@article {pmid41563484,
year = {2026},
author = {Pardy, F and Reblova, K and Svozilova, H and Tichy, B and Pospisilova, S and Kotaskova, J and Navrkalova, V},
title = {Assessment of long-read strategies for the enrichment of clinically relevant breakpoints in lymphomas: towards a diagnostic implementation.},
journal = {Annals of hematology},
volume = {105},
number = {2},
pages = {47},
pmid = {41563484},
issn = {1432-0584},
mesh = {Humans ; *Translocation, Genetic ; *Lymphoma/genetics/diagnosis ; *Chromosome Breakpoints ; Cell Line, Tumor ; High-Throughput Nucleotide Sequencing/methods ; CRISPR-Cas Systems ; },
abstract = {Recurrent chromosomal translocations are hallmarks of many hematological malignancies, including lymphomas and leukemias. Accurate breakpoint detection is essential for diagnostics, treatment optimization, and disease monitoring. Long-read sequencing (Oxford Nanopore Technologies) enables unambiguous mapping and translocation identification. We designed a Cas9-based enrichment panel targeting common translocations in lymphoid malignancies. To accommodate both well-defined and promiscuous translocation partners, we employed single-side and dual-side sequencing strategies. Using well-established lymphoid cell lines, we benchmarked three enrichment approaches: (i) Cas9 read-out, (ii) Cas9 excision with multiplexing, and (iii) adaptive sampling. Cas9-mediated enrichment achieved superior on-target coverage, particularly in densely targeted regions (such as the IGH locus), while single-probe targets showed lower coverage depth. Adaptive sampling offered higher throughput, flexibility, and better pore occupancy, however with limited breakpoint detection. Cas9 excision has been demonstrated as a fast and reliable method to detect canonical translocation partners in clinical lymphoma samples. Our findings indicate that long-read enrichment strategies are suitable for targeting breakpoint hotspots, although the choice of approach depends heavily on the laboratory's specific goal. We propose a decision algorithm for selecting the optimal method based on experimental and clinical needs: Cas9-mediated enrichment suits focused diagnostic intent, while adaptive sampling is preferable for broader research use.},
}

Humans
*Translocation, Genetic
*Lymphoma/genetics/diagnosis
*Chromosome Breakpoints
Cell Line, Tumor
High-Throughput Nucleotide Sequencing/methods
CRISPR-Cas Systems

@article {pmid41491310,
year = {2026},
author = {Arena, KA and Kearns, CA and Ahmed, M and O'Rourke, R and Sagerström, CG and Franco, SJ and Appel, B},
title = {Gsx2 regulates oligodendrocyte precursor formation in the zebrafish spinal cord.},
journal = {Developmental biology},
volume = {531},
number = {},
pages = {30-44},
doi = {10.1016/j.ydbio.2026.01.001},
pmid = {41491310},
issn = {1095-564X},
mesh = {Animals ; *Zebrafish/embryology/genetics/metabolism ; *Spinal Cord/embryology/cytology/metabolism ; *Zebrafish Proteins/metabolism/genetics ; *Oligodendroglia/metabolism/cytology ; Cell Differentiation/genetics ; Gene Expression Regulation, Developmental ; *Oligodendrocyte Precursor Cells/metabolism/cytology ; Neural Stem Cells/metabolism/cytology ; CRISPR-Cas Systems ; Transcription Factors/metabolism/genetics ; *Homeodomain Proteins/genetics/metabolism ; Gene Regulatory Networks ; Cell Lineage ; },
abstract = {Nervous system development relies on sequential and coordinated formation of diverse neurons and glia from neural progenitor cells (NPCs). In the spinal cord, NPCs of the pMN domain produce neurons early in development followed by oligodendrocyte precursor cells (OPCs), which subsequently differentiate as oligodendrocytes (OLs), the myelinating glia of the central nervous system. The mechanisms that specify neural progenitor cells to the OL lineage are not yet well understood. Using zebrafish as an experimental model system, we generated single-cell RNA sequencing and single-nuclei ATAC sequencing data that identified a subpopulation of NPCs, called pre-OPCs, that appeared fated to produce OPCs. pre-OPCs uniquely express several genes that encode transcription factors specific to the OL lineage, including Gsx2, which regulates OPC formation in the mouse forebrain. To investigate Gsx2 function in zebrafish OPC specification, we used CRISPR/Cas9 genome editing to create gsx2 loss-of-function alleles. gsx2 homozygous mutant embryos initiated OPC formation prematurely and produced excess OPCs without altering OL differentiation. Using our single-nuclei multi-omics dataset, we predicted a gene regulatory network centered around gsx2 and identified genes that might be transcriptionally regulated by Gsx2. Taken together, our studies suggest that Gsx2 expression in pre-OPCs contributes to the timing of OPC specification.},
}

Animals
*Zebrafish/embryology/genetics/metabolism
*Spinal Cord/embryology/cytology/metabolism
*Zebrafish Proteins/metabolism/genetics
*Oligodendroglia/metabolism/cytology
Cell Differentiation/genetics
Gene Expression Regulation, Developmental
*Oligodendrocyte Precursor Cells/metabolism/cytology
Neural Stem Cells/metabolism/cytology
CRISPR-Cas Systems
Transcription Factors/metabolism/genetics
*Homeodomain Proteins/genetics/metabolism
Gene Regulatory Networks
Cell Lineage

@article {pmid41443566,
year = {2026},
author = {Ghosh, P and Wadsworth, BC and Terry, L and Evans, TA},
title = {Evolutionary conservation of midline axon guidance activity between Drosophila and Tribolium Frazzled.},
journal = {Developmental biology},
volume = {531},
number = {},
pages = {1-9},
doi = {10.1016/j.ydbio.2025.12.015},
pmid = {41443566},
issn = {1095-564X},
mesh = {Animals ; *Axon Guidance/physiology/genetics ; *Tribolium/embryology/genetics/metabolism ; *Drosophila Proteins/genetics/metabolism ; *Drosophila melanogaster/embryology/genetics/metabolism ; Axons/metabolism ; Signal Transduction ; Netrin Receptors/genetics/metabolism ; Gene Expression Regulation, Developmental ; Biological Evolution ; CRISPR-Cas Systems ; },
abstract = {The regulation of midline crossing of axons is of fundamental importance for the proper development of nervous system connectivity in bilaterian animals. A number of conserved axon guidance signaling pathways coordinate to attract or repel axons at the nervous system midline to ensure the proper regulation of midline crossing. The attractive Netrin-Frazzled/DCC (Net-Fra) signaling pathway is widely conserved among bilaterians, but it is not clear whether the mechanisms by which Net and Fra promote midline crossing are also conserved. In Drosophila, Fra can promote midline crossing via Netrin-dependent and Netrin-independent mechanisms, by acting as a canonical midline attractive receptor and also through a non-canonical pathway to inhibit midline repulsion via transcriptional regulation. To examine the conservation of Fra-dependent axon guidance mechanisms among insects, in this paper we compare the midline attractive roles of the Frazzled receptor in the fruit fly (Drosophila melanogaster) and flour beetle (Tribolium castaneum) using CRISPR/Cas9-mediated gene editing. We replace the Drosophila fra gene with sequences encoding Drosophila Fra (DmFra) or Tribolium Fra (TcFra) and examine midline crossing of axons in the ventral nerve cord of embryos carrying these modified alleles. We show that Tribolium Fra can fully substitute for Drosophila Fra to promote midline crossing of axons in the embryonic nervous system, suggesting that the mechanisms by which Frazzled regulates midline axon guidance are evolutionarily conserved within insects.},
}

Animals
*Axon Guidance/physiology/genetics
*Tribolium/embryology/genetics/metabolism
*Drosophila Proteins/genetics/metabolism
*Drosophila melanogaster/embryology/genetics/metabolism
Axons/metabolism
Signal Transduction
Netrin Receptors/genetics/metabolism
Gene Expression Regulation, Developmental
Biological Evolution
CRISPR-Cas Systems

@article {pmid41435996,
year = {2026},
author = {Gopalakrishnan, R and Kannan, K and Gunasekaran, R and Ramachandran, P and Ganapathy, D and Pitchiah, S},
title = {A comparative review of vector insertion techniques in Saccharomyces cerevisiae.},
journal = {Journal of microbiological methods},
volume = {241},
number = {},
pages = {107378},
doi = {10.1016/j.mimet.2025.107378},
pmid = {41435996},
issn = {1872-8359},
mesh = {*Saccharomyces cerevisiae/genetics ; *Genetic Vectors/genetics ; CRISPR-Cas Systems ; Homologous Recombination ; *Genetic Engineering/methods ; *Mutagenesis, Insertional/methods ; Synthetic Biology/methods ; Gene Editing/methods ; Genome, Fungal ; },
abstract = {Saccharomyces cerevisiae, a model organism in genetics and molecular biology has been extensively engineered using various vector insertion techniques. This review compares and contrasts three prominent techniques: In vivo homologous recombination (HR), Cre-lox recombination and CRISPR/Cas9. In vivo HR leverages the organism's innate DNA repair machinery for easy vector integration and targeted genome modifications. Cre-lox recombination offers high specificity and efficiency at loxP sites, making it ideal for targeted gene excision or integration. CRISPR/Cas9 has revolutionized genome engineering with its precision and ability to target multiple loci simultaneously. Each technique has its strengths and limitations, including site dependency, off-target effects, and strain-specific variability. This review provides a comprehensive overview of these vector insertion techniques, highlighting their applications, advantages, and limitations in S. cerevisiae genome engineering and synthetic biology.},
}

*Saccharomyces cerevisiae/genetics
*Genetic Vectors/genetics
CRISPR-Cas Systems
Homologous Recombination
*Genetic Engineering/methods
*Mutagenesis, Insertional/methods
Synthetic Biology/methods
Gene Editing/methods
Genome, Fungal

@article {pmid41420208,
year = {2025},
author = {Holmlund, H and Yamauchi, Y and Tekayev, M and Jakobs, S and Robin, A and Fujii, W and Ward, MA},
title = {CRISPR/Cas9-mediated knock-in of the murine Y chromosomal genes Zfy1 and Zfy2.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {88},
pmid = {41420208},
issn = {1471-2164},
support = {NIH F31HD111279/GF/NIH HHS/United States ; HD114645/GF/NIH HHS/United States ; 17CON-86294//Hawai'i Community Foundation/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Male ; Mice ; *Gene Knock-In Techniques ; Spermatogenesis/genetics ; *Y Chromosome/genetics ; Testis/metabolism ; Gene Editing ; Zinc Fingers/genetics ; },
abstract = {BACKGROUND: The Y-linked mouse zinc finger genes Zfy1 and Zfy2 are critical fertility factors in mice but the mechanisms by which they regulate spermatogenesis remain unclear. We recently produced Zfy1/2 double knock-out mice and observed a complete loss in fertility. However, the biochemical mechanism by which Zfy regulates spermatogenesis is unknown, and ZFY expression has not yet been confirmed at the protein level. As both Zfy homologues share ~ 95% sequence similarity, it is difficult to produce an anti-ZFY antibody specific to either homologue.

RESULTS: To overcome this technical challenge, we used CRISPR/Cas9 genome editing to develop tagged Zfy1 knock-in (XY[Zfy1-HA]), Zfy2 knock-in (XY[Zfy2-FLAG], XY[Zfy2-3xFLAG], and XY[Zfy2-HA]), and Zfy1/2 double knock-in (XY[Zfy1-HA,Zfy2-MYC]) mice. Successful targeting was confirmed by genotyping and sequencing. The knock-in lines were fertile with normal sperm parameters. Using Western blot on testes, knock-in specific bands were detected matching the predicted ZFY expression patterns. Using immunofluorescence on testis sections from knock-in males, ZFY1 and ZFY2 expression was detected in zygotene spermatocytes, and ZFY2 expression was also detected in spermatids step 7-8 and 9.

CONCLUSIONS: These novel knock-in mice can be used in future investigations to determine how ZFY controls spermatogenesis.},
}

Animals
*CRISPR-Cas Systems
Male
Mice
*Gene Knock-In Techniques
Spermatogenesis/genetics
*Y Chromosome/genetics
Testis/metabolism
Gene Editing
Zinc Fingers/genetics

@article {pmid41160201,
year = {2026},
author = {Wang, YY and Lin, YH and Ke, CC and Lai, TH and Yu, IS and Au, CF and Singh, R and Lin, YH},
title = {Human teratozoospermia-related AGTPBP1 R791H mutation is associated with sperm head and tail defects in a CRISPR-engineered murine model.},
journal = {Journal of assisted reproduction and genetics},
volume = {43},
number = {1},
pages = {133-142},
pmid = {41160201},
issn = {1573-7330},
support = {NSTC 111-2320-B-030 -007-MY3//National Science and Technology Council/ ; CTH-109A-2215//Cardinal Tien Hospital/ ; },
mesh = {Animals ; Male ; *Teratozoospermia/genetics/pathology ; Mice ; *Sperm Head/pathology ; Humans ; *Sperm Tail/pathology ; Disease Models, Animal ; Spermatozoa/pathology ; CRISPR-Cas Systems/genetics ; Mutation ; *Infertility, Male/genetics/pathology ; *Poly-ADP-Ribose Binding Proteins/genetics ; },
abstract = {BACKGROUND: Infertility is a pervasive global health concern affecting millions of couples worldwide. Approximately 7% of the male population is infertile. Teratozoospermia, defined by > 96% abnormal sperm morphology, is a major cause of infertility often linked to genetic defects. In our previous study, we identified three AGTPBP1 mutations (p.Glu423Asp, p.Pro631Leu, and p.Arg811His) in teratozoospermia cases. AGTPBP1 is a key enzyme involved in regulating tubulin polyglutamylation and generating Δ-2 tubulin, a major structural component of the sperm tail and an essential structure for sperm head differentiation. However, functional proof of the impact of AGTPBP1 Arg811His on sperm head and tail impairment remained unestablished.

METHODS: Knock-in mice carrying the equivalent mutation, Arg791His (R791H) corresponding to the human mutation (R811H), in the Agtpbp1 gene were generated and analyzed for sperm morphological abnormalities.

RESULTS: Sperm morphological evaluation revealed a significant increase in the proportion of morphologically abnormal sperm in the Agtpbp1[R791H/R791H] mice. Detailed morphological analysis revealed a significantly higher incidence of sperm head abnormalities and abnormal attachment of the head to the midpiece in the Agtpbp1[R791H/R791H] mice relative to wild-type controls. Further, sperm with head defects from Agtpbp1[R791H/R791H] mice exhibited abnormal accumulation of polyglutamylated tubulin within the sperm head. The mutant mice showed exactly the same morphological defects as seen in human patients and those displayed by mice lacking the complete carboxypeptidase A domain of AGTPBP1 but at a relatively lesser frequency.

