pluripotent Stem Cells ; },
abstract = {BACKGROUND: The prevalence of heart failure is increasing globally, with poor prognosis, highlighting the need for novel therapeutic strategies. PKCα (protein kinase C alpha), encoded by PRKCA, plays a central role in heart failure pathogenesis. Phosphorylation of PKCα at threonine 497 (T497) triggers a series of intramolecular phosphorylation events, leading to its activation. Ablation of T497 phosphorylation leads to reduced stability and activity of PKCα.

METHODS: We generated mice harboring a phospho-resistant PKCα (T497A) mutation in the germline using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-mediated homology-directed repair. To assess the clinical feasibility of postnatal genome editing, we used CRISPR-Cas9 adenine base editing delivered by adeno-associated virus 9 to introduce the T497A substitution into the Prkca gene (Prkca[T497A]) in wild-type mice. Mice underwent transverse aortic constriction to model heart failure. Cardiac function, hypertrophy, fibrosis, and transcriptional changes were evaluated by echocardiography, wheat germ agglutinin staining, Masson's trichrome staining, and RNA-sequencing. The editing efficiency of Prkca[T497A] was assessed using Sanger sequencing and deep amplicon sequencing. To further explore its clinical potential, we introduced the PRKCA[T497A] mutation into human induced pluripotent stem cells by nucleofection-mediated adenine base editing. Ca[2+] homeostasis was analyzed in Fura-2-loaded human induced pluripotent stem cell-derived cardiomyocytes with PRKCA[T497A] under chronic AngII (angiotensin II) stimulation.

RESULTS: The T497A mutation in PKCα prevented its subsequent phosphorylation and led to PKCα protein degradation. Four weeks after transverse aortic constriction surgery, wild-type mice showed impaired cardiac function, cardiac remodeling, and increased lung weight. In contrast, PKCα phospho-resistant mice showed protection against heart failure-related aberrant changes in cardiac hypertrophy, fibrosis, and cardiac gene expression. Mice administered with adeno-associated virus 9 base editors to prevent T497 phosphorylation exhibited similar cardioprotective effects. In vitro, PKCα-edited induced pluripotent stem cell-derived cardiomyocytes were protected from AngII-induced impairments in contractility and Ca[2+] transients.

CONCLUSIONS: The editing of PRKCA[T497A] through adenine base editing represents a potential therapeutic approach for human cardiac diseases.},
}

Animals
*Protein Kinase C-alpha/genetics/metabolism
*Heart Failure/genetics/therapy/enzymology/physiopathology/pathology/metabolism
Phosphorylation
*CRISPR-Cas Systems
*Gene Editing/methods
Mice
Humans
Myocytes, Cardiac/metabolism/enzymology
Mice, Inbred C57BL
Male
Disease Models, Animal
Induced Pluripotent Stem Cells

@article {pmid41834687,
year = {2026},
author = {Yang, J and Huang, Q},
title = {Cas13a/crRNA trans-cleavage triggered primer exchange reaction based self-priming chain extension for sensitive and label-free infantile pneumonia related microRNA analysis.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {12},
pages = {2524-2531},
doi = {10.1039/d6ay00090h},
pmid = {41834687},
issn = {1759-9679},
mesh = {*MicroRNAs/genetics/analysis ; Humans ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Pneumonia/genetics/diagnosis ; Limit of Detection ; *RNA, Circular/genetics ; },
abstract = {Accurate and sensitive detection of microRNAs (miRNAs) is crucial for both pathophysiological studies and clinical diagnostics. Conventional amplification methods often face limitations such as dependence on thermal cycling, susceptibility to contamination, and insufficient specificity to discriminate among closely related miRNA family members. To address these challenges, we developed a label-free isothermal detection platform that integrates the precise RNA-targeting ability of the CRISPR/Cas13a system with a self-priming amplification cascade driven by the primer exchange reaction (PER). In this assay, target miRNA binding directly activates the trans-cleavage activity of the Cas13a/crRNA complex, which subsequently cleaves a uracil-rich toehold region on a stem-loop DNA primer (H1). Following dephosphorylation, the cleaved primer initiates a PER-mediated self-priming amplification process, generating long tandem double-stranded DNA products that can be sensitively detected using the fluorescent dye SYBR Green I. The proposed method demonstrates several key advantages: (i) high specificity enabled by the programmable Cas13a/crRNA complex, allowing clear distinction between the target miRNA and sequences with single-base mismatches or high homology; (ii) exceptional sensitivity, achieving a detection limit of 406 aM and a dynamic range spanning six orders of magnitude, through coupling Cas13a collateral cleavage with exponential isothermal amplification; (iii) excellent reproducibility, reflected by low relative standard deviations and a coefficient of variation of 3.65% in spiked serum samples; and (iv) strong concordance with the reference RT-qPCR method in mock clinical specimens, highlighting its reliability for potential clinical use. In summary, this CRISPR/Cas13a-coupled self-priming amplification strategy provides a robust, accurate, and highly sensitive means for miRNA quantification, offering a promising alternative for point-of-care molecular diagnostic applications.},
}

*MicroRNAs/genetics/analysis
Humans
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*Pneumonia/genetics/diagnosis
Limit of Detection
*RNA, Circular/genetics

@article {pmid41860589,
year = {2026},
author = {Walsh, DJ and Hynes, R and Guo, W and Surgenor, C and Prodöhl, PA and Parle-McDermott, A},
title = {Development and Laboratory Validation of a Field-Deployable CRISPR-Cas12a eDNA Assay for Phylogeographic Lineage Detection in Arctic Char (Salvelinus alpinus).},
journal = {Molecular ecology resources},
volume = {26},
number = {3},
pages = {e70125},
pmid = {41860589},
issn = {1755-0998},
support = {//Higher Education Authority/ ; },
mesh = {Animals ; *Trout/genetics/classification ; Phylogeography/methods ; *CRISPR-Cas Systems ; *DNA, Environmental/genetics/isolation & purification ; Ireland ; Sensitivity and Specificity ; },
abstract = {Environmental DNA (eDNA) tools are increasingly used for biodiversity monitoring, with most existing assays targeting species-level identification. However, the use of eDNA to resolve intraspecific genetic variation remains rare and methodologically underdeveloped. This study presents the development and laboratory validation of a novel molecular assay capable of detecting specific phylogeographic lineages, advancing eDNA applications by enabling resolution below the species level. The assay combines Recombinase Polymerase Amplification (RPA) and CRISPR-Cas12a technologies with a lateral flow platform for field-ready, on-site detection. Irish Arctic char (Salvelinus alpinus) was selected as the model due to its conservation relevance and post-glacial lineage diversity in Ireland. Mitochondrial genome sequencing of known Irish lineages identified a Protospacer Adjacent Motif (PAM) site unique to the Atlantic Subclade 1 lineage, allowing clear discrimination from co-occurring lineages. Two assays were optimised: a species-specific assay detecting all Arctic char lineages and a lineage-specific assay targeting Lineage 1. Both showed high sensitivity and specificity under laboratory conditions, with LbCas12a outperforming AsCas12a at optimised buffer concentrations. The lateral flow adaptation, utilising a dual-labelled FAM-Biotin probe, enabled portable and rapid detection with minimal equipment. Field validation using eDNA from Irish lakes highlighted the need for improved sampling protocols, as lake-edge surface samples failed to yield detections. This assay represents the first reported example of a CRISPR-based eDNA tool for phylogeographic lineage detection in the field. It offers a novel, non-invasive, and scalable approach to fine-scale ecological monitoring and establishes a foundation for future conservation tools targeting intraspecific diversity.},
}

Animals
*Trout/genetics/classification
Phylogeography/methods
*CRISPR-Cas Systems
*DNA, Environmental/genetics/isolation & purification
Ireland
Sensitivity and Specificity

@article {pmid41860948,
year = {2026},
author = {Hong, Y and Si, X and Liu, W and Mai, X and Zhang, Y},
title = {Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {41860948},
issn = {2050-084X},
support = {2021YFA1101002//National Key Research and Development Program of China/ ; 81773304//National Natural Science Foundation of China/ ; 81572795//National Natural Science Foundation of China/ ; 2019A39//the "Hundred, Thousand and Ten Thousand Talent Project" by Beijing municipal government/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Glycosylation ; Mice ; *Galactosyltransferases/metabolism/genetics ; *CD8-Positive T-Lymphocytes/immunology ; *Lymphocyte Activation ; Mice, Inbred C57BL ; Humans ; Receptors, Antigen, T-Cell/metabolism ; },
abstract = {Cytotoxic CD8[+] T-cells play central roles in tumor immunotherapy. Understanding the mechanisms that regulate development, differentiation, and functions of cytotoxic CD8[+] T-cells leads to the development of better immunotherapies. By combining primary T-cell culture and a syngeneic mouse tumor model with both genome-wide and custom CRISPR/Cas9 screenings, we systematically identified genes and pathways that regulate PD-1 expression and functions of CD8[+] T-cells. Among them, inactivation of a key enzyme in glycoconjugate biosynthesis, beta 1,4-galactosyltransferase 1 (B4GALT1), leads to significantly enhanced T-cell receptor (TCR) activation and functions of CD8[+] T-cell. Interestingly, suppression of B4GALT1 enhances functions of TCR-T-cells, but has no effect on chimeric antigen receptor T (CAR-T) cells. We systematically identified the substrates of B4GALT1 on CD8[+] T-cell surface by affinity purification and mass spectrometry analysis, which include protein components in both TCR and its co-receptor complexes. The galactosylation of TCR and CD8 leads to reduced interaction between TCR and CD8 that is essential for TCR activation. Artificially tethering TCR and CD8 by a TCR-CD8 fusion protein could bypass the regulation of B4GALT1 in CD8[+] T-cells. Finally, the expression levels of B4GALT1 normalized to tumor-infiltrated CD8[+] T-cells in tumor microenvironment are significant and negatively associated with prognosis of human patients. Our results reveal the important roles of protein N-glycosylation in regulating functions of CD8[+] T-cells and prove that B4GALT1 is a potential target for tumor immunotherapy.},
}

Animals
*CRISPR-Cas Systems
Glycosylation
Mice
*Galactosyltransferases/metabolism/genetics
*CD8-Positive T-Lymphocytes/immunology
*Lymphocyte Activation
Mice, Inbred C57BL
Humans
Receptors, Antigen, T-Cell/metabolism

@article {pmid41861390,
year = {2026},
author = {Huang, Y and Yi, X and Yang, X and Li, C and Li, Y and Ye, Z and He, J},
title = {Characterization of CRISPR-Cas systems in the Haemophilus genus CRISPR-Cas in Haemophilus spp.},
journal = {Genetics and molecular biology},
volume = {49},
number = {1},
pages = {e20250166},
pmid = {41861390},
issn = {1415-4757},
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) system constitutes a crucial adaptive defense mechanism in prokaryotes against foreign genetic elements. Although CRISPR-Cas systems have been characterized in numerous bacteria, the architecture and function of these systems in the Haemophilus genus remain poorly understood. This study aims to analyze CRISPR-Cas systems in 138Haemophilusstrains and investigate their function, particularly in relation to virulence factors. Results revealed that CRISPR-Cas systems were identified in 31.88% of the Haemophilusstrains. Subtype I-C was the most prevalent, followed by subtypes II-C and III-A. Repeat sequences and thecas1gene were highly conserved within the same subtype. 29.62% of spacer sequences exhibited homology to plasmids or bacteriophages. phiMHaA1 was an important target of the CRISPR-Cas system in Haemophilusgenus. The protospacer adjacent motif sequences (PAM) were determined to be 5'-TTC-3' for subtype I-C and 5'-TTT-3' for subtype II-C. Comparative analysis of virulence genes showed that CRISPR-positive strains carried more ompP2 than CRISPR-negative strains, while the distribution of hmw2C and hmw1C exhibited an opposite trend. These findings provide novel insights into the diversity and function of CRISPR-Cas systems inHaemophilusgenus and propose potential strategies for attenuating the impact ofHaemophilusvirulence factors.},
}

@article {pmid41863808,
year = {2026},
author = {Louis, EM and Fu, L and Luu, N and Sachs, LM and Shi, YB},
title = {Protocol for streamlining genotyping of germline-transmissible mutants from genome editing by using a parallel qPCR-based index and R analysis.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104454},
doi = {10.1016/j.xpro.2026.104454},
pmid = {41863808},
issn = {2666-1667},
abstract = {Targeted genome editing using CRISPR-Cas, ZFNs, or TALENs enables precise gene function studies but often produces point mutations or insertions or deletions (indels) that are difficult to detect by conventional PCR. We developed a parallel qPCR assay with an iGenotype index for simple, reliable genotyping. iGenotype values (1, 0, -1) remained constant across allele-specific primers. qPCR data can be analyzed via an R program, enabling large-scale or automated genotyping. For complete details on the use and execution of this protocol, please refer to Fu et al.[1].},
}

@article {pmid41865126,
year = {2026},
author = {Marei, HE},
title = {Recent Advances in the Non-viral Delivery of Genes to Central Nervous System Disorders.},
journal = {Cellular and molecular neurobiology},
volume = {},
number = {},
pages = {},
doi = {10.1007/s10571-026-01703-z},
pmid = {41865126},
issn = {1573-6830},
abstract = {Disorders of the central nervous system (CNS), neurological disorders, neurodegenerative disorders, genetic disorders) constitute a significant burden on global health, and current treatment options remain challenging. As treatment for CNS disorders is primarily palliative, the underlying causes of disease progression are not addressed through conventional pharmacologic therapies. Gene therapy has the potential to address these root causes of disease progression; however, many of the vectors used in gene therapy (e.g., adeno-associated viruses (AAVs)) have limitations such as immunogenicity, low cargo capacity, and crossing the blood-brain barrier (BBB). These limitations have led to significant progress in the development of non-viral gene delivery systems. Compared with viral vectors, non-viral platforms offer improved safety profiles, greater design flexibility, lower production costs, and superior suitability for repeated administration. This review reports recent advancements in the development of non-viral platforms for CNS gene delivery and focuses on lipid-based nanoparticles, polymeric nanoparticles, exosome-based techniques, and new hybrid technologies. Particular emphasis is placed on nanoparticle modification approaches to enhance BBB penetration and enable delivery of genome-editing technologies (CRISPR/Cas systems). The review provides explanations of clinical trials, regulatory considerations, and manufacturing issues that result from the recent developments noted above. It also explores the emerging role of artificial intelligence in supporting carrier design and enhancing delivery efficiency. Both artificial intelligence and non-viral platforms have the potential to facilitate the advancement of safe, effective, and repeatably administered gene therapies for patients with CNS disorders.},
}

@article {pmid40162765,
year = {2025},
author = {Zeng, X and Jiang, Q and Yang, F and Wu, Q and Lyu, T and Zhang, Q and Wang, J and Li, F and Xu, D},
title = {Establishment and optimization of a system for the detection of Candida albicans based on enzymatic recombinase amplification and CRISPR/Cas12a system.},
journal = {Microbiology spectrum},
volume = {13},
number = {5},
pages = {e0026825},
pmid = {40162765},
issn = {2165-0497},
mesh = {*Candida albicans/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Candidiasis/diagnosis/microbiology ; *Recombinases/metabolism/genetics ; Temperature ; *Molecular Diagnostic Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {UNLABELLED: Invasive candidiasis is a fungal infection caused by various pathogenic yeasts, with Candida albicans as the predominant pathogen. Traditional culturing and identification methods for C. albicans are slow, requiring several days to weeks to produce results, which hampers rapid diagnosis. In this study, we proposed three amplification methods to combine with CRISPR/Cas12a and selected the enzymatic recombinase amplification (ERA) and CRISPR/Cas12a two-step method for the detection of C. albicans in terms of sensitivity, and then the two-step method was optimized to a temperature-controlled one-step method for the detection of C. albicans by enzymatic recombinase amplification (ERA)-CRISPR/Cas12a. The temperature-controlled system employs a combination of liquid and solid paraffin wax to maintain the desired melting point, thus facilitating spatial separation of the ERA amplification system from the CRISPR/Cas12a detection system within a single tube. After a reaction at 37°C, the temperature is raised to 45°C, melting the wax and allowing the amplification system to merge with the detection system, initiating the reaction. This one-step detection platform simplifies and expedites the procedure, achieving a sensitivity level on par with that of two-step methods. The reaction completes in about 30 minutes, detecting as little as 100 ag/µL of genomic DNA from C. albicans pure cultures. It shows high specificity and resistance to clinical nucleic acid interference, without cross-reactivity. Additionally, the method eliminates the need to open the reaction tube, effectively preventing aerosol contamination and providing a stable, thus offering a new tool for the rapid clinical diagnosis of C. albicans.