CONCLUSIONS: We conclude that the R791H mutation in the Agtpbp1 gene impairs sperm head and tail differentiation, resulting in sperm morphological defects.},
}

Animals
Male
*Teratozoospermia/genetics/pathology
Mice
*Sperm Head/pathology
Humans
*Sperm Tail/pathology
Disease Models, Animal
Spermatozoa/pathology
CRISPR-Cas Systems/genetics
Mutation
*Infertility, Male/genetics/pathology
*Poly-ADP-Ribose Binding Proteins/genetics

@article {pmid41128695,
year = {2026},
author = {Plugge, SF and Ma, H and van der Vaart, JY and Sprangers, J and Morsink, FHM and Xanthakis, D and Jamieson, C and Stroot, KBW and Keijzer, AR and Margaritis, T and Candelli, T and Straver, R and de Ridder, J and Holstege, FCP and de Leng, WWJ and Offerhaus, GJA and Merenda, A and Maurice, MM},
title = {Intestinal LKB1 Loss Drives a Premalignant Program Along the Serrated Cancer Pathway.},
journal = {Gastroenterology},
volume = {170},
number = {2},
pages = {298-314},
doi = {10.1053/j.gastro.2025.07.041},
pmid = {41128695},
issn = {1528-0012},
mesh = {Humans ; *Protein Serine-Threonine Kinases/genetics/deficiency/metabolism ; Animals ; AMP-Activated Protein Kinase Kinases ; *Peutz-Jeghers Syndrome/genetics/pathology/enzymology ; Organoids/pathology ; *Colorectal Neoplasms/genetics/pathology/enzymology ; Mice ; *Precancerous Conditions/genetics/pathology/enzymology ; Signal Transduction ; ErbB Receptors/metabolism ; Mice, Knockout ; Colon/pathology/enzymology ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Disease Models, Animal ; Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; *Cell Transformation, Neoplastic/genetics/pathology ; Intestinal Mucosa/pathology ; CRISPR-Cas Systems ; AMP-Activated Protein Kinases ; },
abstract = {BACKGROUND & AIMS: Heterozygous inactivating mutations of Serine Threonine Kinase 11/Liver Kinase B1 (LKB1) are causative to the Peutz-Jeghers syndrome (PJS), a hereditary disease characterized by gastrointestinal hamartomatous polyposis and increased cancer susceptibility. Although LKB1 loss-induced polyp formation has been ascribed to nonepithelial tissues, how LKB1 deficiency increases cancer risk of patients by altering the phenotypical landscape and hierarchical organization of epithelial tissues remains poorly understood.

METHODS: Using CRISPR/Cas9, we generated heterozygous and homozygous Lkb1-deficient mouse small intestinal and human colon organoids. These organoids were characterized by an integrated approach that combines imaging, bulk and single-cell RNA sequencing, and growth factor dependency assays. Our findings were validated in human PJS-derived tissues using immunohistochemistry and linked to colorectal cancer profiles using the Cancer Genome Atlas (TCGA) cancer database.

RESULTS: Our results reveal that heterozygous Lkb1 loss is sufficient to push intestinal cells into a premalignant transcriptional program associated with serrated colorectal cancer, which is further amplified by loss of heterozygosity. This altered epithelial growth state associates with persistent features of regeneration and enhanced EGFR ligand and receptor expression, conferring niche-independent growth properties to Lkb1-deficient organoids. Moreover, our newly generated LKB1-mutant signature is enriched in sporadic serrated colorectal cancer, and synergistic cooperation of Lkb1 deficiency with mutant Kras was experimentally confirmed by assessing organoid growth properties and transcriptomes.

CONCLUSIONS: Heterozygous loss of LKB1 pushes intestinal cells into a chronic regenerative state, which is amplified on loss of heterozygosity. Lkb1 deficiency thereby generates fertile ground for serrated colorectal cancer formation in the intestine, potentially explaining the increased cancer risk observed in PJS.},
}

Humans
*Protein Serine-Threonine Kinases/genetics/deficiency/metabolism
Animals
AMP-Activated Protein Kinase Kinases
*Peutz-Jeghers Syndrome/genetics/pathology/enzymology
Organoids/pathology
*Colorectal Neoplasms/genetics/pathology/enzymology
Mice
*Precancerous Conditions/genetics/pathology/enzymology
Signal Transduction
ErbB Receptors/metabolism
Mice, Knockout
Colon/pathology/enzymology
Cell Proliferation
Gene Expression Regulation, Neoplastic
Disease Models, Animal
Proto-Oncogene Proteins p21(ras)/genetics/metabolism
*Cell Transformation, Neoplastic/genetics/pathology
Intestinal Mucosa/pathology
CRISPR-Cas Systems
AMP-Activated Protein Kinases

@article {pmid39894889,
year = {2026},
author = {Shahid, A and Zahra, A and Aslam, S and Shamim, A and Ali, WR and Aslam, B and Khan, SH and Arshad, MI},
title = {Appraisal of CRISPR Technology as an Innovative Screening to Therapeutic Toolkit for Genetic Disorders.},
journal = {Molecular biotechnology},
volume = {68},
number = {1},
pages = {71-94},
pmid = {39894889},
issn = {1559-0305},
support = {DDWP-2021//Ministry of Science and Technology, Pakistan/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Genetic Diseases, Inborn/diagnosis/therapy/genetics ; *Genetic Therapy/methods ; },
abstract = {The high frequency of genetic diseases compels the development of refined diagnostic and therapeutic systems. CRISPR is a precise genome editing tool that offers detection of genetic mutation with high sensitivity, specificity and flexibility for point-of-care testing in low resource environment. Advancements in CRISPR ushered new hope for the detection of genetic diseases. This review aims to explore the recent advances in CRISPR for the detection and treatment of genetic disorders. It delves into the advances like next-generation CRISPR diagnostics like nano-biosensors, digitalized CRISPR, and omics-integrated CRISPR technologies to enhance the detection limits and to facilitate the "lab-on-chip" technologies. Additionally, therapeutic potential of CRISPR technologies is reviewed to evaluate the implementation potential of CRISPR technologies for the treatment of hematological diseases, (sickle cell anemia and β-thalassemia), HIV, cancer, cardiovascular diseases, and neurological disorders, etc. Emerging CRISPR therapeutic approaches such as base/epigenetic editing and stem cells for the development of foreseen CRIPSR drugs are explored for the development of point-of-care testing. A combination of predictive models of artificial intelligence and machine learning with growing knowledge of genetic disorders has also been discussed to understand their role in acceleration of genetic detection. Ethical consideration are briefly discussed towards to end of review. This review provides the comprehensive insights into advances in the CRISPR diagnostics/therapeutics which are believed to pave the way for reliable, effective, and low-cost genetic testing.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*Genetic Diseases, Inborn/diagnosis/therapy/genetics
*Genetic Therapy/methods

@article {pmid41562469,
year = {2026},
author = {Wan, Z and Xu, C and Wang, Y and Song, L and Yuan, W and Chen, M and Gong, R and Zhang, XE},
title = {An AND-Logic Gate-Based Biosensor for Simultaneous Detection of SARS-CoV-2 Nucleic Acids and Nucleocapsid Proteins.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c07321},
pmid = {41562469},
issn = {1520-6882},
abstract = {Nucleic acids and proteins are recognized as gold standard biomarkers for disease diagnosis and pathogen detection. However, conventional single-analyte detection methods remain susceptible to false positives caused by manual operational errors or sample contamination, thereby undermining diagnostic reliability and increasing the burden on healthcare systems. To address this limitation, we developed a one-pot isothermal amplification and CRISPR-Cas cooperative system (OIACS) that functions as an AND-logic gate biosensor for the simultaneous detection of SARS-CoV-2 RNA and nucleocapsid protein. Unlike conventional methods relying solely on CRISPR RNA (crRNA) recognition, the OIACS employs antibody-mediated target binding with blocker release for target recognition, offering increased flexibility in assay design for different targets. A universal Cas12a-targetable DNA barcode is generated via strand displacement isothermal amplification, enabling signal amplification upon dual-target recognition. The OIACS assay exhibited practical utility by reliably detecting SARS-CoV-2 transcription- and replication-competent virus-like-particles at 5000 copies/mL, and the limit of detection was determined to be as low as 1698 copies/mL, highlighting its robustness and potential for clinical diagnosis.},
}

@article {pmid41562237,
year = {2026},
author = {Mattivi, C and Wang, S and Ji, L and Xiao, Q and Cao, J},
title = {HLA-Knockout: Enabling Allele-Specific Knockout of HLA Class I Genes for Immunogenic Engineering.},
journal = {HLA},
volume = {107},
number = {1},
pages = {e70548},
pmid = {41562237},
issn = {2059-2310},
support = {P30CA072720//Rutgers Cancer Institute of New Jersey/ ; //Nanjing University/ ; //Science Fund Program for Distinguished Young Scholars (Overseas)/ ; 82473308//National Natural Science Foundation of China Grant/ ; 2025300355//Fundamental Research Funds for the Central Universities/ ; },
mesh = {Humans ; *Alleles ; *Gene Knockout Techniques/methods ; CRISPR-Cas Systems ; *Gene Editing/methods ; *Histocompatibility Antigens Class I/genetics/immunology ; Receptors, Antigen, T-Cell/genetics/immunology ; },
abstract = {The interaction between T-cell receptors (TCRs) and antigenic peptides presented by HLA molecules is fundamental to adaptive immunity. However, the extreme polymorphism of HLA genes poses major challenges for transplantation, antigen discovery, immunotherapy and studies of allele-specific function. Although CRISPR/Cas9 has transformed gene editing, existing sgRNA design tools are not optimised for knockout of HLA Class I genes due to their high rates of polymorphism. To address this, we developed HLA-Knockout (https://hlaknockout.rutgers.edu), a novel web-based tool that enables precise, allele-specific targeting of HLA Class I genes. HLA-Knockout retrieves user-defined HLA sequences from the IPD-IMGT/HLA database and applies stringent design criteria, including mismatch filtering and PAM disruption analysis, to ensure high specificity and minimal off-target effects on non-target HLA Class I alleles. Using HLA-Knockout, we achieved efficient single- and double-allele HLA Class I knockouts in human cells without disrupting non-target HLA Class I alleles. Functional assays confirmed allele-specific loss of antigen-specific TCR activation, validating the platform's utility. HLA-Knockout provides a unique resource for dissecting HLA-restricted immune interactions and has broad applications in transplantation biology, autoimmunity and cancer immunotherapy.},
}

Humans
*Alleles
*Gene Knockout Techniques/methods
CRISPR-Cas Systems
*Gene Editing/methods
*Histocompatibility Antigens Class I/genetics/immunology
Receptors, Antigen, T-Cell/genetics/immunology

@article {pmid41562083,
year = {2025},
author = {Liu, J and Hong, W and Sun, Z and Zhang, S and Xue, C and Dong, N},
title = {The gut-lung axis: effects and mechanisms of gut microbiota on pulmonary diseases.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1693964},
pmid = {41562083},
issn = {1664-3224},
mesh = {Humans ; *Gastrointestinal Microbiome/immunology ; Animals ; *Lung/immunology/metabolism/microbiology ; Dysbiosis/immunology ; *Lung Diseases/microbiology/immunology/metabolism/therapy/etiology ; Fatty Acids, Volatile/metabolism ; Fecal Microbiota Transplantation ; },
abstract = {The proposal of the gut-lung axis has profoundly reshaped our understanding of the mechanisms underlying respiratory diseases. As a crucial component of this axis, the gut microbiota plays a central role in pulmonary immune regulation through inter-organ communication mediated by metabolic products. However, a systematic integration of mechanisms explaining how gut microbes achieve precise cross-organ immune regulation remains elusive. Existing research predominantly focuses on descriptive observations, such as the association between early-life microbiota dysbiosis and an increased risk of asthma and chronic obstructive pulmonary disease (COPD), as well as the frequent occurrence of acute respiratory distress syndrome (ARDS) and pulmonary fibrosis (PF), often accompanied by gut microbiome disruption. This paper focuses on three key gut microbial metabolites-short-chain fatty acids (SCFAs), tryptophan metabolites, and polyamines (PAs)-to examine their roles in immune regulation, maintenance of barrier function, and modulation of metabolic signaling networks. Based on the latest experimental and clinical evidence, this study systematically elucidates how dysbiosis of the gut microbiota, a key component of the gut-lung axis, crosses physiological barriers to exacerbate pulmonary inflammation. Regarding intervention strategies, probiotics, fecal microbiota transplantation (FMT), and CRISPR-Cas systems have demonstrated significant therapeutic potential in restoring gut microbial balance. Finally, this paper outlines future research directions, emphasizing the need to further explore non-invasive microbial sampling techniques, molecular interaction mechanisms of the gut-lung axis, and personalized microbiome-based diagnostic and therapeutic strategies to provide new insights for the prevention and treatment of respiratory diseases involving gut microbiota.},
}

Humans
*Gastrointestinal Microbiome/immunology
Animals
*Lung/immunology/metabolism/microbiology
Dysbiosis/immunology
*Lung Diseases/microbiology/immunology/metabolism/therapy/etiology
Fatty Acids, Volatile/metabolism
Fecal Microbiota Transplantation

@article {pmid41561392,
year = {2026},
author = {Choudhary, DK and Turgeman-Grott, I and Robinzon, S and Gophna, U},
title = {CRISPR-Cas targeting in Haloferax volcanii promotes within-species gene exchange by triggering homologous recombination.},
journal = {microLife},
volume = {7},
number = {},
pages = {uqaf047},
pmid = {41561392},
issn = {2633-6693},
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated genes) systems provide adaptive immunity in bacteria and archaea against mobile genetic elements, but the role they play in gene exchange and speciation remains unclear. Here, we investigated how CRISPR-Cas targeting affects mating and gene exchange in the halophilic archaeon Haloferax volcanii. Surprisingly, we found that CRISPR-Cas targeting significantly increased mating efficiency between members of the same species, in contrast to its previously documented role in reducing interspecies mating. This enhanced mating efficiency was dependent on the Cas3 nuclease/helicase and extended beyond the targeted genomic regions. Further analysis revealed that CRISPR-Cas targeting promoted biased recombination in favor of the targeting strain (the strain containing the CRISPR-Cas system) during mating, resulting in an increased proportion of recombinant progeny that are positive for CRISPR-Cas. To test whether an increase in recombination is sufficient to increase mating efficiency, we tested whether strains lacking the Mre11-Rad50 complex, which are known to have elevated recombination activity, also exhibited higher mating success. Indeed, these strains showed higher mating, as did cells that were exposed to DNA damage using methyl methanesulfonate. These findings suggest that CRISPR-Cas systems in archaea play roles beyond their canonical immune function. They may contribute to speciation by facilitating within-species gene exchange while limiting between-species genetic transfer, thereby maintaining species boundaries.},
}

@article {pmid41561390,
year = {2026},
author = {Fehrenbach, A and Mitrofanov, A and Backofen, R and Baumdicker, F},
title = {The complexity of multiple CRISPR arrays in strains with (co-occurring) CRISPR systems.},
journal = {microLife},
volume = {7},
number = {},
pages = {uqaf042},
pmid = {41561390},
issn = {2633-6693},
abstract = {CRISPR and their associated Cas proteins provide adaptive immunity in prokaryotes, protecting against invading genetic elements. These systems are categorized into types and are highly diverse. Genomes often harbor multiple CRISPR arrays varying in length and distance from Cas loci. However, the ecological roles of multiple CRISPR arrays and their interactions with multiple Cas loci remain poorly understood. We present a comprehensive analysis of CRISPR systems that uncovers variation between diverse Cas types regarding the occurrence of multiple arrays, the distribution of their lengths and positions relative to Cas loci, and the diversity of their repeat sequences. Some types tend to occur as the sole Cas locus present in the genome, but typically have two or more associated arrays, especially for types I-E and I-F. Multiple Cas types are also common, with some systems showing a preference for specific co-occurrence. Distinct array distributions and orientations around Cas loci indicate substantial differences in functionality and transcriptional behavior among Cas types. Our analysis suggests that arrays with identical repeats in the same genome acquire new spacers at comparable rates, irrespective of their proximity to the Cas locus. Furthermore, repeat similarities indicate that arrays of systems that often co-occur with other systems tend to have more diverse repeats than those mostly appearing alongside solitary systems. Our results indicate that co-occurring Cas-type pairs might not only collaborate in spacer acquisition but also maintain independent and complementary functions and that CRISPR systems distribute their defensive spacer repertoire equally across multiple CRISPR arrays.},
}

@article {pmid41560876,
year = {2026},
author = {More, R and Yadav, V and Vadakedath, N},
title = {Calyptranema fuscum gen. sp. nov.: a novel cyanobacterial genus within Oculatellaceae based on polyphasic and genomic characterization.},
journal = {Current research in microbial sciences},
volume = {10},
number = {},
pages = {100542},
pmid = {41560876},
issn = {2666-5174},
abstract = {This study presents a comprehensive characterization and classification of a novel cyanobacterial isolate, strain S582, proposed as Calyptranema fuscum gen. sp. nov. within the family Oculatellaceae using an integrated polyphasic approach. Strain S582 was isolated from a lake in the Botanical Garden, Sarangpur, Chandigarh, India. Initial molecular characterization with the 16S rRNA gene revealed ≤ 94.90% of similarity with related genera and showed unique 16S-23S ITS secondary structures, indicating its delineation as a novel genus. Morphological assessment highlighted the presence of a cap-like structure called calyptra at the terminal cells, further distinguishing it from related genera. Furthermore, whole genome sequencing yielded an assembly of 7962,515 bp with GC content of 48.27%. Genome-based analysis encompassing average nucleotide identity (ANI), average amino acid identity (AAI), percentage of conserved proteins (POCP) was subsequently performed. The observed values for ANI (71.15% to 73.00%) and AAI (63.30% to 69.62%) were below the established genus-level thresholds. Phylogenetic analysis using maximum-likelihood and Bayesian inference along with phylogenomic reconstruction based on 1434 single copy core genes supported its taxonomic novelty. Functional classification revealed the presence of biosynthetic gene clusters (BGCs), tRNAs, insertion elements, CRISPR/Cas systems, and genes associated with metabolism, carbon fixation and photosynthesis. Additionally, the pangenome was constructed to study the genomic diversity of the studied isolate and related genera among the Oculatellaceae family and identified core, accessory, and singleton gene clusters. Collectively, these findings establish Calyptranema fuscum gen. sp. nov. as a novel genus within Oculatellaceae while expanding our understanding of cyanobacterial diversity and genomic potential.},
}

@article {pmid41556308,
year = {2026},
author = {Xie, Z and Jin, YS and Miller, MJ},
title = {Exploiting the Endogenous Type II-A CRISPR-Cas System for Functional Engineering of Probiotic Lacticaseibacillus rhamnosus GG.},
journal = {Microbial biotechnology},
volume = {19},
number = {1},
pages = {e70303},
pmid = {41556308},
issn = {1751-7915},
support = {//University of Illinois at Urbana-Champaign/ ; },
mesh = {*CRISPR-Cas Systems ; *Lacticaseibacillus rhamnosus/genetics ; *Gene Editing/methods ; *Probiotics ; Glucuronidase/genetics/metabolism ; },
abstract = {Lacticaseibacillus rhamnosus GG (LGG) is one of the most extensively studied probiotic strains, widely used in food and health applications. However, the absence of efficient, precise genome editing methods has limited its broader potential and functional versatility. Here, we present an endogenous type II-A CRISPR-Cas genome editing workflow for LGG designed for functional strain construction. Using a plasmid interference assay together with single-nucleotide substitutions, we confirm the precise PAM requirement as 5'-NGAAA-3'. We pair a synthetic sgRNA cassette with homology-directed repair donors to enable targeted deletions and insertions across multiple loci, achieving modest but practically relevant editing efficiencies (11.1-25.0% of recovered transformants) that support routine strain construction. Using this optimised and precise genome engineering method, we generated a β-glucuronidase (GUS)-expressing LGG strain for robust strain tracking within complex microbial communities. This work removes barriers to LGG engineering, expands the probiotic CRISPR toolkit, and provides broadly applicable strategies for designing next-generation probiotics with applications in food biotechnology and microbial therapeutics.},
}

*CRISPR-Cas Systems
*Lacticaseibacillus rhamnosus/genetics
*Gene Editing/methods
*Probiotics
Glucuronidase/genetics/metabolism

@article {pmid41556260,
year = {2026},
author = {Neherin, K and Holloway, K and Song, Y and Houston, A and Chen, F and Ding, L and Zhang, H},
title = {Introducing Cellular Senescence in Human Induced Pluripotent Stem Cells and Differentiated Neural Lineage for Modeling of Age-Associated Diseases.},
journal = {Advanced biology},
volume = {10},
number = {1},
pages = {e00468},
doi = {10.1002/adbi.202500468},
pmid = {41556260},
issn = {2701-0198},
support = {RF1AG056302/AG/NIA NIH HHS/United States ; R21AG086894/AG/NIA NIH HHS/United States ; U54AG075934//NIH Common Fund/ ; },
mesh = {Humans ; *Cellular Senescence/genetics/physiology ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Cell Differentiation ; *Neural Stem Cells/cytology/metabolism ; *Aging/genetics ; CRISPR-Cas Systems ; Cell Lineage ; },
abstract = {Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) resets the epigenetic landscapes that mark the aging clock, and consequently cells differentiated from iPSCs resemble fetal cells rather than adult or aged cells. The lack of proper cellular aging in cells differentiated from iPSCs presents a unique challenge in iPSC-based modeling of age-associated diseases such as neurodegeneration. To address this challenge, we seek to introduce cellular senescence, a hallmark of aging, into iPSC-based models in a robust and temporally controlled manner. An inducible CRISPR interference (CRISPRi) is used to suppress the expression of TERF2, a key component of the telomere protecting Shelterin complex. We demonstrate that suppression of TERF2 robustly activates the DNA damage response, p53/p21 signaling, and cellular senescence in iPSCs in a highly homogeneous and synchronous manner. Applying this inducible CRISPRi-TERF2 system to differentiation of iPSCs to neural progenitor cells (NPCs), we show efficient activation of senescence-associated phenotypes in NPCs. This inducible cell model allows isogenic comparisons of the same cell populations over the course of differentiation with or without the activation of cellular senescence in a synchronous and homogeneous manner, and has broad applications in investigating the role of cellular senescence in the progression of age-related diseases.},
}

Humans
*Cellular Senescence/genetics/physiology
*Induced Pluripotent Stem Cells/cytology/metabolism
*Cell Differentiation
*Neural Stem Cells/cytology/metabolism
*Aging/genetics
CRISPR-Cas Systems
Cell Lineage

@article {pmid41555514,
year = {2026},
author = {Xiao, B and Zhang, J},
title = {A Simple Programmable Cas12a/crRNA Induced Walking System for Sensitive Methicillin-Resistant Staphylococcus aureus Detection via Integrated cis- and trans-Cleavage Activity.},
journal = {Journal of microbiology and biotechnology},
volume = {36},
number = {},
pages = {e2511026},
doi = {10.4014/jmb.2511.11026},
pmid = {41555514},
issn = {1738-8872},
mesh = {*Methicillin-Resistant Staphylococcus aureus/isolation & purification/genetics ; *CRISPR-Cas Systems/genetics ; *Bacterial Proteins/genetics/metabolism ; Aptamers, Nucleotide/genetics ; *Staphylococcal Infections/diagnosis/microbiology ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *Biosensing Techniques/methods ; Humans ; },
abstract = {Methicillin-resistant Staphylococcus aureus (MRSA) represents a serious threat to public health due to its strong antibiotic resistance, wide dissemination, and high infection rates. Rapid identification of MRSA strains is essential for accurate diagnosis and timely treatment of related infections. In this study, we propose an analytical method for MRSA that employs a hairpin-structured locker-probe to directly regulate the trans-cleavage activity of Cas12a. This designed locker-probe connects a target-specific aptamer to an inhibitory aptamer of the CRISPR/Cas12a system. Upon binding to the specific target, the probe undergoes a conformational change that abolishes its inhibitory effect on Cas12a. As a result, the structure-switchable probe modulates Cas12a activity in a target-dependent manner. Additionally, the sensing substrate combines a "cis-cleavage trigger" and a "trans-cleavage trigger" to integrate both cis- and trans-cleavage activities of Cas12a/crRNA within a single probe. This design significantly simplifies the probe architecture while maintaining high signal amplification efficiency. The proposed method was successfully applied to detect MRSA, achieving a detection limit as low as 2.5 CFU/ml with high specificity. By exploiting the inhibitory aptamer of Cas12a as a regulatory element for MRSA analysis, this work expands the toolbox of CRISPR/Cas12a-based methodologies and offers a promising strategy for bacterial detection.},
}

*Methicillin-Resistant Staphylococcus aureus/isolation & purification/genetics
*CRISPR-Cas Systems/genetics
*Bacterial Proteins/genetics/metabolism
Aptamers, Nucleotide/genetics
*Staphylococcal Infections/diagnosis/microbiology
*CRISPR-Associated Proteins/genetics/metabolism
*Endodeoxyribonucleases/genetics/metabolism
*Biosensing Techniques/methods
Humans

@article {pmid41555077,
year = {2026},
author = {Yu, L and Yin, M and Zhu, Y and Lu, Z and Xiao, B and Zhou, F and Yu, Y and Huang, Z},
title = {An anti-CRISPR targets the sgRNA to block Cas9 and guides the design of enhanced genome editors.},
journal = {Nature structural & molecular biology},
volume = {},
number = {},
pages = {},
pmid = {41555077},
issn = {1545-9985},
abstract = {Bacteriophages have evolved anti-CRISPR (Acr) proteins to combat the adaptive immunity provided by bacterial CRISPR-Cas systems. Here, we report the cryo-electron microscopy structure of an anti-Cas9 protein AcrIIA27 bound to SpyCas9-sgRNA (single guide RNA) complex. Our structure reveals that AcrIIA27 binds the solvent-exposed phosphate backbone of the sgRNA, acting as a potent inhibitor of diverse Cas9 orthologs. AcrIIA27 in the structure is positioned near the protospacer-adjacent motif DNA-binding pocket on SpyCas9, causing steric hindrance that prevents substrate DNA recognition. This mechanism suggests solvent-exposed regions of sgRNAs (PTP RNAs), prone to nonspecific binding of positively charged components, may compromise CRISPR-Cas genome-editing efficiency. Indeed, truncations of the PTP RNAs in different editing systems significantly enhance genome-editing efficiency in human cells. Overall, our findings reveal a previously uncharacterized inhibition mechanism of an anti-Cas protein and offers a general strategy for developing more efficient genome-editing tools.},
}

@article {pmid41554027,
year = {2025},
author = {Ireri, SW and Cao, M},
title = {CRISPR-Cas9-based Mutagenesis in the Entomopathogenic Nematode Steinernema hermaphroditum and the Maintenance of Mutant Lines.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {226},
pages = {},
doi = {10.3791/68932},
pmid = {41554027},
issn = {1940-087X},
mesh = {Animals ; *CRISPR-Cas Systems ; Mutagenesis ; *Rhabditida/genetics ; Microinjections/methods ; Gene Editing/methods ; },
abstract = {Entomopathogenic nematodes (EPNs) in the genus Steinernema and Heterorhabditis maintain mutualistic interactions with Xenorhabdus and Photorhabdus symbiotic bacteria, respectively. Together, these nematode-bacterium pairs infect and kill insect hosts that are primarily larvae from the orders of Lepidoptera and Coleoptera, forming a tractable tripartite system for dissecting the molecular basis of mutualism and parasitism. A key step towards fully utilizing this model is the development of stable and transgenerational genetic tools in EPNs. Here, we demonstrate a reliable CRISPR-Cas9 genome editing platform in the emerging model Steinernema hermaphroditum, a species that is readily maintained in vivo and in vitro, and is highly amenable to gonadal microinjection. Importantly, its hermaphroditic reproduction greatly streamlines the generation and maintenance of homozygous mutant lines. We provide a detailed protocol for efficient, targeted gene disruption using microinjection-based delivery of Cas9 ribonucleoprotein complexes. As a proof of concept, we modified the conserved muscle-associated gene unc-22, generating a characteristic twitching phenotype that validates targeted mutagenesis in this system. This CRISPR-Cas9 platform opens the door to stable genetic manipulation in S. hermaphroditum, such as transgene expression, and provides a framework that can be extended to additional EPN species of agricultural and ecological importance.},
}