IMPORTANCE: This study established a two-step method through optimization, compared its sensitivity, and then combined the specific detection capabilities of ERA and CRISPR/Cas12a. Furthermore, a one-step method was developed based on the two-step method, creating a one-step system for the detection of Candida albicans. This system does not require the lid to be opened during the reaction process, reducing aerosol contamination and minimizing the risk of false positives. This method does not require advanced instruments or equipment and shows strong specificity without being affected by other pathogens. It can serve as a new method for the detection of Candida albicans and has significant practical application prospects.},
}

*Candida albicans/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
Humans
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
*Candidiasis/diagnosis/microbiology
*Recombinases/metabolism/genetics
Temperature
*Molecular Diagnostic Techniques/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41691189,
year = {2026},
author = {Lv, J and Geng, L and Shi, W and Li, C and Wang, S and Zhang, B and Yu, K and Cui, R and Liu, Y and Xiong, F and Lv, J and Dong, S and Barco, B and Xu, J},
title = {Enhancing heritable genome editing in soybean by optimizing promoter combinations for the LbCas12a system.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {},
pmid = {41691189},
issn = {1471-2229},
abstract = {UNLABELLED: The CRISPR-Cas system, adapted from prokaryotic immune mechanisms, enables programmable DNA targeting and editing. Recent advancements include base editors and prime editors, expanding genetic research applications. The LbCas12a (Cpf1) system offers unique advantages, including simplified operation and multiplex editing, yet achieving stable heritable edits in soybean remains challenging due to its complex paleo-tetraploid genome and transformation constraints. Here we optimized promoter combinations for the LbCas12a editing system to maximize both editing efficiency in the initial (E0) generation and the frequency of homozygous or biallelic mutants in the subsequent (E1) generation. We identified prAtHSP70-1 (At5G02500), prAtEF1αA4 (At5G60390), and prGmUbi1 (Glyma.10G251900) as an effective promoter set, and demonstrated that LbCas12a–crRNA accumulation needs to exceed a defined threshold to achieve efficient target cleavage. These findings provide a practical strategy for enhancing heritable genome editing in soybean and may be broadly applicable to other crops.

SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12870-026-08336-w.},
}

@article {pmid41733889,
year = {2026},
author = {Son, SH and Woo, S and Choi, A and Ahn, S and Yoo, HC},
title = {Advances in Engineered Virus-Like Particles for Genome Editing and Therapy.},
journal = {BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy},
volume = {40},
number = {2},
pages = {317-333},
pmid = {41733889},
issn = {1179-190X},
support = {RS202400403169//Korea Basic Science Institute/ ; 2021R1C1C2006283//National Research Foundation/ ; RS-2024-00412879//National Research Foundation/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; Animals ; Genetic Engineering/methods ; Genetic Vectors ; CRISPR-Cas Systems ; Gene Transfer Techniques ; },
abstract = {Engineered virus-like particles (eVLPs) have emerged as a promising class of delivery systems for genome editing agents. By combining the efficient cellular entry of viral vectors with the safety advantages of nonviral platforms, eVLPs enable transient delivery of ribonucleoproteins such as Cas9, base editors, and prime editors. Successive design strategies, from rational engineering to directed optimization, have progressively improved particle assembly, cargo stability, and editing efficiency. In parallel, pseudotyping approaches have expanded the versatility of eVLPs for cell-type-specific targeting. Recent preclinical studies highlight their potential across diverse applications, ranging from monogenic disease models to complex disorders, and support their advancement toward clinical translation. This review summarizes the structural and production principles of eVLPs, surveys key advances in their development, and discusses therapeutic opportunities and future challenges for their application in genome editing.},
}

*Gene Editing/methods
Humans
*Genetic Therapy/methods
Animals
Genetic Engineering/methods
Genetic Vectors
CRISPR-Cas Systems
Gene Transfer Techniques

@article {pmid41741470,
year = {2026},
author = {Aird, EJ and Serrano-Benitez, A and Siegner, SM and Cannavo, E and Belotserkovskaya, R and Gueorguieva, N and Fielden, J and Cullot, G and Ammann, S and Bader, AS and Gupta, V and Andrieux, G and Raab, R and Del Rey González, M and Cathomen, T and Cejka, P and Corn, JE and Jackson, SP},
title = {ERCC6L2 ensures repair fidelity for staggered-end DNA double-strand breaks.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41741470},
issn = {2041-1723},
support = {855741-DDREAMM-ERC-2019-SyG//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 855741-DDREAMM-ERC-2019-SyG//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; },
mesh = {*DNA Breaks, Double-Stranded ; Humans ; *DNA Helicases/metabolism/genetics ; *DNA Repair ; DNA-Binding Proteins/metabolism/genetics ; Endodeoxyribonucleases/metabolism/genetics ; MRE11 Homologue Protein/metabolism/genetics ; CRISPR-Cas Systems ; Gene Editing ; Cell Cycle Proteins/metabolism/genetics ; DNA Repair Enzymes/metabolism/genetics ; Poly-ADP-Ribose Binding Proteins/metabolism ; HEK293 Cells ; Nuclear Proteins/metabolism ; Bacterial Proteins ; Acid Anhydride Hydrolases ; CRISPR-Associated Proteins ; },
abstract = {DNA double-strand breaks (DSBs) both pose threats to genome integrity and are commonly used for genome editing applications. Structural features of DSB ends play key roles in determining DNA repair pathway usage and outcomes during genome editing, but the cellular factors involved in these processes are only partially known. Through genome-wide CRISPRi screening, we identify ERCC6L2 as critical for repairing Cas12a-induced staggered DSBs but irrelevant for Cas9-induced blunt DSBs. We show that ERCC6L2 acts as a protection factor for staggered DSBs with either 5' or 3' polarity, preventing large deletions and translocations stemming from DNA damage induced by Cas12a, TALENs, or dual Cas9 nicks. Furthermore, ERCC6L2 loss hyper-sensitizes cells to multiple staggered DSBs induced by promiscuous Cas12a activity or etoposide-induced TOP2 trapping. By combining genetics and biochemical reconstitution, we find that ERCC6L2 counteracts MRE11-RAD50-NBS1 (MRN)-mediated resection by binding and melting staggered DNA ends, thereby promoting accurate end joining. Our data reveal a protective role of ERCC6L2 in staggered-end DSB repair, which suggests the molecular underpinnings of pathology in patients with ERCC6L2 mutations and cautions against using overhang-inducing genome editing tools for their treatment.},
}

*DNA Breaks, Double-Stranded
Humans
*DNA Helicases/metabolism/genetics
*DNA Repair
DNA-Binding Proteins/metabolism/genetics
Endodeoxyribonucleases/metabolism/genetics
MRE11 Homologue Protein/metabolism/genetics
CRISPR-Cas Systems
Gene Editing
Cell Cycle Proteins/metabolism/genetics
DNA Repair Enzymes/metabolism/genetics
Poly-ADP-Ribose Binding Proteins/metabolism
HEK293 Cells
Nuclear Proteins/metabolism
Bacterial Proteins
Acid Anhydride Hydrolases
CRISPR-Associated Proteins

@article {pmid41812941,
year = {2026},
author = {Wang, M and Niu, D and Zhang, Q and Tang, Y and Zhao, Y and Chen, F},
title = {CRISPR-based correction of apolipoprotein E4 in Alzheimer's disease: Therapeutic strategies and macromolecular delivery innovations.},
journal = {International journal of biological macromolecules},
volume = {354},
number = {},
pages = {151352},
doi = {10.1016/j.ijbiomac.2026.151352},
pmid = {41812941},
issn = {1879-0003},
mesh = {*Alzheimer Disease/genetics/therapy ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; *Apolipoprotein E4/genetics ; Blood-Brain Barrier/metabolism ; Exosomes/metabolism ; Genetic Therapy/methods ; Nanoparticles/chemistry ; },
abstract = {Alzheimer's disease (AD) is the leading cause of dementia worldwide, with substantial unmet clinical needs. The apolipoprotein E4 (APOE4) allele is the strongest genetic risk factor for late onset AD, with each copy increasing risk approximately two- to three-fold, and homozygous carriers facing up to a 10- to 15-fold higher risk compared to APOE3 carriers. APOE4 contributes to diverse pathogenic mechanisms including lipid dysregulation, neuroinflammation, synaptic dysfunction, and vascular compromise. The precise, allele-specific correction of APOE4 therefore holds transformative therapeutic potential. CRISPR-based genome editing technologies, including nuclease disruption, base editing, and prime editing, offer unprecedented opportunities to directly modify APOE4 at its genomic source. Here, we review mechanistic underpinnings of APOE4 pathology, summarize current gene editing platforms for APOE4 correction, evaluate relevant in vitro and in vivo model systems, and assess delivery strategies with an emphasis on nanoparticle and exosome based approaches. We highlight recent breakthroughs in exosome mediated APOE4 editing while addressing ongoing technical hurdles in allele specificity and translational barriers such as Cas nuclease immunogenicity, limited delivery efficiency across the blood brain barrier (BBB), and concerns over long term genomic safety. This review concludes that overcoming BBB constraints remains the most significant challenge for clinical translation, and that innovations in exosome and nanoparticle based delivery platforms represent the most promising strategies for advancing CRISPR therapeutics for AD.},
}

*Alzheimer Disease/genetics/therapy
Humans
*Gene Editing/methods
*CRISPR-Cas Systems
Animals
*Apolipoprotein E4/genetics
Blood-Brain Barrier/metabolism
Exosomes/metabolism
Genetic Therapy/methods
Nanoparticles/chemistry

@article {pmid41854526,
year = {2026},
author = {Gao, Z and Liu, G},
title = {Advancing Point-of-Care Testing for Helicobacter pylori toward CRISPR-Cas-Enabled Diagnostics.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c00089},
pmid = {41854526},
issn = {2379-3694},
abstract = {Helicobacter pylori (H. pylori) chronically infects nearly half of the global population and is a major risk factor for gastric cancer. Timely and accurate diagnosis is critical to enable targeted eradication therapy and prevent disease progression. However, current gold-standard methods, such as invasive endoscopy and laboratory-based polymerase chain reaction, are costly, time-consuming, and logistically impractical for large-scale screening, particularly in resource-limited settings. Point-of-care testing (POCT) emerges as a transformative solution, offering rapid, user-friendly, and minimally invasive detection at the point of need. In this review, we systematically trace the evolution of H. pylori POCT, with a focus on revolutionary CRISPR-Cas-based diagnostic systems, cutting-edge advancements in substrate engineering (e.g., paper, polymer, hydrogels) and multi-modal signal transduction (e.g., optical, electrochemical). We further outline key design principles for next-generation POCT platforms that strictly align with the World Health Organization's ASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, Deliverable), aiming to accelerate early detection, reduce healthcare disparities, and improve global clinical management of H. pylori infection.},
}

@article {pmid41855975,
year = {2026},
author = {Yong, Q and Ou, X and Zhao, Y and Kang, X and Gao, H and Liu, H and Li, K and Guo, Y},
title = {CRISPR/Cas and isothermal amplification in Pathogen Detection: Applications and future perspectives.},
journal = {Talanta},
volume = {305},
number = {},
pages = {129662},
doi = {10.1016/j.talanta.2026.129662},
pmid = {41855975},
issn = {1873-3573},
abstract = {Conventional pathogen detection methods are often limited by prolonged turnaround times and laboratory dependency. The integration of CRISPR/Cas systems with isothermal amplification (IA) has emerged as a promising approach to enable rapid, accurate, and field-deployable molecular diagnostics. This review systematically outlines the principles, optimization strategies, and recent advances in CRISPR-Cas and IA-integrated platforms. It highlights how synergistic mechanisms enhance detection sensitivity and examines innovative integration strategies-such as physical compartmentalization, chemical regulation, and intelligent system design-that address key compatibility challenges. The role of nanomaterials in enhancing signal amplification and facilitating system integration is thoroughly discussed. Furthermore, the suitability of various readout modalities-including fluorescence, lateral flow assays, electrochemical sensing, and digital detection-is critically evaluated. While challenges remain in terms of stability, cost, and standardization, future advances in intelligent design, portable device development, and quantitative methodologies are expected to establish this technology as a versatile platform for public health control, food safety monitoring, and related fields. This review provides a comprehensive perspective and methodological reference for researchers engaged in point-of-care testing and diagnostic technology development.},
}

@article {pmid41856106,
year = {2026},
author = {Yuan, S and Zhu, H and Yu, M and Jia, H and Peng, S and Ma, Y},
title = {Discovery of human gut phage-encoded anti-CRISPR proteins unveils diverse mechanisms for phages to evade type II CRISPR immunity.},
journal = {Cell host & microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2026.02.017},
pmid = {41856106},
issn = {1934-6069},
abstract = {Phages encode diverse anti-CRISPR (Acr) proteins to counteract bacterial CRISPR-Cas systems. However, gut phage Acrs remain poorly characterized. Using an integrated bioinformatics and high-throughput functional screening approach, we identify 651 phage-encoded positive Acr candidates that target type II CRISPR systems, which predominate in the human gut. Among these, a subset of Acrs is verified through plasmid interference assays, with plaque assays confirming CRISPR-Cas inhibitory activity for 36 Acr candidates. Mechanistic characterization of five Acrs, including the Acr against subtype II-B systems (AcrIIB-1), reveals distinct inhibition strategies. Remarkably, 213 positive Acr candidates, designated here as GutAcraca, exhibit structural convergence by adopting similar folds and exhibit dual functionality: transcription regulation to support their production and inhibition of CRISPR-Cas systems. These GutAcraca are widely distributed across microbial species (detected in 26% of species). Our work uncovers the extensive diversity of phage-encoded Acrs in the human gut and highlights their potential as biotechnology tools.},
}

@article {pmid41856855,
year = {2026},
author = {Tsai, FY and Sternberg, SH},
title = {Memory on demand: how RNA-free Cas9 recharges CRISPR immunity.},
journal = {Trends in biochemical sciences},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibs.2026.01.004},
pmid = {41856855},
issn = {0968-0004},
abstract = {Bacteria and archaea acquire immune memories by integrating foreign DNA into clustered regularly interspaced short palindromic repeats (CRISPR) arrays. Zhou et al. reveal that Cas9-thought to act only with guide RNAs-also functions in its RNA-free form, stimulating spacer acquisition. Rising CRISPR RNA levels shift the equilibrium toward the RNA-bound state, attenuating acquisition and minimizing autoimmunity.},
}

@article {pmid41859925,
year = {2026},
author = {Tibebu, R and Gamo, ME and Ellison, EE and Myers, EA and Sahoo, R and Winecke, SR and Tan, GD and Fischer, JM and Leakey, ADB and Voytas, DF},
title = {Virus-induced gene editing of stomatal regulators in Nicotiana benthamiana enables rapid functional genomics.},
journal = {The Plant journal : for cell and molecular biology},
volume = {125},
number = {6},
pages = {e70805},
pmid = {41859925},
issn = {1365-313X},
support = {SC0023160//U.S. Department of Energy/ ; SC0018277//U.S. Department of Energy/ ; },
mesh = {*Nicotiana/genetics/virology ; *Gene Editing/methods ; *Plant Stomata/genetics/growth & development ; Genomics/methods ; *Plant Viruses/genetics/physiology ; Plants, Genetically Modified ; Plant Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; },
abstract = {Virus-induced gene editing (VIGE) holds promise as a rapid and scalable approach for functional genomics in plants. Here, we apply a tobacco rattle virus (TRV)-based single-guide RNA (sgRNA) delivery system to target key regulators of stomatal development in Nicotiana benthamiana using transgenic Cas9-expressing lines. sgRNAs fused to a mobile RNA element and co-delivered with TRV enabled both somatic and heritable genome editing across orthologs of STOMAGEN, EPF2, YODA, and SPEECHLESS. Somatic editing frequencies reached up to 95%, and heritable tetra-allelic mutations were recovered in multiple target genes. Mutants exhibited significant, gene-specific changes in stomatal density, with corresponding effects on leaf temperature indicative of altered evaporative cooling. Additionally, sgRNAs fused to an AmCyan reporter enabled visualization of virus-infected tissues, allowing stomatal phenotyping in edited M0 sectors. This TRV-based platform facilitates functional assessment of genes influencing stomatal patterning and offers a powerful tool for dissecting gene function in a developmentally and physiologically relevant context.},
}