Animals
*CRISPR-Cas Systems
Mutagenesis
*Rhabditida/genetics
Microinjections/methods
Gene Editing/methods

@article {pmid41540063,
year = {2026},
author = {Port, F and Buhmann, MA and Zhou, J and Stricker, M and Vaughan-Brown, A and Michalsen, AC and Roßmanith, E and Pöltl, A and Großkurth, L and Huber, J and Menendez Kury, LB and Weberbauer, B and Hübl, M and Puscher, E and Heigwer, F and Boutros, M},
title = {Improved in vivo gene knockout with high specificity using multiplexed Cas12a sgRNAs.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {877},
pmid = {41540063},
issn = {2041-1723},
support = {ERC-DECODE//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; SFB1324//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {Animals ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; Gene Editing/methods ; Drosophila melanogaster/genetics ; Gene Targeting/methods ; *Bacterial Proteins/genetics/metabolism ; Drosophila/genetics ; },
abstract = {CRISPR nuclease-mediated gene knock-out is limited by suboptimal sgRNAs, inaccessible target sites, and undesired repair outcomes. Here, we present a Cas12a-based system in Drosophila that targets each gene with four sgRNAs to overcome these limitations. Multiplexed sgRNAs act through redundancy and synergism, frequently creating deletions between target sites and increasing the fraction of loss-of-function mutations. We show that multiplexed gene targeting is well tolerated and does not cause widespread proximity effects. To visualize CRISPR-nuclease activity in living animals, we developed a screening assay and used it to assess Cas12a activity across 33% of the Drosophila genome in combination with over 2000 sgRNAs. This revealed remarkably high on-target (>99%) and very low (<1%) off-target activity of multiplexed Cas12a sgRNA arrays. Quantitative side-by-side comparisons with current Cas9-based systems targeting over 100 genes in parallel demonstrate that multiplexed Cas12a gene targeting achieves superior performance and reveals phenotypes missed by established methods. The system described here provides a framework for reliable gene knock-out in multicellular systems.},
}

Animals
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Gene Knockout Techniques/methods
*CRISPR-Associated Proteins/genetics/metabolism
*Endodeoxyribonucleases/genetics/metabolism
Gene Editing/methods
Drosophila melanogaster/genetics
Gene Targeting/methods
*Bacterial Proteins/genetics/metabolism
Drosophila/genetics

@article {pmid41506457,
year = {2026},
author = {Jiménez Lancho, V and Leitner, K and Agarwal, K and Krishnakumar, A and Khetan, A and Borth, N and Marx, N},
title = {CRISPR-based precise methylation of specific FUT8 promoter regions allows isolation of CHO cells with a fine-tuned glycoprofile.},
journal = {Journal of biotechnology},
volume = {410},
number = {},
pages = {341-352},
doi = {10.1016/j.jbiotec.2026.01.001},
pmid = {41506457},
issn = {1873-4863},
mesh = {CHO Cells ; Cricetulus ; Animals ; *DNA Methylation/genetics ; *Promoter Regions, Genetic/genetics ; *Fucosyltransferases/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Glycosylation ; Gene Editing/methods ; },
abstract = {A major advantage of producing therapeutic proteins in mammalian cells is their ability to tailor proteins with human-like posttranslational modifications such as glycosylation, which ultimately defines aspects like stability, protein folding or immunogenicity. However, producing therapeutic proteins with a consistent and reproducible glycoprofile remains a major challenge for the biopharmaceutical industry, especially with biosimilar production. While the enzymes responsible for glycosylation of proteins have been the subject of various cell engineering approaches, tuning their gene expression to precise levels is still difficult to achieve. While CRISPR/Cas9 enabled the genetic engineering of cells to drastically overexpress or remove a target gene, CRISPR/dCas9-based epigenetic editing by targeted DNA methylation promises to stably change the expression pattern of target genes after transient transfection of the CRISPR-tool. Application of targeted DNA methylation so far has mostly been used to completely silence gene expression by fully methylating the corresponding promoter regions. Here, we aim to tune expression of the associated gene by DNA methylation of confined promoter regions and to apply this technique as a new glycoengineering approach. By coupling CRISPR-based targeted DNA methylation with lectin-FACS assisted sorting we obtained CHO cell lines with a fine-tuned phenotype. First, dCas9-DNMT3A3L in combination with one single gRNA is targeted to the FUT8 promoter to induce confined DNA methylation, resulting in a phenotypically diversified population. Next, a window sorting strategy based on lectin-stained cells using five different sorting gates spanning from low to high FUT8 expression was applied to isolate single clones with a defined phenotype. Isolated clones were phenotypically assessed and re-sorted to obtain a homogenous expression profile. The resulting clonal cell lines showed either tuned or knock-down phenotypes with varying gene expression levels. Two out of seven clones that showed tuned FUT8 gene expression were phenotypically stable over 60 days. Gene expression levels, on the other hand, showed a steady decline over time that in part, however, can be explained by the general variation of FUT8 expression in different growth phases. Importantly, glycan analysis of recombinant EpoFc produced in the tuned clonal cell lines showed ranges of 35-70 % fucosylation, demonstrating that isolated clones can produce recombinant proteins with a distinct glycosylation profile. To understand why some clones showed tuned FUT8 gene expression levels while others were completely knocked-down, we analyzed the DNA methylation status of their respective FUT8 promoter. Critical areas within the FUT8 promoter were identified, with some associated with general repression and others with the tuning of FUT8 gene expression when affected by DNA methylation. Additionally, a combination of histone marks associated with active and repressed promoters was found to potentially define clones with a fine-tuned expression. Combined, the data demonstrates that using targeted DNA methylation in a manner confined to specific promoter regions opens new engineering strategies to fine-tune gene expression in mammalian cells.},
}

CHO Cells
Cricetulus
Animals
*DNA Methylation/genetics
*Promoter Regions, Genetic/genetics
*Fucosyltransferases/genetics/metabolism
*CRISPR-Cas Systems/genetics
Glycosylation
Gene Editing/methods

@article {pmid41496287,
year = {2026},
author = {Chatrousse, L and Poullion, T and Mahiou, H and El-Kassar, L and Giraud-Triboult, K and Boissart, C and Benchoua, A},
title = {Knock-out of specific DMD gene isoforms in the parental hESC line SA001 using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103899},
doi = {10.1016/j.scr.2025.103899},
pmid = {41496287},
issn = {1876-7753},
mesh = {Humans ; *Dystrophin/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; Protein Isoforms/genetics/metabolism ; Cell Line ; Male ; Muscular Dystrophy, Duchenne/genetics ; *Gene Knockout Techniques ; },
abstract = {The DMD gene, which encodes the protein dystrophin, is involved in a group of diseases known as dystrophinopathies, which includes Duchenne Muscular Dystrophy (DMD). DMD is a progressive and lethal muscular disorder mainly affecting boys that results from the loss of function of the longer dystrophin isoform DP427 in skeletal muscles. Dystrophinopathies are also associated with poorly understood neurocognitive and neurodevelopmental disorders. To investigate the role of dystrophin isoforms in neural development, we specifically disrupted three dystrophin isoforms expressed in the brain, namely DP427, DP140 and DP71, in the male human embryonic stem cell line SA001 using the CRISPR/Cas9 system. (100 / 100 words).},
}

Humans
*Dystrophin/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Human Embryonic Stem Cells/metabolism/cytology
Protein Isoforms/genetics/metabolism
Cell Line
Male
Muscular Dystrophy, Duchenne/genetics
*Gene Knockout Techniques

@article {pmid41496285,
year = {2026},
author = {Wang, J and Gao, Y and Jin, K and Jin, ZB and Xu, J},
title = {Generation of RB1 knockout human embryonic stem cell lines derived from H9 using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103897},
doi = {10.1016/j.scr.2025.103897},
pmid = {41496285},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Knockout Techniques ; *Retinoblastoma Binding Proteins/genetics/metabolism/deficiency ; Cell Line ; *Ubiquitin-Protein Ligases/genetics/deficiency/metabolism ; Cell Differentiation ; Animals ; Mice ; },
abstract = {RB1 is a tumor suppressor gene which plays a crucial role in cell cycle and cellular differentiation. Mutations or loss of RB1 are associated with retinoblastoma and a variety of other cancers. We generated RB1 knockout human embryonic stem cell (hESC) lines (H9) using CRISPR/Cas9-mediated gene targeting. These RB1-deficient cell lines maintain typical stem cell morphology, normal karyotype and expression of pluripotent marker genes. Furthermore, they retain their in vivo differentiation capacity, enabling the generation of multiple cell lineages. These RB1 knockout hESC lines provide valuable models for investigating the role of RB1 in tumorigenesis, neurodevelopment, and cell cycle regulation.},
}

Humans
*CRISPR-Cas Systems/genetics
*Human Embryonic Stem Cells/metabolism/cytology
*Gene Knockout Techniques
*Retinoblastoma Binding Proteins/genetics/metabolism/deficiency
Cell Line
*Ubiquitin-Protein Ligases/genetics/deficiency/metabolism
Cell Differentiation
Animals
Mice

@article {pmid41496284,
year = {2026},
author = {Fan, M and Zhao, M and Su, W and Tang, Z and Sun, W and Zhou, T and Liu, P},
title = {Generation of a PLIN2-GFP2-P2A-Puro human induced pluripotent stem cell line (SEUi001-A) via CRISPR/Cas9-mediated gene editing technology.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103896},
doi = {10.1016/j.scr.2025.103896},
pmid = {41496284},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Perilipin-2/genetics/metabolism ; Cell Line ; Green Fluorescent Proteins/metabolism/genetics ; Cell Differentiation ; },
abstract = {Perilipin 2 (PLIN2) dysregulation drives metabolic pathologies including non-alcoholic fatty liver disease (NAFLD). To enable real-time tracking of PLIN2 dynamics, we established a human induced pluripotent stem cell (hiPSC) line with endogenous GFP2 knock-in at the PLIN2 locus via CRISPR/Cas9-mediated non-homologous end joining (NHEJ). This PLIN2-GFP2 reporter line demonstrated synchronous fluorescence and transcriptional expression validated by flow cytometry. Genomic integrity was confirmed by normal diploid karyotype (46, XY). Pluripotency markers (POU5F1, SOX2, NANOG) were stably expressed. Furthermore, the cells possessed the ability to differentiate into three germ layers. As the first reported endogenous PLIN2 reporter in human stem cells, this model overcomes limitations of antibody-based detection and transgenic overexpression systems, preserving native regulatory mechanisms. The model provides a physiologically relevant platform for: (1) live monitoring of LD-mitochondria interactions, (2) high-throughput compound screening for metabolic disorders, and (3) modeling NAFLD pathogenesis in vitro, advancing precision therapeutics and mechanistic disease modeling.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Perilipin-2/genetics/metabolism
Cell Line
Green Fluorescent Proteins/metabolism/genetics
Cell Differentiation

@article {pmid41496283,
year = {2026},
author = {Low, YC and McKnight, CL and Stojanovski, D and Thorburn, DR and Frazier, AE},
title = {Generation of a pluripotent human AGK knockout embryonic stem cell model (WAe009-A-3C) of Sengers syndrome.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103895},
doi = {10.1016/j.scr.2025.103895},
pmid = {41496283},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *Phosphotransferases (Alcohol Group Acceptor)/genetics/metabolism/deficiency ; Cell Line ; CRISPR-Cas Systems ; *Pluripotent Stem Cells/metabolism ; Gene Knockout Techniques ; *Mitochondrial Diseases/genetics/pathology ; Gene Editing ; Cell Differentiation ; },
abstract = {Sengers syndrome is a rare mitochondrial disorder caused by the loss of a nuclear encoded mitochondrial protein, acylglycerol kinase (AGK). Here, we describe the generation of a novel in vitro stem cell model of Sengers syndrome (AGK[KO] C10) using CRISPR/Cas9 gene editing. This cell line displayed normal characteristics of pluripotent stem cells, including colony morphology, expression of pluripotency markers, trilineage potential, and no karyotypic abnormalities. Together with the parental H9 hESC control line, the AGK[KO] C10 line can ultimately be used for investigation of disease mechanisms and drug testing.},
}

Humans
*Human Embryonic Stem Cells/metabolism/cytology
*Phosphotransferases (Alcohol Group Acceptor)/genetics/metabolism/deficiency
Cell Line
CRISPR-Cas Systems
*Pluripotent Stem Cells/metabolism
Gene Knockout Techniques
*Mitochondrial Diseases/genetics/pathology
Gene Editing
Cell Differentiation

@article {pmid41496278,
year = {2026},
author = {Bozaoglu, K and Massie, S and Irion, FE and Davies, KC and Kantor, I and Raabus, M and Haebich, KM and Vlahos, K and Howden, SE and Wright, J and Payne, JM and Lockhart, PJ},
title = {Simultaneous reprogramming and gene correction to generate six iPSC lines and isogenic controls from individuals with neurofibromatosis type 1.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103904},
doi = {10.1016/j.scr.2025.103904},
pmid = {41496278},
issn = {1876-7753},
mesh = {Humans ; *Neurofibromatosis 1/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Cellular Reprogramming ; *Gene Editing ; CRISPR-Cas Systems ; Cell Line ; Male ; Female ; },
abstract = {Neurofibromatosis type 1 (NF1) is a neurodevelopmental disorder that affects ∼ 1: 2700 individuals (Lee et al., 2023) however the underlying pathogenic mechanisms are poorly understood. In this study, we performed simultaneous reprogramming and CRISPR-Cas9 genome editing to generate pluripotent stem cell (iPSCs) lines and their respective isogenic controls from six individuals with different pathogenic NF1 variants. All iPSC lines had a normal karyotype, were pluripotent and able to differentiate into the three embryonic germ layers. These iPSC lines are valuable pre-clinical models to investigate the pathomechanisms of NF1 and can be used for future screening to identify new therapeutic treatments for NF1.},
}

Humans
*Neurofibromatosis 1/genetics/pathology/metabolism
*Induced Pluripotent Stem Cells/metabolism/cytology
*Cellular Reprogramming
*Gene Editing
CRISPR-Cas Systems
Cell Line
Male
Female