*Nicotiana/genetics/virology
*Gene Editing/methods
*Plant Stomata/genetics/growth & development
Genomics/methods
*Plant Viruses/genetics/physiology
Plants, Genetically Modified
Plant Proteins/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems
Gene Expression Regulation, Plant

@article {pmid41860163,
year = {2026},
author = {Wang, B and Liu, W and Li, Y and Ouyang, Q and Wang, Y and Xu, X and Li, B and Xiu, R and Zhang, X and Liu, M},
title = {Consequences of CRISPR-Cas9-Mediated Stromelysin-1 Knockout in Pancreatic Islet Microvascular Endothelial Cells.},
journal = {Journal of cellular and molecular medicine},
volume = {30},
number = {6},
pages = {e71098},
doi = {10.1111/jcmm.71098},
pmid = {41860163},
issn = {1582-4934},
support = {7252093//Beijing Municipal Natural Science Foundation/ ; 81900747//National Natural Science Foundation of China/ ; },
mesh = {*Endothelial Cells/metabolism ; Animals ; *CRISPR-Cas Systems/genetics ; *Islets of Langerhans/blood supply/metabolism/cytology ; Cell Movement/genetics ; Mice ; Cell Proliferation/genetics ; *Microvessels/metabolism/cytology ; Gene Knockout Techniques ; Glucose/toxicity ; Neovascularization, Physiologic ; },
abstract = {The integrity of the pancreatic islet microvasculature is critical for endocrine function, yet it is progressively compromised by glucotoxicity in diabetes. While matrix metalloproteinases are implicated, the role of stromelysin-1 as a potential upstream driver of endothelial dysfunction remains poorly defined. The aim of our study was to elucidate the role of stromelysin-1 in mediating glucotoxic injury to islet microvascular endothelial cells (IMECs). To this end, we employed a CRISPR/Cas9-mediated knockout of stromelysin-1 in IMECs. Cellular functions, including proliferation, migration, and angiogenesis, were assessed using IncuCyte ZOOM live-cell imaging, while endothelial barrier integrity was quantified via a 40 kDa dextran flux assay. Additionally, the secretome was profiled using a cytokine antibody array. We found that genetic ablation of stromelysin-1 conferred protection against glucotoxicity. Stromelysin-1 KO IMECs exhibited significantly enhanced proliferation, migration, and angiogenic capacity compared to wild-type controls. Furthermore, stromelysin-1 deficiency restored endothelial monolayer integrity by attenuating high-glucose-induced hyperpermeability. These functional improvements were linked to a remodelling of the secretome, characterised by decreased secretion of the pro-degradative MMP-2 and increased secretion of the anti-inflammatory cytokine IL-10 and the endogenous inhibitor TIMP-2. Overall, our findings establish stromelysin-1 as a crucial mediator of glucotoxic injury in islet microvascular endothelial cells.},
}

*Endothelial Cells/metabolism
Animals
*CRISPR-Cas Systems/genetics
*Islets of Langerhans/blood supply/metabolism/cytology
Cell Movement/genetics
Mice
Cell Proliferation/genetics
*Microvessels/metabolism/cytology
Gene Knockout Techniques
Glucose/toxicity
Neovascularization, Physiologic

@article {pmid41805859,
year = {2026},
author = {Pang, C and Yang, Q and Zhong, Y and Ye, J and Lv, Y and Xie, S and Tang, Y and Ye, X and Zhang, F and Li, C and Zhang, J and Sun, L and Ai, S and Gao, X},
title = {HAND1 controls the lineage bifurcation of trophoblast and amnion from human pluripotent stem cells.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {83},
number = {1},
pages = {},
pmid = {41805859},
issn = {1420-9071},
support = {32270836//National Natural Science Foundation of China/ ; 32570950//National Natural Science Foundation of China/ ; 82270307//National Natural Science Foundation of China/ ; 32200660//National Natural Science Foundation of China/ ; 2022A1515012664//Basic and Applied Basic Research Foundation of Guangdong Province/ ; 2023A1515010309//Basic and Applied Basic Research Foundation of Guangdong Province/ ; },
mesh = {Humans ; *Trophoblasts/cytology/metabolism ; *Cell Lineage ; Cell Differentiation ; *Amnion/cytology/metabolism ; *Pluripotent Stem Cells/cytology/metabolism ; *Basic Helix-Loop-Helix Proteins/metabolism/genetics ; Wnt Signaling Pathway ; beta Catenin/metabolism ; CRISPR-Cas Systems ; Gene Regulatory Networks ; Female ; },
abstract = {Trophoblast and amniotic lineages, representing key extra-embryonic tissues, can be differentiated from human pluripotent stem cells (hPSCs) under chemically defined conditions. However, the regulatory mechanisms coordinating the fate decision between these lineages during PSC differentiation remain incompletely understood. Leveraging CRISPR/Cas9-mediated loss-of-function screening in lineage-reporter PSCs, we identified the transcription factor HAND1 as a critical determinant controlling the bifurcation of trophoblast and amniotic lineages. Genetic ablation of HAND1 effectively abrogated the amniotic differentiation capacity of PSCs while concomitantly enhancing their trophoblast differentiation potential. Conversely, ectopic HAND1 overexpression impaired trophoblast differentiation. Notably, forced HAND1 expression in human trophoblast stem cells (TSCs) induced transcriptional reprogramming toward an amniotic fate, indicating its lineage-instructive capability. Mechanistic analyses demonstrated that HAND1 interacts with the TCFs and Wnt signaling effectors β-catenin to form a transcriptional complex that antagonistically modulates the balance between trophoblast- and amnion-associated gene regulatory networks. Collectively, our findings establish HAND1 as a master regulator orchestrating the amniotic versus trophoblast lineage choice during human PSC differentiation, thereby illuminating fundamental regulatory mechanism underlying extra-embryonic lineage specification.},
}

Humans
*Trophoblasts/cytology/metabolism
*Cell Lineage
Cell Differentiation
*Amnion/cytology/metabolism
*Pluripotent Stem Cells/cytology/metabolism
*Basic Helix-Loop-Helix Proteins/metabolism/genetics
Wnt Signaling Pathway
beta Catenin/metabolism
CRISPR-Cas Systems
Gene Regulatory Networks
Female

@article {pmid41849360,
year = {2026},
author = {Lin, H and Wang, S and Xie, Y and Cheng, C and Jin, J and Song, X and Zhang, H},
title = {Single-tube two-step RPA-CRISPR/Cas12b platform for detection of Pseudomonas aeruginosa.},
journal = {PloS one},
volume = {21},
number = {3},
pages = {e0340856},
pmid = {41849360},
issn = {1932-6203},
mesh = {*Pseudomonas aeruginosa/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism ; Bacterial Proteins/genetics ; Pseudomonas Infections/diagnosis/microbiology ; DNA, Bacterial/genetics ; },
abstract = {Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen of significant clinical and public health concern, necessitating the development of rapid and reliable detection methods. Traditional diagnostic approaches, which rely on culture-dependent techniques and biochemical identification, are often labor-intensive, time-consuming, and technically demanding. This study describes a novel single-tube, two-step, rapid detection platform that integrates recombinase polymerase amplification (RPA) with clustered regularly interspaced short palindromic repeats-associated protein Cas12b technology. Through systematic experimental optimization, the study identified an optimal RPA primer pair (F2-R1) and single-guide ribonucleic acid 553 that targets the lasR gene of P. aeruginosa, with reaction conditions optimized at 42°C and a primer concentration of 10 μM. The RPA-clustered regularly interspaced short palindromic repeats/Cas12b fluorescence detection system (RPA-Cas12b-Fluo) demonstrated a sensitivity threshold of 10 copies of deoxyribonucleic acid (DNA) per reaction and a bacterial detection limit of 50 colony-forming units (CFU) per reaction. When coupled with a lateral flow strip (RPA-Cas12b-LFS), the sensitivity was slightly reduced but remained robust, achieving detection limits of 10[2] copies and 200 CFU per reaction. Specificity assays confirmed a high discriminatory capacity for P. aeruginosa with no cross-reactivity observed against P. fluorescens, P. putida, or six common foodborne pathogens, thereby validating the specificity profile of the platform. The applicability of the method was further validated by analyzing 20 water samples, which demonstrated 100% concordance with the national standard culture method. These findings have significant implications for improving outbreak surveillance and mitigating the risk of foodborne transmission associated with P. aeruginosa.},
}

*Pseudomonas aeruginosa/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*Recombinases/metabolism
Bacterial Proteins/genetics
Pseudomonas Infections/diagnosis/microbiology
DNA, Bacterial/genetics

@article {pmid41849610,
year = {2026},
author = {Dai, Y and Abudujielili, Z and Ding, Y and Huang, W and Yin, J and Ou, L and Hu, J and Zheng, S and Li, C},
title = {Self-inactivating AAV-CRISPR at different ages enables sustained amelioration of Huntington's disease deficits in BAC226Q mice.},
journal = {Science advances},
volume = {12},
number = {12},
pages = {eaea8052},
pmid = {41849610},
issn = {2375-2548},
mesh = {Animals ; *Huntington Disease/genetics/therapy/pathology ; Mice ; *Gene Editing/methods ; *Huntingtin Protein/genetics/metabolism ; *CRISPR-Cas Systems ; Humans ; Disease Models, Animal ; *Dependovirus/genetics ; Genetic Therapy/methods ; Genetic Vectors/genetics ; },
abstract = {Huntington's disease (HD) is a monogenic autosomal dominant neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the HTT gene, yielding a gain-of-toxic-function mutant Huntingtin protein (mHTT). CRISPR-Cas9 is a potentially powerful therapeutic strategy for HD by eliminating mutant HTT (mHTT) gene. We developed a specific SaCas9 guide RNA to target human mHTT and a self-inactivating gene editing system that abolishes SaCas9 after a short transient expression for high gene editing efficiency and maximal safety to prevent off-target effects. Both conventional and the self-inactivating gene editing systems successfully eliminated mHTT gene, 60 to 90% mHTT protein and 90% of mHTT aggregation in BAC226Q mouse brains, which resulted in significant long-term rescue of neuropathology, motor deficits, weight loss, and shortened life span. These beneficial effects were observed when gene editing was applied before, at, and well after the onset of pathological and behavioral abnormalities. These proof-of-concept data demonstrate that gene editing can be a highly effective therapeutic approach for HD.},
}

Animals
*Huntington Disease/genetics/therapy/pathology
Mice
*Gene Editing/methods
*Huntingtin Protein/genetics/metabolism
*CRISPR-Cas Systems
Humans
Disease Models, Animal
*Dependovirus/genetics
Genetic Therapy/methods
Genetic Vectors/genetics

@article {pmid41849748,
year = {2026},
author = {Wang, Q and Chen, D and Berr, A and Shen, WH},
title = {CRISPR gene editing of AtRING1 unravels a critical role of RAWUL domain in PRC1 repression of transcription.},
journal = {The Plant journal : for cell and molecular biology},
volume = {125},
number = {6},
pages = {e70794},
pmid = {41849748},
issn = {1365-313X},
support = {ANR-12-BSV2-0013-02//Agence National de la Recherche/ ; //Centre National de la Recherche Scientifique/ ; },
mesh = {*Arabidopsis Proteins/genetics/metabolism ; *Arabidopsis/genetics/metabolism ; Gene Editing ; *Polycomb Repressive Complex 1/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; Histones/metabolism ; Protein Domains ; Glucosyltransferases ; },
abstract = {Polycomb Group (PcG) proteins, including members of Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2), regulate many key developmental processes through transcriptional gene repression. While the molecular mechanisms of PRC2 and its histone methyltransferase involved in depositing histone 3 lysine 27 trimethylation (H3K27me3) are well understood, the components and E3 ubiquitin ligase functions of PRC1 in plants remain largely elusive. In Arabidopsis, AtRING1 is a key PRC1 component, containing an N-terminal RING-finger domain and a C-terminal RAWUL domain. Previous studies have relied on T-DNA insertion mutants in the investigation of AtRING1 function. By editing AtRING1A using CRISPR/Cas9 technology in the atring1b-1 background, here we have generated and characterized one N-terminal stop mutant atring1[ko] and two C-terminal deletion mutants atring1[▵C-terminal] lacking the RAWUL domain. We show evidence that atring1[ko] represents the strongest loss-of-function mutant, exhibiting embryonic callus-like structures, demonstrating the essential role of AtRING1 in cell differentiation. Remarkably, the atring1[▵C-terminal] mutants exhibit mild developmental defects, suggesting that the RING domain alone retains partial function, while the RAWUL domain fine-tunes PRC1 activity. Our molecular analyses support a model in which AtRING1/PRC1-mediated H2A monoubiquitination (H2Aub1) often precedes PRC2-mediated H3K27me3 deposition at some target loci. Strikingly, the RAWUL domain is required for efficient H2Aub1 enrichment and influences H3K27me3 deposition in a locus-specific manner. Taken together, our study provides new insights into the molecular mechanism underlying PRC1 E3 ligase activity, supporting that PRC1 function facilitates PRC2 activity in epigenetic gene silencing.},
}

*Arabidopsis Proteins/genetics/metabolism
*Arabidopsis/genetics/metabolism
Gene Editing
*Polycomb Repressive Complex 1/genetics/metabolism
CRISPR-Cas Systems/genetics
Gene Expression Regulation, Plant
Histones/metabolism
Protein Domains
Glucosyltransferases

@article {pmid41850286,
year = {2026},
author = {Jin, S and Zhu, Z and Li, Y and Zhang, S and Liu, Y and Li, D and Li, Y and Luo, Y and Cheng, Z and Zhao, KT and Gao, Q and Yang, G and Li, H and Liang, R and Zhang, R and Qiu, JL and Zhang, YE and Gogo Liu, JJ and Gao, C},
title = {Functional RNA splitting drove the evolutionary emergence of type V CRISPR-Cas systems from transposons.},
journal = {Cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.cell.2026.03.014},
pmid = {41850286},
issn = {1097-4172},
}

@article {pmid41851990,
year = {2026},
author = {Guo, Y and Li, J and Haque, A and Chen, J and Yang, X and Wang, Y and Yao, L and Zhuo, C and Wang, J and He, N and Lin, Y and Xiao, S and Liu, B and Zhuo, C},
title = {Subtypes of Type I-E CRISPR-Cas Systems Distribution in Human Escherichia coli Isolates from China.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {25731599261430828},
doi = {10.1177/25731599261430828},
pmid = {41851990},
issn = {2573-1602},
abstract = {The correlation between CRISPR-Cas systems and plasmid-mediated bacterial antibiotic resistance is increasingly growing attention. However, currently no reports exist on the relationship between the CRISPR-Cas systems and the carriage of blaNDM or plasmids in E.coli. Here, molecular characterization and phylogenetic analysis of 639 E.coli isolated from humans in China were carried out. Depending on similarity in sequence, the type I-E CRISPR-Cas systems in E.coli can be grouped into two distinct clades, which we refer to for descriptive purposes within this study as the type I-E-S1 and I-E-S2, whereas the type I-E-S2 CRISPR-Cas system is further divided into I-E-S2a and I-E-S2b systems based on the presence of cas8e and cas11. ST167 (phylogroup A) and ST410 (phylogroup C) E. coli were observed bearing the type I-E-S1 and I-E-S2b systems, respectively. Compared with strains carrying the I-E-S1 type CRISPR-Cas system, the blaNDM carrying rate, the positive rate of IncX3 plasmid, and the positive rate of IncF plasmid of strains with the I-E-S2a type CRISPR-Cas system were evidently lower (p < 0.05); the blaNDM carrying rate and the positive rate of IncF plasmid of strains with the I-E-S2b type CRISPR-Cas system were evidently higher (p < 0.05). The blaNDM positive rate and IncF plasmid positive rate of strains carrying the I-E-S2a type CRISPR-Cas system were significantly lower than those of strains carrying the I-E-S2b type CRISPR-Cas system (p < 0.001). It proves that the I-E-S1, I-E-S2a, and I-E-S2b type CRISPR-Cas systems are beneficial for spreading blaNDM and IncX3 plasmids. We found significant differences in the cas gene sequences of the I-E-S1 and I-E-S2 type CRISPR loci. The type I-E CRISPR-Cas systems in E. coli isolated from Chinese sources are classified further for the first time, revealing their high correlation with blaNDM, phylogenetic groups, and multilocus sequence typing. This work paves the way for a deeper understanding of the role that CRISPR-Cas systems play in the rise of resistant E.coli ST167 and ST410.},
}