@article {pmid41483428,
year = {2026},
author = {Deng, X and Gao, Q and Shen, K and Mu, W and Ge, T and Gu, J and Yang, X and Cheng, J and Wang, J and Zhang, W and Li, D and Zhou, J and Xiao, M},
title = {TNFRSF13B Variant-Induced TACI Dysregulation Underlies CAEBV Pathogenesis.},
journal = {Journal of clinical immunology},
volume = {46},
number = {1},
pages = {12},
pmid = {41483428},
issn = {1573-2592},
support = {82200259//National Natural Science Foundation of China/ ; 81830008//National Natural Science Foundation of China/ ; 82270203//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Transmembrane Activator and CAML Interactor Protein/genetics/metabolism ; *Epstein-Barr Virus Infections/genetics/immunology/metabolism ; *Herpesvirus 4, Human/physiology/immunology ; Frameshift Mutation ; Signal Transduction ; Exons/genetics ; CRISPR-Cas Systems ; },
abstract = {The tumor necrosis factor (TNF) receptor superfamily member, transmembrane activator and CAML interactor (TACI) encoded by TNFRSF13B, are extensively involved in immune responses. In our previous work, TNFRSF13B exon 2 variants were recurrently identified in chronic active Epstein-Barr virus disease (CAEBV). Here we aim to reveal the roles of TNFRSF13B variants in CAEBV, and investigate the feasibility of targeting TNFRSF13B/TACI as a new approach to control EBV infection. The lymphoblastoid cell lines (LCL) models carrying homozygous TNFRSF13B exon 2 frameshift mutations were constructed using CRISPR/Cas9. Immunological assays, transcriptomic analysis, and gene silencing experiments were performed on LCL models to measure the effect of TNFRSF13B exon 2 variants and explore the underlying mechanisms. TACI ligands and a TLR9 agonist were applied to modulate TACI signaling and EBV activities. Frameshift mutations in exon 2 of TNFRSF13B significantly up-regulated the short isoforms of TACI (TACI-S) at the expense of its long isoforms (TACI-L) in LCLs. The up-regulated TACI-S induced more intense activation of NF-κB, MAPK, and Rho signaling pathways, leading to the switch of EBV activities to lytic reactivation. The subsequent increased viral load and viral IL-10 provide a rational for the susceptibility of variant carriers to CAEBV. The BAFF trimer, an indirect TACI-signaling inhibitor, also significantly suppressed the EBV lytic program. Gene silencing experiments indicated that XBP-1 might be involved in the TACI-mediated regulation of EBV lytic activities in EBV-immortalized B cells. This study underscores the impact of TNFRSF13B variants on EBV infection and host immune responses, offering insights into CAEBV pathogenesis and potential therapeutic strategies.},
}

Humans
*Transmembrane Activator and CAML Interactor Protein/genetics/metabolism
*Epstein-Barr Virus Infections/genetics/immunology/metabolism
*Herpesvirus 4, Human/physiology/immunology
Frameshift Mutation
Signal Transduction
Exons/genetics
CRISPR-Cas Systems

@article {pmid41478438,
year = {2026},
author = {Ma, Q and Wang, N and Qiao, K and Luo, K and Zhao, C and Yan, J and Fan, S and Rong, J and Ma, Q},
title = {The R2R3-MYB transcription factor GhMYB35 governs anther development and pollen viability in upland cotton.},
journal = {Gene},
volume = {984},
number = {},
pages = {149990},
doi = {10.1016/j.gene.2025.149990},
pmid = {41478438},
issn = {1879-0038},
mesh = {*Gossypium/genetics/growth & development/metabolism ; *Pollen/genetics/growth & development ; *Transcription Factors/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *Flowers/genetics/growth & development ; Plant Infertility/genetics ; CRISPR-Cas Systems ; },
abstract = {Male reproductive development is fundamental to the life cycle of flowering plants, culminating in seed production. Aberrations in anther development frequently lead to male sterility, yet the underlying molecular mechanisms in upland cotton (Gossypium hirsutum) remain largely uncharacterized. The R2R3-MYB family of transcription factors are known key regulators of diverse developmental processes, including male fertility in several model species. Here, we identify and functionally characterize GhMYB35, an R2R3-MYB transcription factor that plays an essential role in cotton anther development. CRISPR/Cas9-mediated knockout of GhMYB35 resulted in complete male sterility, with mutants (ghmyb35) exhibiting indehiscent anthers, shorter filaments, and a total absence of viable pollen. Expression analyses reveal that GhMYB35 is predominantly expressed in anthers, with peak expression of its A- and D-subgenome homoeologs occurring at developmental stage 7. Subcellular localization results show that bothGhMYB35_AandGhMYB35_Dare nuclear-localized transcription factors. Furthermore, the total absence of GhMYB35 leads to pollen abortion and subsequent anther collapse without dehiscence. Collectively, our findings establish GhMYB35 as a critical regulator of anther maturation, thereby elucidating a key component of the molecular network governing male fertility in cotton.},
}

*Gossypium/genetics/growth & development/metabolism
*Pollen/genetics/growth & development
*Transcription Factors/genetics/metabolism
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
*Flowers/genetics/growth & development
Plant Infertility/genetics
CRISPR-Cas Systems

@article {pmid41476041,
year = {2025},
author = {Xi, W and Xu, Y and Bao, W and Ding, Z and Xiao, H and Yang, J and Yan, X and Ping, Y},
title = {In vivo chemogenetic RNA editing of macrophages by bioengineered viruses for sepsis treatment.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {927},
pmid = {41476041},
issn = {2041-1723},
support = {82425055//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32261143727//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82504685//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2024M762890//China Postdoctoral Science Foundation/ ; 2025T180980//China Postdoctoral Science Foundation/ ; GZB20240672//China Postdoctoral Science Foundation/ ; LMS25H300002//Natural Science Foundation of Zhejiang Province (Zhejiang Provincial Natural Science Foundation)/ ; },
mesh = {*Sepsis/therapy/genetics/immunology ; Animals ; *Macrophages/metabolism ; Mice ; *RNA Editing/genetics ; *Lentivirus/genetics ; Disease Models, Animal ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics/metabolism ; Mice, Inbred C57BL ; Genetic Vectors/genetics ; Humans ; Genetic Therapy/methods ; Bioengineering ; CRISPR-Cas Systems ; Male ; Chemogenetics ; },
abstract = {Sepsis, a life-threatening condition arising from a dysregulated host response to infection, remains a significant clinical challenge with limited therapeutic options. RNA editing presents a promising avenue for modulating gene expression to attenuate the inflammatory cascade characteristic of sepsis. Here, we introduce an approach utilizing chemogenetic activation of CasRx-based RNA editing via bioengineered lentiviruses for the treatment of sepsis. Our strategy involves the targeted delivery of biomineralized lentiviral vectors encoding RNA-editing enzymes and chemogenetic switches to M1 macrophage populations implicated in sepsis pathogenesis. Upon the administration of a small molecule ligand, the chemogenetic switches activate the RNA-editing tool, CasRx, thereby enabling precise and repeated downregulation of NLRP3 mRNA. We demonstrate the efficacy and repeatability of this viral-based approach in mouse models of sepsis, highlighting its potential as a versatile therapeutic strategy for mitigating sepsis-induced inflammation. This study underscores the utility of chemogenetic technologies in harnessing the power of RNA editing for the treatment of sepsis and other inflammatory disorders.},
}

*Sepsis/therapy/genetics/immunology
Animals
*Macrophages/metabolism
Mice
*RNA Editing/genetics
*Lentivirus/genetics
Disease Models, Animal
NLR Family, Pyrin Domain-Containing 3 Protein/genetics/metabolism
Mice, Inbred C57BL
Genetic Vectors/genetics
Humans
Genetic Therapy/methods
Bioengineering
CRISPR-Cas Systems
Male
Chemogenetics

@article {pmid41422102,
year = {2025},
author = {Zhong, C and Yu, L and Zhao, T and Shen, X and Li, Z and Zhu, W and Hu, Z and Tian, R and Han, Z and He, D},
title = {A Plug-in system for reprogramming the editing patterns of base editors.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {910},
pmid = {41422102},
issn = {2041-1723},
support = {32171465//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32500460//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2023M744121//China Postdoctoral Science Foundation/ ; 2023M734090//China Postdoctoral Science Foundation/ ; 2023M734091//China Postdoctoral Science Foundation/ ; },
mesh = {*Gene Editing/methods ; Zebrafish/genetics/embryology ; Animals ; Humans ; CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; DNA/genetics/metabolism ; HEK293 Cells ; },
abstract = {DNA base editors are transformative genome editing tools that enable nucleotide conversions without inducing double-stranded DNA breaks, making them promising for correcting genetic mutations. Current base editors, however, are limited by fixed editing windows and constrained location of deaminases. To address these constraints, we develop a modular system termed Plug-in Base Editor (Plug-in BE), which dynamically programs deaminase positioning via integrating various epitopes and antibody-fused deaminases. This system expands the editing capabilities of base editors by optimizing deaminase's spatial interaction with DNA, leading to improvements in efficiencies, window restrictions, and safety profiles. We validate Plug-in BE's versatility and high fidelity in cancer gene therapy and zebrafish embryo editing, demonstrating its potential as a powerful and adaptable tool for basic research and therapeutic applications. This innovation can generate a series of base editors without extensive protein evolution, positioning Plug-in BE as a significant advancement in the field of genome editing.},
}

*Gene Editing/methods
Zebrafish/genetics/embryology
Animals
Humans
CRISPR-Cas Systems/genetics
Genetic Therapy/methods
DNA/genetics/metabolism
HEK293 Cells

@article {pmid41421670,
year = {2026},
author = {Fan, X and Li, B and Xu, X and Long, B and Jia, Z and Wang, R and Gao, J and Chen, Y and Peng, M and Zhou, M},
title = {Deciphering the regulatory role of the pfs gene on biofilm formation in Lactobacillus plantarum R: Insights from transcriptome and metabolome.},
journal = {Bioresource technology},
volume = {443},
number = {},
pages = {133833},
doi = {10.1016/j.biortech.2025.133833},
pmid = {41421670},
issn = {1873-2976},
mesh = {*Lactiplantibacillus plantarum/genetics/physiology/metabolism ; *Biofilms/growth & development ; *Transcriptome/genetics ; *Metabolome/genetics ; Gene Expression Regulation, Bacterial ; *Bacterial Proteins/genetics/metabolism ; Quorum Sensing/genetics ; *Genes, Bacterial/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Lactobacillus plantarum is a widely recognized probiotic that forms biofilms to enhance environmental tolerance and probiotic properties, but the mechanisms regulating its biofilm formation remain unclear. This study successfully used CRISPR-Cas9 to delete the pfs gene in the high biofilm-producing strain L. plantarum R, and first investigated its role by integrated transcriptomic and metabolomic analyses. The pfs gene participates in the activated methyl cycle and AI-2 synthesis, which is involved in quorum sensing and biofilm formation. Deletion of pfs increased biofilm biomass by 91% and markedly enhanced matrix accumulation, including exopolysaccharides, extracellular proteins and extracellular DNA (eDNA). Transcriptomic analysis revealed significant perturbation of cysteine and methionine metabolism and altered expression of key genes involved in exopolysaccharide synthesis. Metabolomic profiling identified 223 differentially expressed metabolites, primarily associated with carbon flux and EPS precursor pathways. In summary, pfs deletion enhances biofilm formation via metabolic reprogramming rather than classical AI-2 dependent QS pathways. This study provides new insights into the pfs gene-mediated regulation of biofilm formation in L. plantarum and establishes a foundation for future strategies to manipulate biofilm formation in industrial applications.},
}

*Lactiplantibacillus plantarum/genetics/physiology/metabolism
*Biofilms/growth & development
*Transcriptome/genetics
*Metabolome/genetics
Gene Expression Regulation, Bacterial
*Bacterial Proteins/genetics/metabolism
Quorum Sensing/genetics
*Genes, Bacterial/genetics
CRISPR-Cas Systems/genetics

@article {pmid41419745,
year = {2025},
author = {Ying, Q and Chen, Y and Shen, L and Xu, Y and Tian, R},
title = {SPLiCR-seq: A CRISPR-Based Screening Platform for RNA splicing Identifies Novel Regulators of IRE1α-XBP1 Signaling Under ER Stress.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {911},
pmid = {41419745},
issn = {2041-1723},
support = {82171416//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*X-Box Binding Protein 1/metabolism/genetics ; *Endoribonucleases/metabolism/genetics ; *Protein Serine-Threonine Kinases/metabolism/genetics ; *RNA Splicing/genetics ; Humans ; Signal Transduction/genetics ; Animals ; *Endoplasmic Reticulum Stress/genetics ; Protein Phosphatase 1/metabolism/genetics/antagonists & inhibitors ; Unfolded Protein Response/genetics ; Mice ; CRISPR-Cas Systems ; HEK293 Cells ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {RNA splicing is fundamental to cellular function, yet systematic investigation of its complex regulation has been limited by existing methods. Here, we present SPLiCR-seq (SPLicing regulator identification through CRISPR screening), a high-throughput CRISPR screening platform that enables direct measurement of RNA splicing outcomes for pooled genetic perturbations, overcoming limitations of traditional fluorescence-based approaches. Applying SPLiCR-seq to investigate XBP1 splicing during the unfolded protein response (UPR), we conduct targeted and genome-wide screens across diverse cellular contexts, revealing both common and cell-type specific regulators. Notably, we identify GADD34 (PPP1R15A) as a novel modulator of IRE1α-XBP1 signaling, demonstrating that it directly interacts with IRE1α and functions independently of its canonical role in eIF2α dephosphorylation. Pharmacological inhibition of GADD34 using Sephin1 effectively suppressed XBP1 splicing and alleviated CAR-T cell exhaustion in an ex vivo model, leading to enhanced tumor-killing capacity across multiple cancer models. This work not only establishes a powerful new tool for systematically studying RNA splicing regulation but also uncovers a promising therapeutic strategy for improving CAR-T cell immunotherapy through modulation of the IRE1α-XBP1 pathway.},
}

*X-Box Binding Protein 1/metabolism/genetics
*Endoribonucleases/metabolism/genetics
*Protein Serine-Threonine Kinases/metabolism/genetics
*RNA Splicing/genetics
Humans
Signal Transduction/genetics
Animals
*Endoplasmic Reticulum Stress/genetics
Protein Phosphatase 1/metabolism/genetics/antagonists & inhibitors
Unfolded Protein Response/genetics
Mice
CRISPR-Cas Systems
HEK293 Cells
Cell Line, Tumor
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41418729,
year = {2026},
author = {Wang, Y and Zheng, W and Qiu, B and Chen, Q and Yang, T and Zhou, S and Liu, J and Yang, B},
title = {Generation of a human embryonic stem cell line (SMUDHe010-A-3F) with Cas9 expression cassette integrated at the AAVS1 locus via CRISPR/Cas9-mediated homologous recombination.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103882},
doi = {10.1016/j.scr.2025.103882},
pmid = {41418729},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Homologous Recombination/genetics ; Cell Line ; Gene Editing ; Genetic Loci ; Cell Differentiation ; CRISPR-Associated Protein 9/metabolism ; },
abstract = {Cas9, an RNA-guided nuclease, enables precise genome editing by recognizing sgRNA-complementary sequences and cleaving target DNA. In this study, we used CRISPR/Cas9-mediated homologous recombination to integrate a loxP-flanked STOP cassette-controlled Cas9 expression framework (LSL-Cas9) into the AAVS1 safe-harbor locus of human embryonic stem cells. The resulting cell line, SMUDHe010-A-3F, allows Cre-dependent activation of Cas9 but remains inactive in the absence of Cre recombinase. Karyotype and tri-lineage differentiation confirmed genomic stability and pluripotency. This line provides a valuable platform for organoid gene editing and studies of human development and disease.},
}

Humans
*CRISPR-Cas Systems/genetics
*Human Embryonic Stem Cells/metabolism/cytology
*Homologous Recombination/genetics
Cell Line
Gene Editing
Genetic Loci
Cell Differentiation
CRISPR-Associated Protein 9/metabolism

@article {pmid41406514,
year = {2026},
author = {Ghodrat, R and Ramachandran, H and Hildebrandt, B and Binder, S and Rossi, A and Reichert, AS},
title = {CRISPR/Cpf1-mediated editing of PINK1 in induced pluripotent stem cells.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103887},
doi = {10.1016/j.scr.2025.103887},
pmid = {41406514},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Protein Kinases/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; },
abstract = {The PTEN induced kinase 1 (PINK1) gene is crucial for mitophagy and mitochondrial quality control. Mutations in the PINK1 gene are associated with several neurological disorders. To decipher the role of PINK1-mediated mitophagy in human induced pluripotent stem cells (hiPSCs) and in their differentiated counterparts, we used CRISPR/Cpf1 and generated a human iPSC line with homozygous out-of-frame deletions by targeting exon 6 of the PINK1 gene. The generated homozygous PINK1 mutant cell line showed normal cell morphology, genomic stability, and expression of classical stem cell markers. Furthermore, the cells can be differentiated efficiently into the three germ layers.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology
Humans
*Protein Kinases/genetics/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Cell Differentiation
Cell Line

@article {pmid41398410,
year = {2025},
author = {Chi, H and Hoikkala, V and McMahon, S and Graham, S and Gloster, T and White, MF},
title = {Structure and mechanism of the broad spectrum CRISPR-associated ring nuclease Crn4.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {889},
pmid = {41398410},
issn = {2041-1723},
support = {101018608//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; },
mesh = {Crystallography, X-Ray ; *CRISPR-Cas Systems ; Adenine Nucleotides/metabolism ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Models, Molecular ; *Endonucleases/metabolism/chemistry/genetics ; Bacteriophages/genetics ; Nucleotides, Cyclic/metabolism ; Oligoribonucleotides/metabolism ; },
abstract = {Type III CRISPR systems detect the presence of RNA from mobile genetic elements (MGE) in prokaryotes, providing antiviral immunity. On activation, the catalytic Cas10 subunit conjugates ATP to form cyclic oligoadenylate (cOA) signalling molecules that activate ancillary effectors, providing an immune response. Cellular ring nucleases degrade cOA to reset the system. Here, we describe the structure and mechanism of a new family of ring nucleases, Crn4, associated with type III-D CRISPR systems. The crystal structure of Crn4 reveals a small homodimeric protein with a fold unrelated to any known ring nuclease or, indeed, any known protein structure. Crn4 degrades a wide range of cOA species to linear oligoadenylates in vitro and ameliorates type III CRISPR immunity in vivo. Phage and plasmids also encode Crn4 orthologues that may function as anti-CRISPRs. These observations expand our understanding of ring nucleases and reveal a new protein fold for cyclic nucleotide recognition.},
}

Crystallography, X-Ray
*CRISPR-Cas Systems
Adenine Nucleotides/metabolism
*CRISPR-Associated Proteins/metabolism/chemistry/genetics
Models, Molecular
*Endonucleases/metabolism/chemistry/genetics
Bacteriophages/genetics
Nucleotides, Cyclic/metabolism
Oligoribonucleotides/metabolism

@article {pmid41385899,
year = {2026},
author = {Fathy, K and Bharti, J and Khan Sony, S and Nehra, M and Kaul, R and Rawat, B and Sopory, SK and Agrawal, PK and Prakash, A and Kaul, T},
title = {Triumphing over hidden hunger: Redesigning rice (Oryza sativa L.) for enhanced nutraceutical grain composition utilizing multiplexed genome editing.},
journal = {Journal of plant physiology},
volume = {316},
number = {},
pages = {154667},
doi = {10.1016/j.jplph.2025.154667},
pmid = {41385899},
issn = {1618-1328},
mesh = {*Oryza/genetics/metabolism/chemistry ; *Gene Editing/methods ; *Edible Grain/chemistry/genetics ; Cadmium/metabolism/analysis ; *Dietary Supplements ; Plants, Genetically Modified/genetics ; CRISPR-Cas Systems ; Plant Breeding ; Zinc/metabolism ; Iron/metabolism ; },
abstract = {Rice, a staple food crop, is consumed by most of the world's population. Micronutrient malnutrition is a severe health issue, leading to diseases such as cancer, anemia, diabetes, heart disease, and disorders in physical and psychological development. We aimed to create rice with low cadmium in the grain but having high cadmium in shoots, safe biofortified protein, high iron, and zinc using CRISPR/Cas9 and breeding technologies instead of adding drugs. The triple gene Knockout rice lines for two iron sensors and one negative regulator gene for cadmium were created to offer high Fe/Zn and low Cd content for breeders. Multiplexed gene editing mediated biolistic transformation of rice callus, and genotyping was used to check the genetic stability of the edited rice lines. Rice lines were found to have enhanced iron, zinc, and protein content, with concentrations varying based on growth conditions. These lines can be used as phytoremediators for cadmium by storing Cd on plant shoots. The rice-edited plants possessed excellent agro-morphological traits, photosynthetic, and physiological performance. The developed edited indica rice lines have crucial agronomic traits with more nutritional value. Compared to the other lines and the wild wildtype, the genome-edited free Cas9 line 2 showed better traits: 13.48 μg/g (iron), 22.9 μg/g (zinc), and a high protein content, which depends on how bioavailable metals and nutrients are in the soil. The line also had 20.60 g of seeds per 1000 g of plant, a total plant yield of 102.76 g, and 101 days of 50 % flowering. This work offers efficient and precise multiple gene-editing in rice with an effective, sustainable strategy for multi-trait enhancement. The developed lines could be used in breeding programs for sustainable solutions for malnutrition worldwide. The experimental results can provide reference and support for the safe use of edited crops as a diet.},
}

*Oryza/genetics/metabolism/chemistry
*Gene Editing/methods
*Edible Grain/chemistry/genetics
Cadmium/metabolism/analysis
*Dietary Supplements
Plants, Genetically Modified/genetics
CRISPR-Cas Systems
Plant Breeding
Zinc/metabolism
Iron/metabolism

@article {pmid41285723,
year = {2025},
author = {Morshedi Rad, D and Richards, C and Zhand, S and de Alwis, N and Hannan, NJ and Faiz, A and McClements, L and Ebrahimi Warkiani, M},
title = {CRISPR/Cas9-mediated gene editing in trophoblast cells via mechanoporation for preeclampsia insight.},
journal = {Cell death & disease},
volume = {17},
number = {1},
pages = {61},
pmid = {41285723},
issn = {2041-4889},
support = {106628//National Heart Foundation of Australia (Heart Foundation)/ ; DP200101860//Department of Education and Training | Australian Research Council (ARC)/ ; 2021CDF1148//Cancer Institute NSW (Cancer Institute New South Wales)/ ; },
mesh = {*Trophoblasts/metabolism/pathology ; Female ; *Pre-Eclampsia/genetics/pathology/metabolism ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Pregnancy ; Cell Movement/genetics ; Cell Line ; },
abstract = {Preeclampsia is a severe pregnancy complication marked by impaired trophoblast function and abnormal placental development, leading to significant maternal and fetal morbidity. FK506-binding protein-like (FKBPL) has been identified as a potential biomarker as it is significantly downregulated in early pregnancy stages of women who progress to develop preeclampsia. However, editing the Fkbpl gene in trophoblast cells to create a model of preeclampsia using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is challenging due to inefficient delivery, leading to low editing efficiency and reduced cell viability. To address these challenges, we developed a cost-effective and minimally invasive mechanoporation system using micro-engineered filters to deliver CRISPR/Cas9 plasmid DNA (pDNA) targeting the Fkbpl gene into trophoblast cells. This approach successfully generated cell lines with a 38% knockout (K/O) of Fkbpl expression, significantly reducing cell migration (wildtype (WT): 28.77% ± 4.7 vs. 38% K/O: 4.95% ± 0.8, wound closure, **p < 0.01) and proliferation (WT: 1.26 ± 0.06 vs. 38% K/O: 0.81 ± 0.01, ****p < 0.0001). Lower Fkbpl-K/O efficiency of 17% showed a similar reduction in cell proliferation as the 38% K/O clone. Although a full Fkbpl-K/O in the ACH-3P first-trimester trophoblast cell line was not achieved, the partial K/O provided valuable insights into Fkbpl's role in trophoblast function relevant to preeclampsia pathogenesis. Moreover, treatment with mesenchymal stem cell (MSC)-derived small extracellular vesicles (sEVs) or MSC-sEVs did not restore migratory capacity in Fkbpl-deficient cells (p = 0.14). MSC-sEVs increased proliferation in WT ACH-3P cells at 1 µg (p < 0.05) and 2 µg (p < 0.01) doses, however, were not effective in either 17% or 38% Fkbpl-K/O clones, suggesting that FKBPL is an important mechanism of MSC-sEV-mediated therapeutic effect in trophoblasts in the context of preeclampsia. This study advances gene-editing techniques in placental biology and proposes new therapeutic strategies and mechanisms for pregnancy-related complications. A Schematic overview of CRISPR/Cas9 plasmid delivery using microfiltroporation compared to gold standard electroporation and lipofection technologies in trophoblast cells. A CRISPR/Cas9 plasmid targeting Fkbpl was delivered to the first trimester trophoblast cell line, ACH-3P. Cells were sorted according to green fluorescence protein (GFP) expression, expanded and assessed for changes in cell function using proliferation and migration assays. B Actual images of the isopore silicon nitride (SiN) microfilters used in this study and diagram of cell membrane dynamics in response to mechanoporation. This figure was created with Biorender.com. CRISPR clustered regularly interspaced short palindromic repeats, EP electroporation, MFP microfiltroporation.},
}

*Trophoblasts/metabolism/pathology
Female
*Pre-Eclampsia/genetics/pathology/metabolism
Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Pregnancy
Cell Movement/genetics
Cell Line

@article {pmid41194485,
year = {2026},
author = {Yang, TT and Zhang, JR and Xie, ZH and Ren, ZL and Zhao, MY and Hu, WJ and Yan, JW and Ni, M},
title = {CRISPR-Cas9-Targeted Nanopore Sequencing for STR Typing.},
journal = {Electrophoresis},
volume = {47},
number = {1},
pages = {57-67},
doi = {10.1002/elps.70051},
pmid = {41194485},
issn = {1522-2683},
support = {2021QY2004//We thank the National Key Research and Development Program of China/ ; 82030058//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Nanopore Sequencing/methods ; *Microsatellite Repeats/genetics ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; *DNA Fingerprinting/methods ; Pilot Projects ; *Sequence Analysis, DNA/methods ; DNA/genetics ; },
abstract = {CRISPR-Cas9-targeted sequencing can enrich DNA regions of interest by directing the Cas9 protein to bind and cleave specific DNA sequences via single-guide RNA (sgRNA). It is interesting to explore the efficacy of using CRISPR-Cas9-targeted nanopore sequencing (referred to as Cas9-seq), a polymerase chain reaction (PCR)-free workflow, for forensic short tandem repeats (STR) profiling, and to compare it with the amplification-based approach. In this pilot study, we constructed a Cas9-seq method for profiling seven STR loci, including D18S51, FGA, TPOX, D16S539, vWA, CSF1PO, and TH01. With 3 µg DNA inputs from human NA12878 and 293T cell lines, we achieved 643.45- and 468.34-fold enrichment ratios of the sgRNA-targeted regions by using Cas9-seq, respectively. Compared to nanopore sequencing of PCR amplicon products (amplicon-seq) of the ForenSeq DNA Signature Prep kit, the Cas9-seq reads had an ultralow strand bias. However, surprisingly, Cas9-seq did not show advantages in allele balance and had higher noise in the reads. At the seven STR loci for the two samples, both Cas9-seq and amplicon-seq had three genotyping errors. Additionally, there were no false-positive single-nucleotide polymorphisms (SNPs) introduced by Cas9-seq, whereas amplicon-seq produced three. In sum, we conclude that the PCR-free Cas9-seq might not be favorable for forensic STR genotyping.},
}

Humans
*Nanopore Sequencing/methods
*Microsatellite Repeats/genetics
*CRISPR-Cas Systems/genetics
HEK293 Cells
*DNA Fingerprinting/methods
Pilot Projects
*Sequence Analysis, DNA/methods
DNA/genetics