@article {pmid41854513,
year = {2026},
author = {Han, JH and Kang, YJ and Lee, SY and Jin, HB and Lee, CS and Park, HH},
title = {AcrVA3 Is a Double Strand DNA-Cleaving Anti-CRISPR That Indirectly Inhibits Cas12.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {6},
pages = {e71705},
pmid = {41854513},
issn = {1530-6860},
support = {RS-2025-02316334//National Research Foundation of Korea (NRF)/ ; RS-2026-25470081//National Research Foundation of Korea (NRF)/ ; },
mesh = {*CRISPR-Cas Systems ; *DNA/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry/antagonists & inhibitors ; *Viral Proteins/metabolism/chemistry/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Cleavage ; Bacteriophages/genetics/metabolism ; },
abstract = {CRISPR-Cas12 systems protect bacteria from foreign DNA, but are themselves targeted by anti-CRISPR (Acr) proteins evolved by phages. Among the eight known AcrV proteins that inhibit Cas12-based type V CRISPR-Cas systems, the mechanisms of all except AcrVA3 have been structurally and biochemically characterized. Here, we report the high-resolution structure of AcrVA3 and examine its inhibitory function in vitro. Unexpectedly, AcrVA3 does not directly work on Cas12. Instead, it exhibits double-stranded DNA (dsDNA) cleavage activity, suggesting an indirect mechanism of CRISPR inhibition through DNA degradation. This unique DNA-centric strategy contrasts with previously known Acr mechanisms and expands our understanding of how mobile genetic elements evade CRISPR immunity.},
}

*CRISPR-Cas Systems
*DNA/metabolism/genetics
*CRISPR-Associated Proteins/metabolism/genetics/chemistry/antagonists & inhibitors
*Viral Proteins/metabolism/chemistry/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
DNA Cleavage
Bacteriophages/genetics/metabolism

@article {pmid41549285,
year = {2026},
author = {Peng, X and Huang, J and Lv, S and Zhang, J and Zhang, L and Liu, H and Liu, Y and Qiao, ZA and Wei, F and Su, BL},
title = {TriCON: A Carbon-Based Triple-Modal Nanoplatform for Pancreatic Cancer Therapy.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {17},
pages = {e12978},
doi = {10.1002/advs.202512978},
pmid = {41549285},
issn = {2198-3844},
support = {81972273//National Natural Science Foundation of China/ ; 82273276//National Natural Science Foundation of China/ ; 82473314//National Natural Science Foundation of China/ ; 20200201546JC//Natural Science Foundation of Jilin Province/ ; JJKH20211208KJ//Science and Technology Project of Jilin Provincial Education Science Department/ ; 2018J039//Health and Innovation Technology Project of Jilin Province/ ; 2024KC142//2025 Doctoral Research Innovation Enhancement Project of Jilin Province/ ; },
mesh = {*Pancreatic Neoplasms/therapy/genetics/immunology/drug therapy ; Animals ; Mice ; Humans ; Doxorubicin/pharmacology/administration & dosage ; *Immunotherapy/methods ; Tumor Microenvironment/immunology/drug effects ; Cell Line, Tumor ; Gene Editing/methods ; CRISPR-Cas Systems ; *Carbon/chemistry ; *Carcinoma, Pancreatic Ductal/therapy ; },
abstract = {Pancreatic cancer, recognized for its high malignancy and tumor immunosuppressive microenvironment, has been refractory to conventional therapeutic modalities, necessitating the exploration of novel treatment strategies. Among these, reprogramming of the tumor immunosuppressive microenvironment is a promising strategy to enhance the efficacy of tumor immunotherapy. The CRISPR/Cas9 system-based gene editing further offers a viable approach for precise regulation of endogenous gene expression associated with tumor immunosuppression. Current delivery vectors face a trilemma between biosafety profiles, expansion capacity, and targeting accuracy. To this end, we developed a triple-modality therapeutic platform, termed TriCON (Triple Convergent Oncology Nanotherapy), characterized by three core mechanistic attributes: spatiotemporal convergence, stimuli-responsive controllability, and tumor-microenvironment modulatory conductivity. This orchestrated combination of rationally designed gene editing (targeting poliovirus receptor), nano-encapsulated doxorubicin (DOX) chemotherapy, and checkpoint blockade immunotherapy demonstrated enhanced synergistic antitumor activity in pancreatic ductal adenocarcinoma (PDAC) models, achieving tumor regression through enhanced chemotherapy, immunogenic cell death induction, and natural killer (NK) cells activation. The platform achieved superior in vivo gene editing (14.2% PVR editing efficiency) via optimized endosomal escape and CRISPR system release. This triaxial approach establishes a programmable nanotherapeutic paradigm that synergizes gene editing precision with chemo-immunotherapy, offering a novel framework for PDAC treatment.},
}

*Pancreatic Neoplasms/therapy/genetics/immunology/drug therapy
Animals
Mice
Humans
Doxorubicin/pharmacology/administration & dosage
*Immunotherapy/methods
Tumor Microenvironment/immunology/drug effects
Cell Line, Tumor
Gene Editing/methods
CRISPR-Cas Systems
*Carbon/chemistry
*Carcinoma, Pancreatic Ductal/therapy

@article {pmid41560288,
year = {2026},
author = {Zou, R and Lu, X and Liu, Y and Yang, P and Sun, S and Liu, Y and Mo, Y and Zhu, G and Lee, JS and Guo, Y},
title = {Quantitative Proteomics and CRISPR/Cas9 Editing Reveal UPR-Mediated Control of Immunoglobulin Homeostasis in Hybridomas.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {17},
pages = {e14140},
doi = {10.1002/advs.202514140},
pmid = {41560288},
issn = {2198-3844},
support = {32302389//National Natural Science Foundation of China/ ; 32272574//National Natural Science Foundation of China/ ; 2023M743075//China Postdoctoral Science Foundation/ ; 2024T170787//China Postdoctoral Science Foundation/ ; RS-2024-00397714//National Research Foundation of Korea/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Unfolded Protein Response/genetics/immunology ; Animals ; *Proteomics/methods ; Gene Editing/methods ; Mice ; *Homeostasis/genetics/immunology ; *Hybridomas/metabolism/immunology ; *Immunoglobulins/metabolism/genetics ; Antibodies, Monoclonal/genetics ; },
abstract = {Despite their tremendous economic value, monoclonal antibodies are often compromised by the loss of immunoglobulin (Ig) chains, which disrupts antibody homeostasis and quality control. Through subclone screening and characterization, we identified that the loss of Ig production impaired the recognition ability of hapten-specific hybridomas. Proteomic analysis further highlighted the critical role of the unfolded protein response (UPR) pathway in regulating aberrant Ig chain production. Using CRISPR/Cas9-mediated knockout and rescue experiments, we revealed the importance of the UPR pathway in facilitating hybridoma antibody production by targeting Xbp1s, an active transcription factor downstream of UPR signaling. With CRISPR/HDR, we inserted a fluorescent mGFP tag into the endogenous Hspa5 gene (encoding BiP, the master regulator of the UPR pathway), enabling in situ and real-time monitoring of UPR activation. A strong negative correlation (R[2] = 0.86) was observed between intracellular mGFP signals and IgG levels in the engineered system, indicating a close relationship between UPR activation and Ig production. Fluorescence-activated cell sorting of high-mGFP populations identified two dysfunctional subclones that failed to secret Ig, validating the system's effectiveness in tracing Ig homeostasis. In summary, this study provides new insights into UPR-mediated regulation of Ig synthesis and offers a novel UPR-based reporter system for monitoring antibody stability.},
}

*CRISPR-Cas Systems/genetics
*Unfolded Protein Response/genetics/immunology
Animals
*Proteomics/methods
Gene Editing/methods
Mice
*Homeostasis/genetics/immunology
*Hybridomas/metabolism/immunology
*Immunoglobulins/metabolism/genetics
Antibodies, Monoclonal/genetics

@article {pmid41841497,
year = {2026},
author = {Nusawardhana, A and Hale, A and Straka, J and Nicolae, CM and Moldovan, GL},
title = {Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41841497},
issn = {1362-4962},
support = {R01ES026184/GF/NIH HHS/United States ; R01GM134681/GF/NIH HHS/United States ; R01CA244417/GF/NIH HHS/United States ; F31CA294862/GF/NIH HHS/United States ; 4D01_2024_1002//Four Diamonds Transformative Patient-Oriented Cancer Research/ ; /NH/NIH HHS/United States ; //Penn State University/ ; },
mesh = {*Exodeoxyribonucleases/genetics/metabolism ; *DNA Damage ; Cisplatin/pharmacology ; DNA Repair/genetics ; Humans ; *R-Loop Structures/genetics ; CRISPR-Cas Systems ; *DNA Repair Enzymes/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Synthetic Lethal Mutations ; },
abstract = {DNA repair is critical for cellular homeostasis under both normal conditions as well as in response to genotoxic agents such as chemotherapeutics. EXO1 is a 5'-3' exonuclease with multiple roles in DNA biology. To better understand these roles, we employed CRISPR loss-of-function genome-wide screening to identify genes required for proliferation and cisplatin sensitivity in EXO1-deficient cells. We uncovered differential regulators of cisplatin sensitivity between wildtype (WT) and EXO1-deficient cells. By analyzing the genetic networks that these regulators belong to, we found that DNA repair was the main biological process suppressing cisplatin sensitivity in WT cells, but this was not the case in EXO1-deficient cells, indicating that EXO1 is critical for the repair of cisplatin-induced DNA damage. Moreover, synthetic lethality screens identified a genetic interaction between EXO1 and the histone chaperone CAF-1. Mechanistically, we show that EXO1 and CAF-1 are independently recruited to R-loops and participate in separate, synergistic pathway of R-loop suppression. Even in the absence of DNA damage treatment, concomitant loss of EXO1 and CAF-1 causes R-loop accumulation and increased R-loop-associated DNA damage. Our work sheds light on the critical roles of EXO1 in genomic stability.},
}

*Exodeoxyribonucleases/genetics/metabolism
*DNA Damage
Cisplatin/pharmacology
DNA Repair/genetics
Humans
*R-Loop Structures/genetics
CRISPR-Cas Systems
*DNA Repair Enzymes/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats
Synthetic Lethal Mutations

@article {pmid41843435,
year = {2026},
author = {Wang, W and Lin, S and Luo, Y and Shao, C and Shi, Q and Shao, J and Chen, Y and Hu, H and Wan, S and Song, X and Jin, D and Jin, Y},
title = {Genomic and phenotypic insight into Clostridioides difficile RT027 isolates from China reveals diverse virulence associated with clinical symptoms.},
journal = {Emerging microbes & infections},
volume = {15},
number = {1},
pages = {2637287},
pmid = {41843435},
issn = {2222-1751},
mesh = {*Clostridioides difficile/genetics/pathogenicity/isolation & purification/classification/drug effects ; *Clostridium Infections/microbiology/epidemiology ; China/epidemiology ; Humans ; Virulence/genetics ; *Genome, Bacterial ; Phylogeny ; Phenotype ; Anti-Bacterial Agents/pharmacology ; Whole Genome Sequencing ; Bacterial Proteins/genetics ; Bacterial Toxins/genetics ; Virulence Factors/genetics ; Genomics ; Ribotyping ; },
abstract = {Clostridioides difficile (C. difficile) ribotype 027 (RT027) has caused severe outbreaks in North America and Europe over the past 20 years. However, RT027 infections are rare in Asia, particularly in China, with limited severe cases. To clarify its molecular and phenotypic features, we investigated 11 RT027 isolates collected from Shandong, China. Whole-genome sequencing, comparative transcriptomics, CRISPR-Cas analysis, and pan-genome profiling were combined with phenotypic assays of toxin production, sporulation, antimicrobial resistance, and motility. Evolutionary analysis demonstrated that isolates from China clearly diverged from the FQR2 lineage and were phylogenetically closer to FQR1, forming a distinct sublineage. All isolates from Shandong, China encoded a complete tcd and cdt locus and harboured rifamycin and aminoglycoside resistance genes. However, transcriptomic profiling demonstrated significantly reduced expression of binary toxin genes (cdtAB, cdtR), decreased spo0A transcription, and downregulation of flagellar pathways. Phenotypic assays confirmed impaired sporulation, motility and cytotoxicity, while TcdB production and adhesion was comparable to reference strains. CRISPR-Cas elements were conserved but showed reduced transcriptional activity, suggesting diminished host-pathogen interactions. The pan-genome revealed high genomic conservation, consistent with limited functional diversity. Together, these data indicate that RT027 isolates circulating in China possess unique evolutionary trajectories and attenuated virulence traits, helping to explain the rarity of severe RT027 infections in Asia. These findings provide important insights into the regional epidemiology of C. difficile and inform strategies for diagnosis, treatment, and prevention.},
}

*Clostridioides difficile/genetics/pathogenicity/isolation & purification/classification/drug effects
*Clostridium Infections/microbiology/epidemiology
China/epidemiology
Humans
Virulence/genetics
*Genome, Bacterial
Phylogeny
Phenotype
Anti-Bacterial Agents/pharmacology
Whole Genome Sequencing
Bacterial Proteins/genetics
Bacterial Toxins/genetics
Virulence Factors/genetics
Genomics
Ribotyping

@article {pmid41843552,
year = {2026},
author = {Leix, K and Serrano-Zayas, C and Vyas, HS and Graham, SE and Emmer, BT},
title = {Functional interrogation of candidate cis-regulatory elements at the LDLR locus.},
journal = {PLoS genetics},
volume = {22},
number = {3},
pages = {e1012082},
doi = {10.1371/journal.pgen.1012082},
pmid = {41843552},
issn = {1553-7404},
mesh = {*Receptors, LDL/genetics/metabolism ; Humans ; Enhancer Elements, Genetic/genetics ; Gene Expression Regulation ; Promoter Regions, Genetic/genetics ; Genome-Wide Association Study ; *Regulatory Sequences, Nucleic Acid/genetics ; Introns/genetics ; CRISPR-Cas Systems ; Cholesterol, LDL/genetics ; },
abstract = {Regulation of LDLR gene expression plays an important role in the development of atherosclerotic diseases including heart attack and stroke. Although LDLR regulation by sterol response elements has been well characterized, the functional significance of other noncoding regions at the LDLR locus remains poorly defined. In this study, we developed and applied a high throughput CRISPR screen to test the functional importance of candidate LDLR cis-regulatory elements (CREs) in their native genomic context. In total, we found 25 discrete regions to exhibit a significant impact on LDLR expression. For one of these regions with particularly strong activity in the first intron, we validated the presence of an enhancer by confirming that its disruption reduced endogenous LDLR expression while its insertion upstream of a minimal promoter augmented reporter gene expression. We then applied a massively parallel reporter assay to fine map enhancer activity within this region to a 129 bp interval that is highly conserved among vertebrates, exhibits biochemical hallmarks of enhancer activity, is enriched for transcription factor binding motifs, and contains a common genetic variant (rs57217136) that has been associated with human LDL cholesterol levels by genome-wide association studies. Overall, these findings demonstrate the power of CRISPR screening to interrogate candidate CREs and clarify the functional landscape of noncoding sequences at the LDLR locus.},
}

*Receptors, LDL/genetics/metabolism
Humans
Enhancer Elements, Genetic/genetics
Gene Expression Regulation
Promoter Regions, Genetic/genetics
Genome-Wide Association Study
*Regulatory Sequences, Nucleic Acid/genetics
Introns/genetics
CRISPR-Cas Systems
Cholesterol, LDL/genetics

@article {pmid41845915,
year = {2026},
author = {Liu, S and Geng, S and Dreisigacker, S},
title = {Genomics in wheat improvement: Progress and perspectives.},
journal = {The plant genome},
volume = {19},
number = {1},
pages = {e70176},
pmid = {41845915},
issn = {1940-3372},
support = {32201878//National Natural Science Foundation of China/ ; 2024SFGC0404//Key R&D Program of Shandong Province, China/ ; 2023ZD0406802//Biological Breeding-National Science and Technology Major Project/ ; 202403250025//China Scholarship Council (CSC)/ ; },
mesh = {*Triticum/genetics ; *Genomics ; *Genome, Plant ; Plant Breeding ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {Bread wheat (Triticum aestivum) remains a major source of food and calories globally, yet its vast genome, polyploidy, and high number of repetitive sequences make genomic research challenging in this crop. In this review, we discuss the progress and future perspectives of genome research in wheat. Current efforts focus on the establishment of genome assemblies, advances in functional genomics, advances in epigenetics, translational genetics, and CRISPR-Cas9 genome editing offers a powerful tool for site-specific genome editing for wheat improvement and functional genetic analysis. These approaches have elucidated the genetic basis of many important agronomic traits such as grain yield, biotic and abiotic stress, and wheat quality. Future aims are expected to expand to pan-genomics, the mechanism of wheat domestication, funnel the outputs of functional genomics for deployment in wheat breeding, multi-omics studies facilitate genetic dissection, and the era of big data: creation, integration and utilization, and artificial intelligence breeding.},
}

*Triticum/genetics
*Genomics
*Genome, Plant
Plant Breeding
Gene Editing
CRISPR-Cas Systems

@article {pmid41847198,
year = {2026},
author = {D, J and Mulavagili, S and Vijayasimha, M},
title = {Commentary: CRISPR-Cas systems against carbapenem resistance-from proof-of-concept to clinical translation.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1773181},
pmid = {41847198},
issn = {1664-302X},
}

@article {pmid41731282,
year = {2026},
author = {du Rand, A and Buttle, B and Sheppard, H},
title = {Developing CRISPR-Based Therapies for Epidermolysis Bullosa: A Comprehensive Review of Current Strategies.},
journal = {Drugs},
volume = {86},
number = {4},
pages = {465-483},
pmid = {41731282},
issn = {1179-1950},
mesh = {Humans ; *Gene Editing/methods ; *Epidermolysis Bullosa/therapy/genetics ; *Genetic Therapy/methods ; *CRISPR-Cas Systems/genetics ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Currently, there is no permanent treatment for the group of severe monogenic fragile skin conditions epidermolysis bullosa (EB). The recent US Food and Drug Administration (FDA)-approved in vivo gene replacement therapy beremagene geperpavec (Vyjuvek[®]) provides a promising solution, but it requires ongoing application and is not applicable to all forms of EB. Targeted gene editing approaches directly addressing pathogenic mutations hold great promise for the development of durable personalized therapies. Here, we comprehensively describe the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) gene editing landscape for EB, critically review the advantages and limitations of emerging therapeutic strategies, and present some future perspectives. We find that the widespread application of Cas9 nuclease is currently hindered by off-target genotoxicity, which can be mitigated using Cas9 nickases. Further, new tools including prime editing have recently emerged and hold significant potential for EB gene therapy. Ongoing developments in gene editing technologies focused on improving safety and editing precision offer significant promise for the future clinical translation of potentially lifelong treatments for people with EB.},
}

Humans
*Gene Editing/methods
*Epidermolysis Bullosa/therapy/genetics
*Genetic Therapy/methods
*CRISPR-Cas Systems/genetics
Animals
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41836275,
year = {2026},
author = {Otero, CP and Qi, LS},
title = {Rewriting the epigenome: CRISPR tools for biological discovery and therapeutics.},
journal = {Current opinion in biomedical engineering},
volume = {38},
number = {},
pages = {},
pmid = {41836275},
issn = {2468-4511},
support = {R21 AG077193/AG/NIA NIH HHS/United States ; },
abstract = {The eukaryotic epigenome plays a central role in regulating gene expression, cellular identity, and development through dynamic, multilayered biochemical modifications to DNA, histones, and chromatin architecture. Disruption of these regulatory mechanisms contributes to a wide range of human diseases, including cancer, neurodegenerative disorders, and immunological conditions. Targeted epigenome editing offers promising discovery and therapeutic strategies by enabling the correction of aberrant epigenetic states without the need for permanent changes to the DNA sequence. The catalytically inactive CRISPR-Cas (dCas) molecule fused to epigenetic effector domains has emerged as a versatile platform for programmable, locus-specific modulation of chromatin states. These CRISPR-based epigenetic editors can deposit or remove desired epigenetic marks and alter three-dimensional genome organization to fine-tune gene expression with high specificity. Recent developments have expanded the CRISPR epigenome editing toolbox by introducing new effector domains, improving multiplexing capabilities, and enabling large-scale genetic screening, leading to novel insights into the functional genomics across various cellular contexts. However, clinical translation remains challenged by inefficient delivery and suboptimal editing efficacy in vivo. This review highlights recent advances in CRISPR-based epigenetic editing, with a focus on applications in primary cells, new tool development, and the translational potential of epigenome modulation for safe, durable, and precise therapies.},
}

@article {pmid41836767,
year = {2026},
author = {Luo, L and Yang, Y and Zhang, Y and Mao, G},
title = {Advances in nanozyme-assisted CRISPR diagnostic technology.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {14},
number = {},
pages = {1796403},
pmid = {41836767},
issn = {2296-4185},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) system has significant potential in biological diagnostics because of its precise nucleic acid identification abilities. Traditional CRISPR diagnostics, however, have limitations such as insufficient signal output, dependence on exogenous enzymes, and high equipment demands. Nanozymes, as nanomaterials with enzyme-mimetic catalytic activity, integrate the catalytic efficiency of natural enzymes with the stability and modifiability of nanomaterials, providing a viable resolution to the limitations in CRISPR diagnostics. This article comprehensively evaluates the advancements in nanozyme-enhanced CRISPR diagnostic technologies. Furthermore, it delineates the fundamental attributes of the CRISPR diagnostic system and nanozymes, as well as the necessity of their integration. Moreover, the coupling mechanisms between the CRISPR/Cas system and nanozymes, including the regulation of nanozyme catalytic activity by Cas protein function and CRISPR signal amplification facilitated by nanozymes, were also comprehensively evaluated. The application of this technique in detecting nucleic acid and non-nucleic acid targets was assessed. Further, this study discusses the current limitations of this technology, such as complex separation of heterogeneous systems, laborious reaction protocols, and slow detection rates. The future advancements, such as the establishment of homogenous systems, the creation of integrated devices, and the utilization of single-atom nanozymes, have also been discussed in this review. The results of this study will provide references for the comprehensive integration of nanozymes and CRISPR technology, together with their diagnostic applications.},
}

@article {pmid41840824,
year = {2026},
author = {Thalib, HI and Khan, S and Hanin Shaikh, A and Alawi, KM and Alabdrabalrasol, ZH and Mehveen, S and Haidar, S and ElSayed Hassan, F and Shaik, NA},
title = {CRISPR Gene Editing for Nucleotide Repeat Expansion Disorders: A Systematic Review of Preclinical and Clinical Evidence.},
journal = {Genetic testing and molecular biomarkers},
volume = {30},
number = {3},
pages = {71-80},
doi = {10.1177/19450265261434900},
pmid = {41840824},
issn = {1945-0257},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Myotonic Dystrophy/genetics/therapy ; Genetic Therapy/methods ; *Trinucleotide Repeat Expansion/genetics ; Muscular Dystrophy, Duchenne/genetics/therapy ; Huntington Disease/genetics/therapy ; Animals ; },
abstract = {BACKGROUND: Incurable hereditary diseases such as Duchenne muscular dystrophy (DMD), Huntington's disease (HD), and myotonic dystrophy type 1 (DM1) fall into the nucleotide repeat expansion disorder (NRED) category. The discovery of CRISPR-Cas genome editing has paved the way toward hopeful strategies for accurate DNA-level repair. This systematic review presents preclinical data on the efficacy, molecular effects, and limitations of CRISPR-based treatments for NREDs.

METHODS: As per Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2020 guidelines, systematic PubMed, Scopus, and Embase searches up to June 2025 identified studies that evaluated CRISPR-Cas systems in human-derived in vitro models of NREDs. Methodological Index for Non-Randomized Studies tool was used to score eligible studies by methodological quality. CRISPR platforms, delivery systems, gene targets, molecular endpoints, and functional rescue data were extracted and synthesized descriptively.

RESULTS: Twenty-four out of 6510 records screened were included. They employed most of them to target specific DMD (n = 9), HD (n = 6), and DM1 (n = 3) with patient-derived induced pluripotent stem cells or differentiated myogenic/neuronal cells. Streptococcus pyogenes CRISPR-associated protein 9 as a nuclease was the most frequently used, although engineered Cas9 enzymes and dCas9 fusion proteins were also utilized to control transcription. Delivery was achieved through viral vectors (adeno-associated virus, lentivirus) and nonviral routes (plasmid, lipofection, electroporation). Uniform genomic editing, transcript rescue, and protein restoration were seen in CRISPR-mediated editing studies, and functional restoration was demonstrated for splicing correction and dystrophin restoration. Methodological flaws such as the absence of blinding, failure to follow up, and lack of full reporting of off-target effects limited robustness.

CONCLUSION: CRISPR-Cas systems exhibit reproducible molecular and functional correction in NRED models with their translational potential. Methodological strength, whole safety profiling, and in vivo verification remain a necessity, however, before clinical translation.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Myotonic Dystrophy/genetics/therapy
Genetic Therapy/methods
*Trinucleotide Repeat Expansion/genetics
Muscular Dystrophy, Duchenne/genetics/therapy
Huntington Disease/genetics/therapy
Animals

@article {pmid41841492,
year = {2026},
author = {Li, Z and Kong, J and Wu, W and Duan, Y and Zhu, Z and Hua, C and Yan, P and Cao, C and Cao, X and Xiao, Y and Lu, M and Chen, M},
title = {Structural and functional insights into the adenosine deaminase of the type III-B CRISPR-Cas system.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41841492},
issn = {1362-4962},
support = {2023YFC3402300//National Key Research and Development Program of China/ ; 2021ZD0203400//National Key Research and Development Program of China/ ; 82473977//National Natural Science Foundation of China/ ; 32271330//National Natural Science Foundation of China/ ; 32471316//National Natural Science Foundation of China/ ; 82304614//National Natural Science Foundation of China/ ; 82373892//National Natural Science Foundation of China/ ; SBK2024010634//Basic Research Program of Jiangsu/ ; BK20250200//Basic Research Program of Jiangsu/ ; //State Key Laboratory of Natural Medicines/ ; SKLNMZZ2024JS31//China Pharmaceutical University/ ; 2632025TD02//Fundamental Research Funds for the Central Universities/ ; 2023YFC3402300//National Key Research and Development Program of China/ ; 2021ZD0203400//National Key Research and Development Program of China/ ; 82473977//National Natural Science Foundation of China/ ; 32271330//National Natural Science Foundation of China/ ; 32471316//National Natural Science Foundation of China/ ; 82304614//National Natural Science Foundation of China/ ; SBK2024010634//Basic Research Program of Jiangsu/ ; BK20250200//Basic Research Program of Jiangsu/ ; //Project Program of State Key Laboratory of Natural Medicines/ ; SKLNMZZ2024JS31//China Pharmaceutical University/ ; 2632025TD02//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Adenosine Deaminase/chemistry/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; Adenine Nucleotides/metabolism ; Adenosine Triphosphate/metabolism ; },
abstract = {Type III CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) systems confer antiviral immunity via cyclic oligoadenylate (cOA) signaling. Here, we elucidate a cooperative bacterial defense strategy involving two cOA-activated CRISPR-associated Rossmann fold (CARF)-containing effectors, adenosine deaminase CAAD and ribonuclease Csx1, in Thermoanaerobaculum aquaticum. Genomic analyses indicate widespread co-occurrence of CRISPR-associated adenosine deaminase (CAAD) with ancillary CARF-containing effectors in type III CRISPR systems, suggesting that multiple CARF-containing proteins may contribute to a coordinated cOA-dependent defense. Biochemical and structural studies reveal the intrinsic dynamics of CAAD hexamer, and demonstrate that cA4/cA6 binding stabilizes CAAD hexamers, triggering metal-ion-dependent conversion of ATP into inosine triphosphate. Concurrently, the downstream Csx1 is exclusively activated by cA4 to cleave single-stranded RNA. Strikingly, we found that both effectors are capable of degrading cA4, suggesting that this CAAD-Csx1 pair may be cross-regulated and achieve immunity through a dual-targeting mechanism: in response to infection, Csx1 degrades viral RNA while CAAD disrupts nucleotide metabolism via ATP deamination, which can be relieved via cA4 degradation when infection has been eliminated. This study proposes an enhanced defense mechanism through coordinated activation and regulation of multiple CRISPR effectors by a single signaling molecule, unveiling unprecedented complexity in CRISPR immunoregulation.},
}

*Adenosine Deaminase/chemistry/metabolism/genetics
*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/metabolism/chemistry/genetics
*Bacterial Proteins/chemistry/metabolism/genetics
Adenine Nucleotides/metabolism
Adenosine Triphosphate/metabolism

@article {pmid41530266,
year = {2026},
author = {Freedman, BS and Bulte, JWM and Conklin, BR and Judge, LM and Dwinell, MR and Geurts, AM and Sitton, MJ and Mahajan, V and Kiani, S and Gersbach, CA and Ebrahimkhani, MR and Kelly, JJ and Ronald, JA and Morizane, R and Gupta, N and Shakeri-Zadeh, A and Vo, N and Saha, K and Saxena, S and Gamm, DM and Sinha, D and Tarantal, AF and Vandsburger, M and Matsubara, A and Fu, H and Tsai, SQ and , and , },
title = {Monitoring biological effects of somatic cell genome editing.},
journal = {Nature reviews. Genetics},
volume = {27},
number = {4},
pages = {323-342},
pmid = {41530266},
issn = {1471-0064},
support = {UH2 EB028904/EB/NIBIB NIH HHS/United States ; U01 EB028892/EB/NIBIB NIH HHS/United States ; R21 DK129909/DK/NIDDK NIH HHS/United States ; U01 AI176460/AI/NIAID NIH HHS/United States ; S10 RR025063/RR/NCRR NIH HHS/United States ; U19 NS132296/NS/NINDS NIH HHS/United States ; R35 GM119644/GM/NIGMS NIH HHS/United States ; S10 OD018102/OD/NIH HHS/United States ; DP2 DK133821/DK/NIDDK NIH HHS/United States ; U01 DK127587/DK/NIDDK NIH HHS/United States ; S10 OD016261/OD/NIH HHS/United States ; U01 EB028899/EB/NIBIB NIH HHS/United States ; UG3 NS111688/NS/NINDS NIH HHS/United States ; UH3 EB028904/EB/NIBIB NIH HHS/United States ; U01 HL145792/HL/NHLBI NIH HHS/United States ; S10 OD028713/OD/NIH HHS/United States ; R01 DK141567/DK/NIDDK NIH HHS/United States ; U01 DK127553/DK/NIDDK NIH HHS/United States ; U42 OD027094/OD/NIH HHS/United States ; UH3 EB028910/EB/NIBIB NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; Genetic Therapy/methods ; },
abstract = {CRISPR-based genome editing therapeutics are entering the clinic, offering transformative potential but also presenting potential risks. Preclinical-to-clinical toolkits are needed to assess the safety and efficacy of these new therapies and accelerate progress. Emerging technologies to monitor the biological effects of genome editors cover a range of biological scales, from the direct measurement of editing outcomes in DNA, to human microphysiological systems, and non-invasive in vivo imaging. Measurements of on-target and off-target editing outcomes, including sequences unique to humans, provide essential benchmarks to understand functional responses. Microphysiological systems, including organoids and organs-on-chips, enable phenotypic evaluations of editing strategies in varied organ lineages and disease states. Non-invasive imaging modalities can track the biodistribution and activities of genome editors and edited cells in vivo. Collectively, these technologies provide complementary insights across different scales, from the single nucleotide to the whole organism, bridging preclinical therapeutics development with clinical trials.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems/genetics
Animals
Genetic Therapy/methods