@article {pmid40522172,
year = {2025},
author = {Xue, B and Zhang, MX and Bi, XC and Lai, SP and Bie, XT and Dong, Y and Li, JF and Gao, F and Zhang, X and Wang, Y},
title = {An Amygdala-hippocampus Circuit for Endocannabinoid Modulation of Anxiety Avoidance.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {34},
pages = {e05121},
pmid = {40522172},
issn = {2198-3844},
support = {2021ZD0203000 (2021ZD0203002//STI2030-Major/ ; 82090033//National Natural Science Foundation of China/ ; 81830035//National Natural Science Foundation of China/ ; 82003729//National Natural Science Foundation of China/ ; ZR2019ZD35//Natural Science Foundation of Shandong Province/ ; ZR2020QH357//Natural Science Foundation of Shandong Province/ ; 202205AF150068//Zhang Xia Expert Workstation of the Yunnan Province/ ; ZYGD23034//West China Hospital, Sichuan University/ ; },
mesh = {*Amygdala/physiology ; *Hippocampus/physiology ; *Endocannabinoids/metabolism ; *Anxiety/metabolism/physiopathology ; Male ; Animals ; Mice ; Receptor, Cannabinoid, CB1/metabolism ; Mice, Inbred C57BL ; Basolateral Nuclear Complex/physiology ; Neurons/physiology ; Avoidance Learning/physiology ; CRISPR-Cas Systems ; Brain/physiology ; },
abstract = {Recent studies indicate a therapeutic potential of increased brain endocannabinoids (eCBs) in anxiety disorders, but the underlying brain circuits are still elusive. Here, it is observed that optogenetic inhibition and activation of anterior basolateral amygdala (aBLA) - ventral hippocampus (vHPC) glutamatergic projections respectively decrease and increase anxiety avoidance behaviors. Then, the contributions of eCBs in aBLA-vHPC projections to anxiety avoidance are investigated by employing three newly developed synapse- and circuit-specific eCB-targeted viral strategies to achieve real-time monitoring of eCB release, in vivo optogenetic activation of CB1 receptors, and CRISPR-Cas9 gene knockdown of eCB biosynthesis enzymes. Prominent eCB release are surprisingly found at aBLA-vHPC glutamatergic synapses during anxiety avoidance, suggesting inhibitory effects of increased eCBs in aBLA-vHPC projections on anxiety avoidance. This idea is further supported by findings that specific activation of CB1 receptors at aBLA-vHPC synapses inhibit presynaptic glutamate release and reduce anxiety avoidance. In contrast, specific knockdown of eCB biosynthesis enzymes at aBLA-vHPC synapses reduce eCB levels at aBLA-vHPC glutamatergic synapses and increase anxiety avoidance. Additionally, inhibition of aBLA-innervated vHPC glutamatergic neurons alleviates anxiety avoidance. Together, these findings reveal counteracting effects of increased eCB signaling in aBLA-vHPC circuits on anxiety avoidance.},
}

*Amygdala/physiology
*Hippocampus/physiology
*Endocannabinoids/metabolism
*Anxiety/metabolism/physiopathology
Male
Animals
Mice
Receptor, Cannabinoid, CB1/metabolism
Mice, Inbred C57BL
Basolateral Nuclear Complex/physiology
Neurons/physiology
Avoidance Learning/physiology
CRISPR-Cas Systems
Brain/physiology

@article {pmid40434188,
year = {2026},
author = {Wu, F and Li, N and Xiao, Y and Palanki, R and Yamagata, H and Mitchell, MJ and Han, X},
title = {Lipid Nanoparticles for Delivery of CRISPR Gene Editing Components.},
journal = {Small methods},
volume = {10},
number = {2},
pages = {e2401632},
pmid = {40434188},
issn = {2366-9608},
support = {XDB0570000//Strategic Priority Research Program of the Chinese Academy of Science/ ; 32471401//National Natural Science Foundation of China/ ; ssIII-2024B01//Shanghai Sci-Tech Inno Center for Infection & Immunity/ ; },
mesh = {*Gene Editing/methods ; *Nanoparticles/chemistry ; Humans ; *Lipids/chemistry ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Transfer Techniques ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Liposomes ; },
abstract = {Gene editing has emerged as a promising therapeutic option for treating genetic diseases. However, a central challenge in the field is the safe and efficient delivery of these large editing tools, especially in vivo. Lipid nanoparticles (LNPs) are attractive nonviral vectors due to their low immunogenicity and high delivery efficiency. To maximize editing efficiency, LNPs should efficiently protect gene editing components against multiple biological barriers and release them into the cytoplasm of target cells. In this review, the widely used CRISPR gene editing systems are first overviewed. Then, each component of LNPs, as well as their effects on delivery, are systematically discussed. Following this, the current LNP engineering strategies to achieve non-liver targeting are summarized. Finally, preclinical and clinical applications of LNPs for in vivo genome editing are highlighted, and perspectives for the future development of LNPs are provided.},
}

*Gene Editing/methods
*Nanoparticles/chemistry
Humans
*Lipids/chemistry
Animals
*CRISPR-Cas Systems/genetics
*Gene Transfer Techniques
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Liposomes

@article {pmid41553913,
year = {2026},
author = {Garnica, M and San Martin-Uriz, P and Rodriguez-Marquez, P and Calleja-Cervantes, ME and Rodriguez-Diaz, S and Martinez-Turrillas, R and Hernaez, M and Prosper, F and Rodriguez-Madoz, JR},
title = {Improving CRISPR-Cas9 Screens in CAR T Cells: A Refined Method for Library Preparation.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {227},
pages = {},
doi = {10.3791/69721},
pmid = {41553913},
issn = {1940-087X},
mesh = {Humans ; *CRISPR-Cas Systems ; *T-Lymphocytes/immunology ; *Receptors, Chimeric Antigen/genetics/immunology ; *Gene Library ; Immunotherapy, Adoptive/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {Chimeric antigen receptor (CAR) T cell therapies have demonstrated remarkable efficacy in several hematological malignancies, yet their success has not been fully replicated in solid tumors. Moreover, even in hematological cancers, relapse after CAR T cell infusion continues to compromise long-term outcomes. These challenges highlight the urgent need to develop strategies that enhance CAR T cell efficacy, persistence, overcoming tumor and microenvironment-mediated resistance. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-based screening platforms provide a powerful approach to systematically identify genes that regulate CAR T cell function. By linking genetic perturbations to phenotypic outcomes, these assays enable the discovery of pathways controlling activation, proliferation, memory formation, and cytotoxicity. Standard workflows involve transduction of substantial numbers of cells with a single guide RNA (sgRNA) library, Cas9-mediated editing, selection of edited cells, and PCR amplification of sgRNA cassettes from genomic DNA (gDNA) prior to sequencing. However, PCR amplification using large amounts of gDNA poses significant challenges and often fails to selectively amplify and retrieve sgRNAs. Here, we describe an optimized CRISPR-Cas9 knockout screening protocol, which we have tested on primary human CAR T cells. The method here incorporates an intermediate step during sgRNA library preparation that reduces gDNA carryover through enzymatic digestion and selective pulldown of the sgRNA cassette, thereby increasing the efficiency of the first PCR amplification. This modification allowed us to retrieve sgRNA information across our CAR T cell screens, which had remained elusive in our previous attempts using traditional 1 and 2-step PCR amplification protocols. In conclusion, this optimized workflow facilitates CRISPR screening library preparation in challenging samples and enables the identification of key genetic determinants that can be targeted to improve therapeutic efficacy.},
}

Humans
*CRISPR-Cas Systems
*T-Lymphocytes/immunology
*Receptors, Chimeric Antigen/genetics/immunology
*Gene Library
Immunotherapy, Adoptive/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing/methods

@article {pmid41551929,
year = {2026},
author = {Mir-Pedrol, J and Kuhlburger, L and Sanvicente-García, M and Yazar, M and Ryan, CJ and Krakau, S and Gabernet, G and Güell, M and Bonfanti, M and Nf-Core Community, and Nahnsen, S},
title = {nf-core/crisprseq: a versatile pipeline for comprehensive analysis of CRISPR gene editing and screening assays.},
journal = {NAR genomics and bioinformatics},
volume = {8},
number = {1},
pages = {lqaf214},
pmid = {41551929},
issn = {2631-9268},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Software ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {In recent years, CRISPR technology has become widely applied in scientific research, being simpler, cheaper, and more precise than previous gene-editing techniques. This editing technology can be used for various applications, such as gene knockout, gene knock-in, CRISPR activation (CRISPRa), CRISPR interference (CRISPRi), CRISPR screens, base editing, and prime editing. The share of pipelines to analyze the variety of CRISPR editing methods is low, and until now, none of them caters to both gene editing and CRISPR-based functional genomics. Here, we introduce nf-core/crisprseq, a Nextflow DSL2 pipeline for the assessment of CRISPR gene editing and screening assays. The workflow is written in a modularized fashion to allow the easy incorporation of new steps. nf-core/crisprseq is the first generic pipeline enabling the analysis of the broad spectrum of CRISPR designs. We show the performance and usability of the software using publicly available datasets.},
}

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

@article {pmid41550651,
year = {2025},
author = {Yan, W and Weng, X},
title = {Targeted RNA base editing for therapeutic: mechanisms and advances.},
journal = {Pharmaceutical science advances},
volume = {3},
number = {},
pages = {100089},
pmid = {41550651},
issn = {2773-2169},
abstract = {RNA base editing, which enables RNA base modification through effector proteins guided by targeting systems, is a powerful technology to correct disease-associated point mutations. Although overshadowed by CRISPR-based genome editing, RNA editing has seen rapid development in recent years, with significant improvements in efficiency and precision. In this review, we summarize the core components of RNA base editing systems (RNA-targeting systems and effector proteins) and describe major RNA editing types, including A-to-I, C-to-U, A-to-m[6]A/m[6]A-to-A, and U-to-Ψ base editors, along with their research progress. In addition, we systematically summarize the delivery methods of the developed RNA editors and their initial exploration in treating diseases caused by nonsense mutations. Finally, combining the current development status of the RNA editing related field, we reflect on the problems encountered in the current development of the RNA editing field and offer our own insights on the future development direction.},
}

@article {pmid41550334,
year = {2025},
author = {Lin, S and Li, H and Bai, S and Han, X},
title = {Mobile RNAs as systemic signaling beyond boundaries for plant stress resistance.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1712714},
pmid = {41550334},
issn = {1664-462X},
abstract = {Plant mobile RNAs-including small RNAs (miRNAs, siRNAs), mRNAs, lncRNAs, and tRNA fragments-function as systemic signaling molecules that traverse cellular, tissue, and species boundaries to coordinate plant adaptation to environmental stresses. Here, we summarize the critical roles of mobile RNAs in mediating systemic adaptation to abiotic challenges and biotic interactions. Crucially, we highlight the diverse transport mechanisms enabling their movement and discuss the emerging functional versatility of mobile RNAs, which extends beyond transcriptional regulation to encompass epigenetic modifications, resource allocation, and cross-species communication. These fundamental insights are driving transformative applications: Mobile RNAs provide the foundation for developing systemic RNAi-based biopesticides and are being integrated with CRISPR-Cas technologies to overcome delivery barriers and enable heritable, transgene-free genome editing in crops. Understanding and harnessing mobile RNA networks offers unprecedented potential for engineering resilient crops and implementing precise, sustainable crop protection strategies to address global food security challenges.},
}

@article {pmid41549062,
year = {2026},
author = {Mukherjee, P and Benicky, J and Panigrahi, A and Ailles, L and Goldman, R},
title = {SULF1 in Cancer Associated Fibroblasts Promotes Invasion in Head and Neck Cancer Cell Lines.},
journal = {Cancer medicine},
volume = {15},
number = {1},
pages = {e71540},
pmid = {41549062},
issn = {2045-7634},
support = {R01CA238455/GF/NIH HHS/United States ; 2P30CA051008/CA/NCI NIH HHS/United States ; /NH/NIH HHS/United States ; 1S10OD028623-01A1//NIH Office of the Director/ ; S10OD032420//NIH Office of the Director/ ; //George Washington University/ ; //School of Medicine and Health Sciences/ ; },
mesh = {Humans ; *Cancer-Associated Fibroblasts/metabolism/pathology ; *Head and Neck Neoplasms/pathology/genetics/metabolism ; *Sulfotransferases/metabolism/genetics ; Cell Movement ; Neoplasm Invasiveness ; Cell Line, Tumor ; *Squamous Cell Carcinoma of Head and Neck/pathology/genetics/metabolism ; Cell Proliferation ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Gene Expression Regulation, Neoplastic ; },
abstract = {BACKGROUND: Cancer-associated fibroblasts (CAFs) significantly influence tumor behavior in head and neck squamous cell carcinoma (HNSCC) and other malignancies. We identified the extracellular sulfatase SULF1 as a key stromal factor highly expressed in CAFs and associated with poor prognosis.

METHODS AND RESULTS: Using CRISPR/Cas9-edited SULF1-knockout primary HNSCC CAFs, we demonstrate that loss of SULF1 reduces fibroblast proliferation and markedly impairs cancer cell migration and invasion in vitro. Two-photon microscopy in 3D spheroid cocultures revealed that SULF1-deficient CAFs fail to support invasiveness of Cal33 cells, resulting in spheroids with fewer invasive projections and altered morphology. Proteomic analysis confirmed the absence of SULF1 in the knockout cell cultures and revealed that SULF2, expressed in tumor cells, does not compensate for its loss.