@article {pmid41673016,
year = {2026},
author = {Theerapanon, T and Intarak, N and Rattanapornsompong, K and Thaweesapphithak, S and Sriwattanapong, K and Prommanee, S and Kulvitit, S and Skrinjaric, T and Samaranayake, L and Pongpanich, M and Yeetong, P and Chaivoravitsakul, N and Mehl, NS and Assawapitaksakul, A and Srichomthong, C and Chetruengchai, W and Porntaveetus, T and Shotelersuk, V},
title = {A missense variant in ASCL5 leads to lobodontia.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41673016},
issn = {2041-1723},
mesh = {Animals ; Humans ; Mice ; Female ; Male ; *Basic Helix-Loop-Helix Proteins/genetics/metabolism ; *Mutation, Missense ; Pedigree ; Gene Knock-In Techniques ; Transcription Factors/genetics/metabolism ; Homeodomain Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Lobodontia, a rare dental anomaly marked by supernumerary cusps and a single pyramid-shaped molar root, has been previously linked to a variant in the CACNA1S gene without definitive evidence. This study investigates 17 patients with lobodontia from Thai and Croatian families. Microsatellite genotyping defines a 15.4 Mbp critical region encompassing CACNA1S and ASCL5 among Thai families. While genome sequencing confirms the CACNA1S variant only in the Thai patients, all 17 patients harbor the ASCL5 c.274 G > A (p.Glu92Lys) variant, which is absent in 12 unaffected members. Functional studies using CRISPR/Cas9-generated Ascl5 knock-in mutant mice demonstrate the dental anomalies resembling lobodontia in Ascl5[Mut/WT], while Ascl5[Mut/Mut] display severe defects in tooth and jaw development, underscoring the essential role of ASCL5 in craniofacial patterning. Transcriptomic analysis of E17.5 mandibular dental arches reveals differential expression of key craniofacial developmental genes in Ascl5[Mut/Mut] compared to Ascl5[WT/WT], including Dlx1as, and Dlx2. Luciferase assay shows that the p.Glu92Lys ASCL5 impairs DLX2 activation, further supporting the variant's pathogenicity. This study establishes ASCL5 as the gene responsible for lobodontia, revising its previously understood genetic basis, and highlights its crucial role in craniofacial development.},
}

Animals
Humans
Mice
Female
Male
*Basic Helix-Loop-Helix Proteins/genetics/metabolism
*Mutation, Missense
Pedigree
Gene Knock-In Techniques
Transcription Factors/genetics/metabolism
Homeodomain Proteins/genetics/metabolism
CRISPR-Cas Systems

@article {pmid41673027,
year = {2026},
author = {Melore, SM and McRoberts Amador, CD and Hamilton, MC and Gersbach, CA and Reddy, TE},
title = {dHyperCas12a enables multiplexed CRISPRi screens.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41673027},
issn = {2041-1723},
support = {R01 MH125236/MH/NIMH NIH HHS/United States ; UM1 HG012053/HG/NHGRI NIH HHS/United States ; U01 AI146356/AI/NIAID NIH HHS/United States ; R01 CA289574/CA/NCI NIH HHS/United States ; RM1 HG011123/HG/NHGRI NIH HHS/United States ; RM1 HG011123/HG/NHGRI NIH HHS/United States ; UM1 HG012053/HG/NHGRI NIH HHS/United States ; UM1 HG012053/HG/NHGRI NIH HHS/United States ; R01 MH125236/MH/NIMH NIH HHS/United States ; R01 MH125236/MH/NIMH NIH HHS/United States ; R01 CA289574/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Induced Pluripotent Stem Cells/metabolism/cytology ; Animals ; RNA Polymerase II/genetics/metabolism ; Mice ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation ; Regulatory Sequences, Nucleic Acid/genetics ; Gene Editing/methods ; },
abstract = {Interactions between genes or cis-regulatory elements (CREs) underlie many biological processes. High-throughput CRISPR screens have allowed researchers to assess the impact of activation or repression of gene and regulatory elements on many phenotypes. However, assessment of interactions between those genes or elements remains limited. To enable efficient highly-multiplexed control of regulatory element activity, we combine a hyper-efficient version of Lachnospiraceae bacterium dCas12a (dHyperLbCas12a) with RNA Polymerase II expression of long CRISPR RNA (crRNA) arrays. We demonstrate this system with several activation and repression domains, in cultured primary immune cells, and to differentiate induced pluripotent stem cells. We also develop approaches to use dCas12a for simultaneous activation and repression. Lastly, we demonstrate that dHyperLbCas12a effectors can be used to dissect the independent and combinatorial contributions of CREs to gene expression. These tools create possibilities for highly multiplexed control of gene expression in many biological systems.},
}

*CRISPR-Cas Systems/genetics
Humans
Induced Pluripotent Stem Cells/metabolism/cytology
Animals
RNA Polymerase II/genetics/metabolism
Mice
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Expression Regulation
Regulatory Sequences, Nucleic Acid/genetics
Gene Editing/methods

@article {pmid41673416,
year = {2026},
author = {Gao, H and Li, Y and Chen, Y and Liu, X and Fang, M and Zhang, S and Ding, J and Zhu, D and Tan, A and Sheng, S},
title = {Genomic landscape and genetic manipulation of an ectoparasitoid wasp, Gregopimpla kuwanae.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41673416},
issn = {2399-3642},
support = {BK20241864//Natural Science Foundation of Jiangsu Province (Jiangsu Provincial Natural Science Foundation)/ ; 32400380//National Natural Science Foundation of China (National Science Foundation of China)/ ; 31925007//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Wasps/genetics ; *Genome, Insect ; CRISPR-Cas Systems ; RNA Interference ; Genomics ; Female ; Insect Proteins/genetics ; Pest Control, Biological ; Gene Editing ; },
abstract = {Parasitoid wasps are important biological control resources, yet their genetic manipulation has long been constrained by small body size and parasitization behavior, limiting their broader application in pest management. Here we report a chromosome-level genome assembly of the ectoparasitoid Gregopimpla kuwanae (322.87 Mb, 24 chromosomes), a relatively large species that parasitizes various lepidopteran pests. In the first part of this study, we established a foundational genomic resource and experimental platform by producing a high-quality genome and demonstrating the feasibility of functional genetics: RNA interference successfully silenced the cinnabar gene, while CRISPR/Cas9 editing generated vestigial knockout mutants, thus establishing G. kuwanae as a tractable system for gene manipulation. In the second part, we applied comparative genomics to identify lineage-specific gene-family expansions linked to parasitism, including venom-related genes, immune suppression factors, and detoxification enzymes (cytochrome P450s and UDP-glucosyltransferases), and we identified eight HGT candidates; one candidate (JSFChr12G01362) showed pre-feeding expression in females and caused increased adult mortality upon RNAi. Our study provides both the means and the candidates for mechanistic dissection of parasitoid adaptations, laying a foundation for the broader application of parasitoid wasps in sustainable biocontrol programs.},
}

Animals
*Wasps/genetics
*Genome, Insect
CRISPR-Cas Systems
RNA Interference
Genomics
Female
Insect Proteins/genetics
Pest Control, Biological
Gene Editing

@article {pmid41680160,
year = {2026},
author = {Wang, B and Zhou, S and Zhang, X and Wang, R and Huang, S and Li, A and He, H and Zhang, Y and Wei, Y and Yang, Z and Song, F and Li, X and Wan, X and Shen, Y and Ma, C and Mao, H and Ledesma-Amaro, R and Yin, D and Wei, Q and Deng, R and Yang, T and Liu, S and Wan, Y and Mao, C},
title = {PRICE: direct and robust detection of microRNAs at single-nucleotide resolution.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41680160},
issn = {2041-1723},
support = {14208723//Research Grants Council, University Grants Committee (RGC, UGC)/ ; },
mesh = {*MicroRNAs/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Polymorphism, Single Nucleotide ; *Peptide Nucleic Acids/genetics/metabolism ; },
abstract = {Accurate single-nucleotide discrimination of miRNA is clinically vital because small sequence variations can have significant phenotypic and clinical consequences, yet existing techniques can only detect single nucleotide variations (SNVs) at specific loci. Here, we present a generalized peptide nucleic acid (PNA) mediated CRISPR/Cas13a system (PRICE), enabling detection of SNVs in miRNA sequence without sacrificing the sensitivity. PRICE utilizes PNA blockers fully complementary to non-target miRNAs (e.g., miRNAs containing SNVs at loci of no interest) but not to the target miRNA. These blockers selectively hybridize with and inhibit non-target sequences in samples (serum, cells, or tissues). Only the unhybridized target miRNA then binds to crRNA within the Cas13a complex, activating Cas13a to cleave a fluorescent reporter-quencher linker, generating a detectable signal (~10 fM limit). By designing a panel of PNAs against SNVs, PRICE provides a versatile, amplification-free platform for precise miRNA analysis, advancing cancer diagnosis, prognosis, and biology.},
}

*MicroRNAs/genetics
Humans
*CRISPR-Cas Systems/genetics
*Polymorphism, Single Nucleotide
*Peptide Nucleic Acids/genetics/metabolism

@article {pmid41808567,
year = {2026},
author = {Ding, X and Liu, Y and Luo, L and Cai, Y and Wang, C and Jin, J and Chen, Y},
title = {Review Genomic Hotspot Mining and Characterization for Stable Expression of Therapeutic Protein in Chinese Hamster Ovary Cells.},
journal = {ACS synthetic biology},
volume = {15},
number = {3},
pages = {1241-1247},
doi = {10.1021/acssynbio.5c00776},
pmid = {41808567},
issn = {2161-5063},
mesh = {Animals ; CHO Cells ; Cricetulus ; *Recombinant Proteins/genetics/biosynthesis ; CRISPR-Cas Systems/genetics ; Chromosome Mapping ; Cricetinae ; Promoter Regions, Genetic ; Genomics/methods ; Lentivirus/genetics ; },
abstract = {The development of rCHO cell lines that stably express therapeutic proteins is crucial for pharmaceutical protein industrial production. In this study, a systematic method was established to identify genomic hotspots for exogenous protein expression in CHO cells and construct stable recombinant CHO cell strains. Four stable monoclonal cell lines (1b7, 1d2, 2d9, and 2f7) were obtained by using the lentiviral random integration reporter gene. Chromosome mapping analysis found four stable integration sites: chr1_0 (7,30,83,299-7,32,45,508 bp) in 1b7, chr1_0 (17,69,68,187-17,69,68,191 bp) in 1d2, chr3 (4,08,81,262-4,08,99,858 bp) in 2d9, and chr5 (1,69,77,575-1,70,61,744 bp) in 2f7. Based on these sites, we developed recombinant CHO cells capable of long-term stable expression of foreign proteins through the combined application of CRISPR/Cas9 technology and Bxb1 recombinase-mediated cassette exchange. Utilizing "promoter capture technology", all screened LP cell monoclonal lines can express exogenous proteins, with the entire construction process completed in just 2∼3 weeks.},
}

Animals
CHO Cells
Cricetulus
*Recombinant Proteins/genetics/biosynthesis
CRISPR-Cas Systems/genetics
Chromosome Mapping
Cricetinae
Promoter Regions, Genetic
Genomics/methods
Lentivirus/genetics

@article {pmid41832076,
year = {2026},
author = {Gundra, SR and Jiang, W and Aouida, M and Wang, Q and Kazlak, AM and Elbehery, AHA and Saleh, A and Masood, M and Ghouneimy, A and Mahfouz, M},
title = {Characterization and engineering of highly efficient Cas12j genome editors.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2026.02.001},
pmid = {41832076},
issn = {1879-3096},
abstract = {The large size of widely used CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) enzymes limits their delivery for therapeutic applications. Cas12j nucleases offer a hypercompact alternative but show modest editing efficiency. To overcome this limitation, we identified eight novel Cas12j orthologs from viral metagenomes, which in their native form exhibit low editing activity in mammalian cells. We therefore engineered T5 exonuclease-Cas12j fusions, resulting in substantially enhanced genome-editing activity across multiple mammalian cell types, reaching levels comparable to established compact CRISPR-Cas editors. Intriguingly, robust cellular editing occurred in the presence of a previously unrecognized trinucleotide sequence context within the target DNA. Furthermore, we developed Cas12j-based adenine base editors by coupling catalytically inactive Cas12j orthologs with adenine deaminase, enabling efficient A-to-G base conversion in mammalian cells. This study expands the CRISPR toolbox by establishing engineering principles that convert compact Cas12j nucleases into efficient and modular genome-editing platforms well suited for delivery-constrained therapeutic applications.},
}

@article {pmid41832226,
year = {2026},
author = {Lee, SY and Park, HH},
title = {AcrIIA7 hijacks tracrRNA to block CRISPR-Cas system.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-70749-w},
pmid = {41832226},
issn = {2041-1723},
support = {RS-2025-02316334//National Research Foundation of Korea (NRF)/ ; },
abstract = {The CRISPR-Cas9 system provides adaptive immunity against invading genetic elements through a dual-RNA-guided DNA cleavage mechanism. This system relies on the precise assembly of a ribonucleoprotein (RNP) complex composed of the Cas9 endonuclease, a CRISPR-derived RNA (crRNA), and a trans-activating CRISPR RNA (tracrRNA). Around 100 anti-CRISPR proteins that inhibit CRISPR-Cas systems have been identified, and the mechanisms by which they act are increasingly being elucidated. However, the inhibitory mechanisms of many Acrs, including AcrIIA7, remain poorly understood. Here, we present the structure of AcrIIA7 and uncover a previously unrecognized mechanism by which it inhibits Cas9 function. Structural and biochemical analyses reveal that AcrIIA7 specifically binds to tracrRNA, preventing its association with crRNA and thereby blocking formation of the active Cas9 RNP complex. This tracrRNA hijacking mechanism represents a unique strategy for CRISPR inhibition, in which an anti-CRISPR protein targets an RNA scaffold essential for Cas9 activation rather than interacting directly with the Cas9 protein. Our findings provide the first structural insight into tracrRNA-targeted anti-CRISPR activity and highlight RNA-RNA interaction interfaces as vulnerable nodes in CRISPR-Cas immunity.},
}

@article {pmid41834690,
year = {2026},
author = {Marpaung, DSS and Yap Sinaga, AO and Damayanti, D and Utari, NWA and Harmiansyah, and Karangan, A and Kusmali, M},
title = {Integration of CRISPR/Cas12a and Toehold-Mediated Strand Displacement for Alternative Conventional miRNA Detection.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {27},
number = {6},
pages = {e202500932},
doi = {10.1002/cbic.202500932},
pmid = {41834690},
issn = {1439-7633},
mesh = {*MicroRNAs/analysis/genetics/metabolism ; *CRISPR-Cas Systems ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {MicroRNAs (miRNAs) are short, noncoding RNAs that regulate gene expression and serve as powerful biomarkers for cancer and other diseases. Conventional detection methods such as RT-qPCR, Northern blotting, microarrays, and next-generation sequencing provide robust analytical capabilities but remain limited by complexity, cost, and poor suitability for point-of-care diagnostics. CRISPR/Cas12a has emerged as a versatile nucleic acid detection platform with high specificity and sensitivity. However, its intrinsic preference for DNA substrates restricts direct application to miRNA sensing. Early CRISPR/Cas12a-based assays relied on enzymatic amplification, direct RNA-induced activation, or split-component designs, each offering proof-of-concept feasibility but facing trade-offs in sensitivity, workflow complexity, or robustness. Toehold-mediated strand displacement (TSD) provides a powerful alternative by converting miRNA inputs into DNA activators or crRNAs that efficiently trigger Cas12a. This integration enables enzyme-free amplification, programmable logic operations, and enhanced sensitivity, while reducing reliance on multienzyme cascades. This review critically evaluates conventional, enzymatic, direct, and split-based CRISPR/Cas12a strategies and emphasizes emerging TSD-assisted platforms as next-generation solutions for sensitive, specific, and portable miRNA detection.},
}

*MicroRNAs/analysis/genetics/metabolism
*CRISPR-Cas Systems
Humans
*Endodeoxyribonucleases/metabolism/genetics
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid41834849,
year = {2026},
author = {Tenea, GN},
title = {Comprehensive genomic and metabolomic profiling of Weissella confusa UTNCys2-2 highlights bioactive potential.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1779198},
pmid = {41834849},
issn = {1664-302X},
abstract = {INTRODUCTION: The genus Weissella comprises a diverse group of lactic acid bacteria (LAB) widely distributed across plant- and food-associated ecosystems and recognized for their functional and technological versatility. Weissella confusa UTNCys2-2, a plant-derived strain isolated from Amazonian spiral ginger (Costus sp.), that produces exopolysaccharides (EPS) with documented antioxidant activity and promising probiotic properties.

METHODS: Whole-genome sequencing of UTNCys2-2 was performed to establish its taxonomic assignment, phylogenomic analysis, while genome mining was conducted to evaluate safety, metabolic potential, and biosynthetic capabilities. Carbohydrate-active enzymes (CAZymes), Kyoto Encyclopedia of Genes and Genomes (KEGG), and MetaCyc pathways were analyzed for functional insights. Moreover, the metabolite composition of the cell-free supernatant (CFS) was examined using liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with Sequential Windowed Acquisition of all Theoretical Fragment Ion Mass Spectra (SWATH-MS).

RESULTS: The genome consists of a 2.32 Mb circular chromosome (44.59% GC) encoding 2,194 proteins, 76 tRNAs, and 10 rRNAs, with no plasmids. Phylogenomic analyses assigned the strain to the W. confusa clade, clustering closely with the reference strain DSM 20196. UTNCys2-2 harbors a complete Type II-A CRISPR-Cas system, intact prophages, and mobile elements, while lacking virulence determinants and transferable antimicrobial resistance genes. Functional annotation revealed 118 CAZymes supporting EPS biosynthesis, polysaccharide utilization, and carbohydrate metabolism. KEGG and MetaCyc pathways highlighted glycogen and riboflavin biosynthesis, stress tolerance, and metabolic versatility. Genome mining identified a Type III polyketide synthase (T3PKS) gene cluster with low similarity to known pathways, suggesting potential for novel secondary metabolites. Pangenome analysis showed extensive strain-specific genes linked to carbohydrate metabolism and EPS production. Metabolomic profiling of the CFS detected alkaloids, bioactive peptides, functional carbohydrates, and phenolics, supporting antimicrobial, probiotic, and host-interactive activities.

CONCLUSION: W. confusa UTNCys2-2 represents a biosafe and metabolically versatile strain with strong genomic capacity for EPS production, potential for novel secondary metabolite biosynthesis, and diverse bioactive properties, supporting its applicability in food fermentation, probiotic development, and microbial biotechnology.},
}

@article {pmid41834871,
year = {2026},
author = {Abdul Rehman, Y and Fayyaz, A and Alblooshi, AS and Muhammad, K and Mundra, S and Alam, MT},
title = {Molecular adaptations and engineering of extremophiles for synthetic biology and biotechnological applications.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1754802},
pmid = {41834871},
issn = {1664-302X},
abstract = {Extremophiles are microorganisms that thrive in environments previously thought to be uninhabitable, including extreme temperature, salinity, pH, pressure, and radiation. These organisms, found in Archaea, Bacteria, and Eukarya, exhibit distinct structural, metabolic, and genetic adaptations, such as enhanced enzyme stability, efficient DNA repair mechanisms, and robust stress-response systems that enable survival under extreme conditions. Understanding these adaptation mechanisms is key to engineering similar traits in mesophilic organisms. This review discusses the diversity of extremophiles and presents phylogenetic and comparative genomic insights which may provide insights into the origins and evolution of early life on Earth We highlight recent advances in CRISPR/Cas-based genome editing, genome-scale metabolic modeling (GEM), and synthetic biology that have expanded the use of extremophiles in sustainable industrial biotechnology. The exceptional stability and catalytic efficiency of extremozymes under harsh conditions underscore their potential in various biotechnological applications. Finally, we discuss the ecological significance of extremophiles in climate change mitigation and outline current challenges and future directions in extremophile research.},
}

@article {pmid40692150,
year = {2026},
author = {Sun, Z and Cui, M and Deng, Z and Zhou, L and Zeng, F and Liu, Z and Liu, Y},
title = {Loss of CD99L2 Contributed to Temozolomide Resistance and Glioblastoma Tumorigenesis Based on Genome-scale CRISPR/Cas9 Screening.},
journal = {Current pharmaceutical design},
volume = {32},
number = {9},
pages = {696-709},
pmid = {40692150},
issn = {1873-4286},
support = {81974512//National Natural Science Foundation of China/ ; 2020JJ4894//Hunan Natural Science Foundation/ ; },
mesh = {Humans ; *Temozolomide/pharmacology ; *Glioblastoma/drug therapy/genetics/pathology/metabolism ; *Drug Resistance, Neoplasm/drug effects/genetics ; *Brain Neoplasms/drug therapy/genetics/pathology/metabolism ; *CRISPR-Cas Systems/genetics ; *Antineoplastic Agents, Alkylating/pharmacology ; *12E7 Antigen/genetics/metabolism ; Cell Proliferation/drug effects ; *Carcinogenesis/drug effects/genetics ; Cell Line, Tumor ; },
abstract = {INTRODUCTION: Glioblastoma Multiforme (GBM) is a highly aggressive and fatal brain malignancy, with Temozolomide (TMZ) serving as the first-line chemotherapeutic treatment. However, over 50% of patients do not respond to TMZ, and the underlying mechanisms remain unclear. This study utilized the GeCKO library to identify novel genes involved in TMZ resistance and to explore their functions.

METHODS: Loss-of-function genes related to TMZ resistance in GBM cells were identified using the GeCKO library and Next-Generation Sequencing (NGS), validated by qPCR and CCK-8 assays. CD99L2 function was assessed through proliferation, migration, and EdU assays in U251 and U87 cells. Tumor samples from 55 stage IV GBM patients were analyzed to explore the correlation between CD99L2 expression and Progression- Free Survival (PFS).

RESULTS: GeCKO library screening identified seven genes associated with TMZ resistance. After validation, CD99L2 was confirmed as a key contributor to TMZ resistance. Knockdown of CD99L2 increased the IC50 of U251 and U87 cells by 1.39- and 1.54-fold, respectively. Conversely, CD99L2 overexpression reduced the IC50 by 0.52- and 0.58-fold. CD99L2 knockdown also promoted tumor proliferation and aggressiveness. Additionally, higher CD99L2 expression was associated with longer PFS in GBM patients (median PFS: 7.87 months vs. 2.7 months, P=0.0003).

DISCUSSION: The functions of CD99L2 remain poorly understood. A few studies have reported that CD99L2 may serve as an adhesion molecule modulating inflammatory responses. One study has shown that CD99L2 is highly expressed in the brain and affects neuronal excitability. These findings suggest that CD99L2 may play a positive role in the body's defense against glioma.

CONCLUSION: This study demonstrated that CD99L2 knockdown promotes TMZ resistance and tumorigenesis in GBM, suggesting its potential as a novel biomarker for TMZ resistance.},
}

Humans
*Temozolomide/pharmacology
*Glioblastoma/drug therapy/genetics/pathology/metabolism
*Drug Resistance, Neoplasm/drug effects/genetics
*Brain Neoplasms/drug therapy/genetics/pathology/metabolism
*CRISPR-Cas Systems/genetics
*Antineoplastic Agents, Alkylating/pharmacology
*12E7 Antigen/genetics/metabolism
Cell Proliferation/drug effects
*Carcinogenesis/drug effects/genetics
Cell Line, Tumor

@article {pmid41319833,
year = {2026},
author = {Hu, H and Ke, X and Xiao, H and Shang, X and Yin, Q and Li, J and Li, X and Hu, Z and Qian, P and Wang, M},
title = {Genomic assembly, rescue, and characterization of a functional pseudorabies virus.},
journal = {Virologica Sinica},
volume = {41},
number = {1},
pages = {97-106},
doi = {10.1016/j.virs.2025.11.010},
pmid = {41319833},
issn = {1995-820X},
mesh = {Animals ; *Herpesvirus 1, Suid/genetics ; *Genome, Viral ; Chlorocebus aethiops ; Vero Cells ; Swine ; Mice ; Pseudorabies/virology ; Gene Editing ; Mice, Inbred BALB C ; CRISPR-Cas Systems ; Swine Diseases/virology ; },
abstract = {With recent advances in synthetic biology methods, the genomes of several large DNA viruses have been de novo synthesized and assembled, leading to the functional rescue of the respective viruses. Pseudorabies virus (PRV), a large DNA virus belonging to the family Herpesviridae, causes severe diseases in swine, resulting in significant economic losses to the global pig farming industry. Genome editing is crucial for attenuating virulence and developing safer vaccines for PRV. However, its complex repetitive sequences and extremely high GC-rich genome pose significant challenges for genetic manipulation. In this study, we developed a PRV genome assembly platform using yeast-based transformation-associated recombination (TAR) technology. The genome of a prevalent genotype II variant strain, PRV-GX-2011 (GenBank number PV405324.1), was divided into nine A-level fragments and cloned into vectors via TAR. Subsequently, three B-level fragments were generated by recombining three A-level fragments each. In vitro CRISPR/Cas9-mediated editing was introduced to insert an egfp gene into the non-coding intergenic region between UL23 and UL22 genes. Infectious viruses were rescued by co-transfection of linearized B-level fragments in Vero cells, and an isolated virus, PRV-GX-Syn1, was purified via plaque assay. While PRV-GX-Syn1 exhibited reduced viral titer and smaller plaque size compared to the parental strain, its morphological characteristics remained indistinguishable from the parental virus. In BALB/c mice, PRV-GX-Syn1 caused lethal infection, producing lung pathology comparable to the parental strain. This TAR-based platform offers faster and more flexible genomic modification of PRV, facilitating both basic research and PRV-based vaccine vectors.},
}

Animals
*Herpesvirus 1, Suid/genetics
*Genome, Viral
Chlorocebus aethiops
Vero Cells
Swine
Mice
Pseudorabies/virology
Gene Editing
Mice, Inbred BALB C
CRISPR-Cas Systems
Swine Diseases/virology

@article {pmid41611887,
year = {2026},
author = {Gordon, A and Yoon, SJ and Bicks, LK and Martín, JM and Pintacuda, G and Arteaga, S and Wamsley, B and Guo, Q and Elahi, L and Dolmetsch, RE and Bernstein, JA and O'Hara, R and Hallmayer, JF and Lage, K and Pasca, SP and Geschwind, DH},
title = {Developmental convergence and divergence in human stem cell models of autism.},
journal = {Nature},
volume = {651},
number = {8106},
pages = {707-719},
pmid = {41611887},
issn = {1476-4687},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/pathology/cytology ; Organoids/metabolism/pathology/cytology ; *Autism Spectrum Disorder/genetics/pathology ; Mutation/genetics ; Cell Differentiation/genetics ; Neural Stem Cells/metabolism/cytology/pathology ; Protein Interaction Maps/genetics ; *Models, Biological ; CRISPR-Cas Systems/genetics ; Female ; Transcriptome/genetics ; Male ; Cell Line ; },
abstract = {Two decades of genetic studies in autism spectrum disorder (ASD) have identified more than 100 genes harbouring rare risk mutations[1-13]. Despite this substantial heterogeneity, transcriptomic and epigenetic analyses have identified convergent patterns of dysregulation across the ASD postmortem brain[14,15-17]. To identify shared and distinct mechanisms of ASD-linked mutations, we assembled a large patient collection of human induced pluripotent stem (hiPS) cells, consisting of 70 hiPS cell lines after stringent quality control representing 8 ASD-associated mutations, idiopathic ASD, and 20 lines from non-affected control individuals. Here we used these hiPS cell lines to generate human cortical organoids, profiling by RNA sequencing at four distinct time points up to 100 days after in vitro differentiation. Early time points harboured the largest mutation-specific changes, but different mutations converged on shared transcriptional changes as development progressed. We identified a shared RNA and protein interaction network, which was enriched in ASD risk genes and predicted to drive the observed downstream changes in gene expression. CRISPR-Cas9 screening of these candidate transcriptional regulators in induced human neural progenitors validated their downstream convergent molecular effects. These data illustrate how risk associated with genetically defined forms of ASD can propagate by means of transcriptional regulation to affect convergently dysregulated pathways, providing new insight into the convergent impact of ASD genetic risk on human neurodevelopment.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/pathology/cytology
Organoids/metabolism/pathology/cytology
*Autism Spectrum Disorder/genetics/pathology
Mutation/genetics
Cell Differentiation/genetics
Neural Stem Cells/metabolism/cytology/pathology
Protein Interaction Maps/genetics
*Models, Biological
CRISPR-Cas Systems/genetics
Female
Transcriptome/genetics
Male
Cell Line

@article {pmid41667457,
year = {2026},
author = {He, M and Wang, W and Zhou, H and Liu, C and Zhao, C and Li, J and Han, Y and Qin, Y and Chen, M},
title = {A synthetic system for RNA-responsive pyroptosis based on type III-E CRISPR nuclease-protease.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41667457},
issn = {2041-1723},
support = {//National Key R&D Program of China/ ; //National Natural Science Foundation of China/ ; //Natural Science Foundation of Wuhan/ ; },
mesh = {*Pyroptosis/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *RNA/genetics/metabolism ; Animals ; Mice ; HEK293 Cells ; },
abstract = {Pyroptosis plays a crucial role in immune defense against infections and endogenous threats by eliminating harmful cells and modulating the immune response through inflammation. However, the natural activation of pyroptosis involves intricate signaling pathways, posing significant challenges for its artificial manipulation in research and therapies. Here, we present DAMAGE (Death Manipulation Gene), an innovative system that integrates gasdermins within the type III-E CRISPR framework, enabling the specific recognition of target RNA (tgRNA) and triggering pyroptosis. This mechanism allows DAMAGE to selectively identify and eliminate virus-infected, cancerous, and senescent cells, all of which exhibit altered RNA transcriptomes. Additionally, DAMAGE exhibits considerable promise as a platform for mRNA-LNP therapy. Our study highlights the potential of this CRISPR-based system in the controllable induction of pyroptosis, offering an innovative therapeutic strategy for treating RNA-heterogeneous diseases.},
}

*Pyroptosis/genetics
Humans
*CRISPR-Cas Systems/genetics
*RNA/genetics/metabolism
Animals
Mice
HEK293 Cells

@article {pmid41679545,
year = {2026},
author = {Bahlmann, N and Alshawabkeh, M and Tsoukas, R and Schröer, K and Schellhorn, S and Sieler, M and Dittmar, T and Ehrke-Schulz, E and Ehrhardt, A and Zhang, W},
title = {Desmoglein 2 (DSG2)-knockout human respiratory epithelial cell model to study species B adenovirus receptor usage.},
journal = {Virologica Sinica},
volume = {41},
number = {1},
pages = {172-181},
doi = {10.1016/j.virs.2026.02.007},
pmid = {41679545},
issn = {1995-820X},
mesh = {Humans ; *Desmoglein 2/genetics/metabolism ; *Receptors, Virus/genetics/metabolism ; Gene Knockout Techniques ; Membrane Cofactor Protein/genetics/metabolism ; *Adenoviruses, Human/genetics/physiology ; CRISPR-Cas Systems ; *Epithelial Cells/virology/metabolism ; *Coxsackie and Adenovirus Receptor-Like Membrane Protein/genetics/metabolism ; Cell Line ; Cell Proliferation ; },
abstract = {With an increasing number of human adenoviruses identified, the selection of potential therapeutic vectors broadens. For safety reasons, achieving cell-specific gene delivery is crucial to minimize off-target effects. Therefore, it is essential to gain a systematic understanding of adenovirus receptor-usage. Our aim is to establish a human-originated in vitro model for comparative analysis of human adenoviruses receptor usage. Based on our previous work of human CD46 and coxsackievirus and adenovirus receptor (CAR) knockout cell lines, we generated desmoglein 2 (DSG2) knockout cell lines using genome-engineering technology based on Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 (CRISPR/Cas9). All together, we established a panel of cell lines that carry a single, double, or triple knockout of the three major human adenovirus receptors: CAR, CD46 and DSG2. Notably, cell proliferation speed was affected by the CAR-knockout, but not the DSG2-or CD46-knockouts. In addition, the spheroid formation ability was sharply reduced in CAR- or DSG2-knockout cells, but not the CD46-knockout cells. With this receptor-knockout model, we confirmed the receptor usage of nine species B adenoviruses. Furthermore, adenovirus vectors containing a previously identified DSG2-binding affinity-enhanced mutation showed DSG2-dependent cell entry within this cell model, which indicates that they are de-targeted from CD46 - the ubiquitously expressed receptor on all nucleated cells. Collectively, our findings show that the adenovirus major receptor knockout cell lines can serve as an in vitro model to help select adenovirus types suitable for individual applications and to better understand adenovirus infection biology.},
}

Humans
*Desmoglein 2/genetics/metabolism
*Receptors, Virus/genetics/metabolism
Gene Knockout Techniques
Membrane Cofactor Protein/genetics/metabolism
*Adenoviruses, Human/genetics/physiology
CRISPR-Cas Systems
*Epithelial Cells/virology/metabolism
*Coxsackie and Adenovirus Receptor-Like Membrane Protein/genetics/metabolism
Cell Line
Cell Proliferation

@article {pmid41691970,
year = {2026},
author = {Anders, M and Hoppe, S and Eberl, H and Rebs, S and Seedorf, A and Maurer, W and Schill, T and Zibat, A and Unsöld, JK and Yigit, G and Wollnik, B and Vollmann, D and Sossalla, S and Streckfuss-Bömeke, K},
title = {Generation of pluripotent stem cell line (IPWi001-A) and a corresponding CRISPR/Cas9 modified isogenic rescue control (IPWi001-A-1) from a patient with arrhythmia-induced cardiomyopathy harboring a KCNQ1 truncating mutation.},
journal = {Stem cell research},
volume = {92},
number = {},
pages = {103921},
doi = {10.1016/j.scr.2026.103921},
pmid = {41691970},
issn = {1876-7753},
mesh = {Humans ; *KCNQ1 Potassium Channel/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Arrhythmias, Cardiac/genetics/complications/pathology ; *Cardiomyopathies/genetics/pathology ; *Mutation/genetics ; Cell Line ; Myocytes, Cardiac/metabolism ; Cell Differentiation ; Male ; },
abstract = {KCNQ1 functions as a slow rectifying potassium channel during the repolarization of the cardiac action potential, with mutations causing long-QT syndrome 1 and arrhythmias. A genetic link between KCNQ1 mutations and arrhythmia-induced cardiomyopathy (AIC) has not been identified, and the underlying pathways remain elusive. We generated human induced pluripotent stem cells (hiPSCs) from an AIC patient harboring the heterozygous truncating mutation p.W15* in KCNQ1 and corrected the mutation to wildtype using CRISPR/Cas9. The hiPSCs retained full pluripotency, genomic integrity, and differentiation ability. They were differentiated into hiPSC-cardiomyocytes (hiPSC-CM), establishing a patient-specific model to explore potential genetic connections to AIC.},
}

Humans
*KCNQ1 Potassium Channel/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Induced Pluripotent Stem Cells/metabolism/cytology
*Arrhythmias, Cardiac/genetics/complications/pathology
*Cardiomyopathies/genetics/pathology
*Mutation/genetics
Cell Line
Myocytes, Cardiac/metabolism
Cell Differentiation
Male

@article {pmid41693245,
year = {2026},
author = {Kou, S and Chua, LC and Tan, JQ and Lau, OS},
title = {Stomatal XVE: an inducible system for cell-stage-specific gene expression and editing in the stomatal lineage.},
journal = {The New phytologist},
volume = {250},
number = {2},
pages = {1330-1347},
pmid = {41693245},
issn = {1469-8137},
support = {NRF-CRP22-2019-0001//National Research Foundation Singapore/ ; },
mesh = {*Plant Stomata/genetics/cytology ; *Arabidopsis/genetics/cytology ; *Gene Expression Regulation, Plant/drug effects ; *Gene Editing/methods ; *Cell Lineage/genetics ; Arabidopsis Proteins/metabolism/genetics ; Promoter Regions, Genetic/genetics ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; Phenotype ; },
abstract = {Stomatal development has emerged as a valuable model for studying developmental processes. Examining gene function along the stomatal lineage often requires gene perturbation in a controlled and cell-stage-specific manner, but this remains tedious without a dedicated genetic tool. Here, we describe Stomatal XVE, a modular, two-component XVE-based inducible system that enables user-controlled gene overexpression and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based knockout at defined stomatal cell stages in Arabidopsis thaliana. The system consists of a collection of estrogen-responsive XVE driver lines under cell-stage-specific promoters and effector vectors responsive to activated XVE. This design simplifies cloning and allows users to scale their investigation. We validated the cell-stage specificity and inducibility of the XVE driver lines and characterized key induction parameters. To test the system functionally, we employed it to study MAPKKK YODA and a pathogen effector AvrPtoB. While YODA overexpression reproduced known early- and late-stage phenotypes, stage-specific knockouts argued against its late-stage role in guard cell (GC) differentiation. Furthermore, AvrPtoB expression during later stages triggered striking disruptions in GC morphology and viability, revealing cell-type-specific effects of the pathogen protein. Overall, our Stomatal XVE system enables precise functional analysis of genes across defined stages of stomatal development and is particularly well suited for investigating genes with pleiotropic effects.},
}

*Plant Stomata/genetics/cytology
*Arabidopsis/genetics/cytology
*Gene Expression Regulation, Plant/drug effects
*Gene Editing/methods
*Cell Lineage/genetics
Arabidopsis Proteins/metabolism/genetics
Promoter Regions, Genetic/genetics
CRISPR-Cas Systems/genetics
Plants, Genetically Modified
Phenotype

@article {pmid41722369,
year = {2026},
author = {Boumpoureka, I and Gorgogietas, V and Petkovski, E and Massart, F and Mellick, GD and Krüger, R},
title = {Generation of two isogenic control iPSC lines (LCSBi001-A-2 and LCSBi001-A-3) from a Parkinson's disease patient line (LCSBi001-A) carrying the pathogenic VPS35 p.D620N mutation.},
journal = {Stem cell research},
volume = {92},
number = {},
pages = {103944},
doi = {10.1016/j.scr.2026.103944},
pmid = {41722369},
issn = {1876-7753},
mesh = {Humans ; *Parkinson Disease/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology/pathology ; *Vesicular Transport Proteins/genetics ; *Mutation/genetics ; Cell Line ; CRISPR-Cas Systems ; Cell Differentiation ; },
abstract = {The pathogenic mutation VPS35 p.D620N has been identified to cause autosomal dominant, late-onset Parkinson's disease (PD) in multiple individuals and families worldwide. Here, we describe the generation of two new isogenic control lines (LCSBi001-A-2 and LCSBi001-A-3) from an already established patient-derived line (LCSBi001-A) carrying the heterozygous VPS35 p.D620N mutation. The control lines were generated with CRISPR/Cas9 technology, and the correction of the mutation was verified with Sanger sequencing. Both lines express pluripotency markers, are capable of in vitro differentiation into the three germ layers, and have a normal karyotype. The mutant and control iPSC lines are available for research purposes.},
}

Humans
*Parkinson Disease/genetics/pathology/metabolism
*Induced Pluripotent Stem Cells/metabolism/cytology/pathology
*Vesicular Transport Proteins/genetics
*Mutation/genetics
Cell Line
CRISPR-Cas Systems
Cell Differentiation

@article {pmid41747624,
year = {2026},
author = {Ming Wu, SC and Boheler, KR},
title = {Generation of an induced pluripotent stem cell and isogenic control line from a vascular Ehlers-Danlos Syndrome (vEDS) patient harboring a pathogenic c.755G>T in the COL3A1 gene.},
journal = {Stem cell research},
volume = {92},
number = {},
pages = {103945},
doi = {10.1016/j.scr.2026.103945},
pmid = {41747624},
issn = {1876-7753},
mesh = {Humans ; *Ehlers-Danlos Syndrome/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology/pathology ; *Collagen Type III/genetics/metabolism ; Cell Line ; Cell Differentiation ; Fibroblasts/metabolism ; CRISPR-Cas Systems ; Ehlers-Danlos Syndrome, Type IV ; },
abstract = {We report the generation and characterization of a collagen III-mutant human iPSC line (JHUi007-A) and an isogenic gene-edited control (JHUi007-A-1). Reprogramming of dermal fibroblasts, obtained from a patient with vascular Ehlers-Danlos syndrome (vEDS) carrying the COL3A1 c.755G>T variant, was performed using integration-free Sendai virus. Isogenic controls were produced by CRISPR/Cas9 gene editing. Both lines displayed typical morphologies, expressed stemness factors, formed derivatives of all three germ layers, and maintained a normal karyotype. These lines readily differentiated into vascular smooth muscle cells with cytoskeletal differences between vEDS and control cells, confirming the utility of this resource to study disease processes.},
}

Humans
*Ehlers-Danlos Syndrome/genetics/pathology/metabolism
*Induced Pluripotent Stem Cells/metabolism/cytology/pathology
*Collagen Type III/genetics/metabolism
Cell Line
Cell Differentiation
Fibroblasts/metabolism
CRISPR-Cas Systems
Ehlers-Danlos Syndrome, Type IV

@article {pmid41747626,
year = {2026},
author = {Yoo, DH and Bayarsaikhan, D and Lee, J and Im, YS and Bayarsaikhan, G and Kang, HA and Lee, B and Kim, YO},
title = {Generation and characterization of SOX17-specific EGFP expressing human induced pluripotent stem cell line, KSCBi017-A-4, using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {92},
number = {},
pages = {103943},
doi = {10.1016/j.scr.2026.103943},
pmid = {41747626},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *SOXF Transcription Factors/metabolism/genetics ; *Green Fluorescent Proteins/metabolism/genetics ; Cell Line ; Cell Differentiation ; Endoderm/cytology/metabolism ; },
abstract = {We generated a human induced pluripotent stem cell (hiPSC) reporter line in which EGFP was inserted in-frame at the C-terminus of the endogenous SOX17 locus using CRISPR/Cas9-mediated homologous recombination. The targeted clone, KSCBi017-A-4, was isolated by puromycin selection and validated by PCR and Sanger sequencing. This SOX17-EGFP hiPSC line retains a normal karyotype and pluripotency and displays specific EGFP expression upon directed definitive endoderm differentiation. This reporter line provides a reliable tool for monitoring SOX17 expression during human endoderm specification.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*SOXF Transcription Factors/metabolism/genetics
*Green Fluorescent Proteins/metabolism/genetics
Cell Line
Cell Differentiation
Endoderm/cytology/metabolism

@article {pmid41763219,
year = {2026},
author = {Aravind, KM and Del Vecchio, D},
title = {Resource competition shapes CRISPR-mediated gene activation.},
journal = {Cell systems},
volume = {17},
number = {3},
pages = {101511},
doi = {10.1016/j.cels.2025.101511},
pmid = {41763219},
issn = {2405-4720},
mesh = {*CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; Gene Editing/methods ; *Transcriptional Activation/genetics ; },
abstract = {CRISPR-mediated gene activation (CRISPRa) allows concurrent transcriptional control of many genes and is widely used in genome-wide screening, bioproduction, and therapeutics. Multi-gene control is possible due to the sequence specificity by which guide RNAs (gRNAs) recruit dCas9 and an activator protein to target genes. Still, the optimization of CRISPRa systems remains difficult. Here, we show that, despite sequence specificity, different gRNAs interfere with each other by competing for dCas9 and the activator protein. This competition breaks modularity and hinders CRISPRa. We also discover that gene activation is biphasic, wherein increased level of a gRNA leads to target repression instead of activation. We introduce a chemical reaction-network model that captures these effects and use it for improving the dynamic range of CRISPRa. Our results demonstrate that CRISPRa is not as modular or scalable as previously thought and establish a predictive modeling tool that enables systematic design and optimization of multi-gRNA CRISPRa systems. A record of this paper's transparent peer review process is included in the supplemental information.},
}

*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Humans
Gene Editing/methods
*Transcriptional Activation/genetics

@article {pmid41814120,
year = {2026},
author = {Simonneau, B and Baghdoyan, S and Cailleret, M and Simon, S and Ruckebusch, O and Vrablikova, B and Giraud-Triboult, K and Kassar, LE and Fanen, P and Duriez, B},
title = {CRISPR-Cas9 genome editing in the parental iPSC line PCIi033-A to introduce the homozygous mutation p.F508del (c.1521_1523del) in the CFTR gene.},
journal = {Stem cell research},
volume = {92},
number = {},
pages = {103947},
doi = {10.1016/j.scr.2026.103947},
pmid = {41814120},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Homozygote ; Cell Line ; *Mutation/genetics ; Cystic Fibrosis/genetics/pathology ; Cell Differentiation ; },
abstract = {Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene. Patients carrying the most common mutation, p.F508del, benefit from the triple therapy Kaftrio®. We genome-edited the commercially available iPSC line PCIi033-A (wild-type CFTR) to generate the subclone PCIi033-A-5, which is homozygous for the mutation c.1521_1523del (p.F508del), using CRISPR-SpCas9 tools. PCIi033-A-5 has a normal karyotype and stem cell morphology, is pluripotent, and differentiates into the three germ layers. Introducing this mutation in a parental isogenic iPSC line is essential to demonstrate the feasibility of modeling CF disease after differentiation of the iPS cells into bronchial epithelium.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Cystic Fibrosis Transmembrane Conductance Regulator/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Homozygote
Cell Line
*Mutation/genetics
Cystic Fibrosis/genetics/pathology
Cell Differentiation

@article {pmid41826758,
year = {2026},
author = {Deol, KK and Harris, CA and Tomlinson, SJ and Delaney, CJ and Al-Farhan, A and Mathiowetz, AJ and Doubravsky, CE and Pratt, DA and Olzmann, JA},
title = {Vitamin B2 metabolism promotes FSP1 stability to prevent ferroptosis.},
journal = {Nature structural & molecular biology},
volume = {33},
number = {3},
pages = {525-536},
pmid = {41826758},
issn = {1545-9985},
mesh = {*Ferroptosis ; Humans ; *Riboflavin/metabolism ; *S100 Calcium-Binding Protein A4/metabolism/genetics/chemistry ; Protein Stability ; Flavin-Adenine Dinucleotide/metabolism ; CRISPR-Cas Systems ; HEK293 Cells ; },
abstract = {Ferroptosis, a regulated form of cell death driven by excessive lipid peroxidation, has emerged as a promising therapeutic target in cancer. Ferroptosis suppressor protein 1 (FSP1) is a critical regulator of ferroptosis resistance, yet the mechanisms controlling its expression and stability remain mostly unexplored. To uncover regulators of FSP1 abundance, we conducted CRISPR-Cas9 screens using a genome-edited, dual-fluorescent FSP1 reporter cell line, identifying both transcriptional and post-translational mechanisms that determine FSP1 levels. Notably, we identified riboflavin kinase and flavin adenine dinucleotide (FAD) synthase, enzymes that are essential for synthesizing FAD from vitamin B2, as key contributors to FSP1 stability. Biochemical and cellular analyses revealed that FAD binding is critical for both FSP1 activity and stability. FAD deficiency and mutations blocking FSP1-FAD binding triggered FSP1 degradation through a ubiquitin-proteasome pathway involving the E3 ligase RNF8. Unlike other vitamins that inhibit ferroptosis by scavenging radicals, vitamin B2 supports ferroptosis resistance through FAD cofactor binding, ensuring proper FSP1 stability and function. This study provides a rich resource detailing mechanisms that regulate FSP1 abundance and highlights a novel connection between vitamin B2 metabolism and ferroptosis resistance, with implications for therapeutic strategies targeting FSP1 in cancer.},
}

*Ferroptosis
Humans
*Riboflavin/metabolism
*S100 Calcium-Binding Protein A4/metabolism/genetics/chemistry
Protein Stability
Flavin-Adenine Dinucleotide/metabolism
CRISPR-Cas Systems
HEK293 Cells