CONCLUSION: These findings highlight the importance of CAF-derived SULF1 in regulating tumor invasion and suggest that SULF1 is a promising therapeutic target in HNSCC.},
}

Humans
*Cancer-Associated Fibroblasts/metabolism/pathology
*Head and Neck Neoplasms/pathology/genetics/metabolism
*Sulfotransferases/metabolism/genetics
Cell Movement
Neoplasm Invasiveness
Cell Line, Tumor
*Squamous Cell Carcinoma of Head and Neck/pathology/genetics/metabolism
Cell Proliferation
CRISPR-Cas Systems
Gene Knockout Techniques
Gene Expression Regulation, Neoplastic

@article {pmid41548978,
year = {2026},
author = {Zheng, SH and Liu, Y and Xia, XX and Liu, YM},
title = {Advances in base editing technology and the construction of precise zebrafish disease models.},
journal = {Yi chuan = Hereditas},
volume = {48},
number = {1},
pages = {46-60},
doi = {10.16288/j.yczz.25-157},
pmid = {41548978},
issn = {0253-9772},
mesh = {Animals ; *Zebrafish/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Disease Models, Animal ; Humans ; },
abstract = {Single nucleotide variants (SNVs) are among the primary pathogenic factors of human genetic diseases, accounting for a significant proportion of all mutation types. Conducting in-depth research on the pathogenic significance of these mutations in animal models is essential for understanding disease mechanisms and developing therapeutic strategies. The progress of such research largely depends on the continuous innovation and advancement of gene editing technologies. In recent years, base editing technology based on the CRISPR/Cas9 system has emerged, enabling precise conversion of individual nucleotides. Owing to its efficiency and convenience, base editing has been widely applied in gene therapy, the construction of animal models, and molecular breeding, bringing new breakthroughs and opportunities to life sciences and medical research. Zebrafish, with their advantages of small size, high fecundity, transparent embryos, and external development, have become an ideal model organism for studying disease mechanisms and drug screening. In this review, we summarize the development of CRISPR/Cas9-based base editing technologies, highlight the emergence of novel editing tools, and explore the application and progress of base editing in constructing precise zebrafish disease models.},
}

Animals
*Zebrafish/genetics
*Gene Editing/methods
CRISPR-Cas Systems/genetics
*Disease Models, Animal
Humans

@article {pmid41546796,
year = {2026},
author = {Borgohain, T and Suma, R and Muttappagol, M and Saikia, B and Keithellakpam, A and Laskar, A and Hiremath, SS and Basu, U and Velmurugan, N and Palakolanu, SR and Chikkaputtaiah, C},
title = {Precision breeding in a changing climate: unlocking resilience through omics and gene editing.},
journal = {Functional & integrative genomics},
volume = {26},
number = {1},
pages = {26},
pmid = {41546796},
issn = {1438-7948},
support = {31/0025 (15358) 2022-EMR-I//Human Resource Development Centre, Council of Scientific And Industrial Research/ ; DST/WISE-PDF/LS-112/2024(G)//Department of Science and Technology, Ministry of Science and Technology, India/ ; MMP025301//Council of Scientific and Industrial Research, India/ ; },
mesh = {*Gene Editing/methods ; *Plant Breeding/methods ; *Climate Change ; *Crops, Agricultural/genetics/growth & development ; *Genomics ; Genome-Wide Association Study ; },
abstract = {Climate change, rising global food demand, and shrinking resources require transformative innovations in crop breeding. This review outlines recent advances in new breeding technologies (NBTs), including molecular markers, genome-wide association studies (GWAS), genomic selection (GS), next-generation sequencing (NGS), and gene editing (GE) tools such as the clustered regularly interspaced short palindromic repeat (CRISPR/Cas), base editing, and prime editing. These methods enable the accurate improvement of traits, thereby accelerating the development of crops resistant to both abiotic and biotic stresses. The integration of multi-omics platforms, including genomics, transcriptomics, proteomics, metabolomics, and phenomics, provides a comprehensive framework for deciphering and manipulating complex trait architectures. Artificial intelligence (AI) and machine learning (ML) enhance precision breeding by providing data-driven insights and enabling the forecasting of traits. Emphasis is also placed on combining gene editing with other strategies, such as speed breeding, to accelerate the development of traits. This review underscores the importance of an integrated systems biology approach that combines multi-omics, gene editing, AI, and speed breeding to accelerate the development of climate-resilient, high-yielding, and nutritionally enhanced crops. The integration of these innovative technologies holds great promise for addressing global food security, environmental sustainability, and agricultural resilience in the face of climate change. A strategic framework for the future of plant breeding is outlined, emphasizing the importance of interdisciplinary collaboration in building a sustainable agricultural future.},
}

*Gene Editing/methods
*Plant Breeding/methods
*Climate Change
*Crops, Agricultural/genetics/growth & development
*Genomics
Genome-Wide Association Study

@article {pmid41543903,
year = {2026},
author = {Yin, S and Jarosz, DF and Ting, AY},
title = {Towards CRISPR-based editing of the mitochondrial genome in yeast.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {3},
pages = {e2505894123},
pmid = {41543903},
issn = {1091-6490},
support = {NA//Stanford University (SU)/ ; NA//Chan Zuckerberg Initiative (CZI)/ ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *Gene Editing/methods ; *Genome, Mitochondrial/genetics ; *CRISPR-Cas Systems/genetics ; Mitochondria/genetics/metabolism ; DNA, Mitochondrial/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Mitochondria, which evolved from symbiotic bacteria, possess their own genomes (mtDNA) and support independent transcription and translation within the organelle. Given the essential role of mtDNA in energy production, metabolism, as well as cellular homeostasis, and the high density of confirmed pathogenic mutations that map to mtDNA, there is a pressing need for versatile methods to study and manipulate this genome. Although CRISPR technology has revolutionized the editing of nuclear genomes, it has not been successfully extended to mtDNA, primarily due to the challenge of delivering single guide RNAs (sgRNAs) across both outer and inner mitochondrial membranes. Here we develop a survival-based reporter in Saccharomyces cerevisiae to screen for potential RNA import motifs. We identify a 40-nucleotide aptamer (IM83) that facilitates sgRNA entry into the mitochondrial matrix, enabling CRISPR editing by a mitochondrially-localized adenine base editor. We show that mitochondrial import of IM83 is ATP-dependent and enhanced by the tRNA synthetase Msk1. Further investigations identify barriers to efficient CRISPR editing of mtDNA, including loss of membrane potential associated with mitochondrial targeting of the base editor. These insights lay the groundwork for future improvements in CRISPR-based editing of mtDNA in eukaryotes.},
}

*Saccharomyces cerevisiae/genetics/metabolism
*Gene Editing/methods
*Genome, Mitochondrial/genetics
*CRISPR-Cas Systems/genetics
Mitochondria/genetics/metabolism
DNA, Mitochondrial/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Saccharomyces cerevisiae Proteins/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41543528,
year = {2026},
author = {O'Connor-Moneley, J and Lange, T and Flanagan, PR and Brunke, S and Fischer, L and Sharma, R and Puri, S and Hube, B and Sullivan, DJ and Moran, GP},
title = {Depletion of the Candida albicans TLO gene family reveals a requirement for alpha TLO genes for wild-type virulence.},
journal = {Microbiology (Reading, England)},
volume = {172},
number = {1},
pages = {},
doi = {10.1099/mic.0.001654},
pmid = {41543528},
issn = {1465-2080},
mesh = {*Candida albicans/genetics/pathogenicity/growth & development ; Animals ; Virulence/genetics ; Mice ; *Fungal Proteins/genetics/metabolism ; Hyphae/growth & development/genetics ; Disease Models, Animal ; Candidiasis, Oral/microbiology ; Humans ; Macrophages/microbiology ; Female ; Gene Expression Regulation, Fungal ; CRISPR-Cas Systems ; *Mediator Complex/genetics/metabolism ; Multigene Family ; },
abstract = {Candida albicans uniquely possesses an expanded family of genes (the TLO gene family) that encodes 10-15 paralogues of the Med2 component of the transcriptional regulator Mediator. Previous studies have shown that TLO null mutants are unable to form hyphae and are hypersensitive to environmental stress. However, the reason for the TLO gene expansion remains unclear, and the current study aimed to determine if reduction in the TLO family copy number affected virulence. In order to investigate this, we used CRISPR-Cas9 mutagenesis to generate two TLO-depleted mutants: one mutant retaining only TLOβ2 (CaTLO2) and the second mutant containing only TLOγ5 (CaTLO5). Both TLO-depleted mutants exhibited increased filamentous growth, increased susceptibility to specific stresses and reduced virulence in a murine model of oropharyngeal candidiasis (OPC). In vitro, the CaTLO5 mutant also exhibited impaired hyphal escape from macrophages and reduced hyphal invasion of oral keratinocytes. We then investigated if complementation with TLOα1, a gene previously shown to restore wild-type growth in a Δtlo null mutant, could restore virulence. In vitro infection models showed that TLOα1 could restore true hypha formation, epithelial invasion and hyphal escape from macrophages in the CaTLO5 background. The murine OPC model showed that TLOα1 could restore wild-type virulence in both CaTLO2 and CaTLO5 strains, suggesting an essential role for α-TLO in oral mucosal infection. Together, these findings highlight the functional specialization between the α, β and γ TLO gene groups and establish α-TLO as a major regulator of virulence in C. albicans.},
}

*Candida albicans/genetics/pathogenicity/growth & development
Animals
Virulence/genetics
Mice
*Fungal Proteins/genetics/metabolism
Hyphae/growth & development/genetics
Disease Models, Animal
Candidiasis, Oral/microbiology
Humans
Macrophages/microbiology
Female
Gene Expression Regulation, Fungal
CRISPR-Cas Systems
*Mediator Complex/genetics/metabolism
Multigene Family

@article {pmid41543272,
year = {2026},
author = {Lummerstorfer, M and Lächelt, U},
title = {Non-Viral CRISPR carriers: transient delivery with lasting effects.},
journal = {Drug delivery},
volume = {33},
number = {1},
pages = {2614125},
pmid = {41543272},
issn = {1521-0464},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Therapy/methods ; *Gene Transfer Techniques ; Animals ; Genetic Vectors ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR-Cas9 has revolutionized the field of genome editing. While conventional gene supplementation therapies and the market of related gene therapy products are dominated by viral vectors, non-viral delivery strategies are increasingly being explored for in vivo CRISPR applications. Given the permanent nature of genome editing, prolonged expression of the CRISPR machinery is not required, and transient delivery nevertheless can achieve lasting therapeutic effects. In contrast, short-term availability of genome editing components is rather considered advantageous to reduce the risk of off-target effects in a 'hit-and-run' fashion. In this article, we provide a systematic survey of the current clinical trial landscape with focus on in vivo CRISPR therapies and discuss utilized delivery strategies. As of December 2025, 136 CRISPR trials are ongoing, including 36 based on in vivo delivery of CRISPR components which show a clear shift towards non-viral vectors. The article describes the clinically employed CRISPR technologies and non-viral delivery platforms, highlighting both the present opportunities and key challenges associated with CRISPR delivery in the future.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Genetic Therapy/methods
*Gene Transfer Techniques
Animals
Genetic Vectors
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41414648,
year = {2026},
author = {Wang, LR and Zhu, ST and Liao, ZH and Wu, N and Nie, ZK and Ye, C and Shi, TQ},
title = {Establishing a CRISPR/Cas9 Genome Editing System Combined with URA3-Blaster in Botrytis cinerea for Enhanced Abscisic Acid Production.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {2},
pages = {2207-2217},
doi = {10.1021/acs.jafc.5c14153},
pmid = {41414648},
issn = {1520-5118},
mesh = {*Botrytis/genetics/metabolism ; *Abscisic Acid/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Metabolic Engineering ; *Fungal Proteins/genetics/metabolism ; },
abstract = {Abscisic acid (ABA) is a key plant growth regulator widely used in agriculture and ecological restoration. Although metabolic engineering of the fungus Botrytis cinerea can enhance ABA production, it has been hindered by inefficient genetic tools. In this study, we first established a recyclable selection marker system in B. cinerea based on orotidine-5'-phosphate decarboxylase. Subsequently, the CRISPR/Cas9 system was optimized, achieving up to 100% editing efficiency, far surpassing traditional homologous recombination. Based on this platform, multiple metabolic engineering strategies were systematically explored to enhance ABA biosynthesis. Increasing acetyl-CoA supply, inhibiting squalene synthesis, and knocking out key secondary metabolism genes Bcpks12 and Bcphs1 all significantly promoted ABA accumulation. Notably, co-overexpression of Bcacly1 and Bcacly2 combined with 1 g/L citrate increased ABA production to 1.36 g/L, representing a 38.66% improvement. Overall, this study provides an efficient genetic toolkit and a solid foundation for the industrial-scale production of ABA via engineered B. cinerea.},
}

*Botrytis/genetics/metabolism
*Abscisic Acid/metabolism
*CRISPR-Cas Systems
*Gene Editing/methods
Metabolic Engineering
*Fungal Proteins/genetics/metabolism

@article {pmid41397984,
year = {2025},
author = {Chen, J and Hu, L and Vernuccio, R and Shi, N and Tian, J and Zhang, Y and Tian, S and Cao, X and Ha, Z and Lu, J and Battini, L and Raynal, B and Haouz, A and Xue, J and Cai, Q and Zhao, Y and Lu, Y and Smith, GL and Xie, Y and Lu, H and Guardado-Calvo, P and Zhang, P and Zhang, R},
title = {Development of a replication-defective mpox virus platform for fundamental and therapeutic research.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {798},
pmid = {41397984},
issn = {2041-1723},
mesh = {*Virus Replication/drug effects/genetics ; Humans ; *Antiviral Agents/pharmacology/chemistry ; Animals ; CRISPR-Cas Systems ; Chromosomes, Artificial, Bacterial/genetics ; Genome, Viral ; *Orthopoxvirus/genetics/drug effects/physiology ; Virion/genetics/drug effects ; *Defective Viruses/genetics/drug effects ; Virus Assembly/drug effects/genetics ; },
abstract = {The recent global outbreaks of mpox highlight the urgent need for both fundamental research and antiviral development. However, studying the mpox virus (MPXV), with its large and complex genome, remains challenging due to the requirement for high-containment facilities. Here, we describe a strategy for de novo assembly of MPXV clade IIb genomes in bacterial artificial chromosomes using transformation-associated recombination cloning. Leveraging CRISPR-Cas9 and Lambda Red recombination, we engineer replication-defective MPXV particles with dual deletions of OPG96 (M2R) and OPG158 (A32.5 L)-genes essential for virion assembly, that are capable of recapitulating key stages of the viral life cycle. We apply this system to screen a compound library and identify G243-1720, a potent anti-poxvirus inhibitor with broad activity in vitro and in vivo. G243-1720 blocks the formation of extracellular enveloped virions and cell-cell spread. Resistance mutation selection, crystallographic analysis, analytical ultracentrifugation, and mass photometry reveal that, despite its distinct chemical structure, G243-1720 shares a mode of action with tecovirimat, both functioning by affecting dimerization of protein OPG57 (F13). Our findings underscore the potential of G243-1720 as a promising broad-spectrum anti-poxvirus lead compound and demonstrate the utility of replication-defective MPXV particles as a reliable platform for viral biology studies and antiviral development.},
}

*Virus Replication/drug effects/genetics
Humans
*Antiviral Agents/pharmacology/chemistry
Animals
CRISPR-Cas Systems
Chromosomes, Artificial, Bacterial/genetics
Genome, Viral
*Orthopoxvirus/genetics/drug effects/physiology
Virion/genetics/drug effects
*Defective Viruses/genetics/drug effects
Virus Assembly/drug effects/genetics

@article {pmid41365122,
year = {2026},
author = {Kim, WN and Kim, HU},
title = {Precise DGAT1 base editing and in-frame deletion reveal motif-specific regulation of seed oil biosynthesis in Arabidopsis.},
journal = {Plant physiology and biochemistry : PPB},
volume = {230},
number = {},
pages = {110861},
doi = {10.1016/j.plaphy.2025.110861},
pmid = {41365122},
issn = {1873-2690},
mesh = {*Arabidopsis/genetics/metabolism ; *Diacylglycerol O-Acyltransferase/genetics/metabolism ; *Seeds/metabolism/genetics ; *Arabidopsis Proteins/genetics/metabolism ; *Gene Editing ; *Plant Oils/metabolism ; Triglycerides/biosynthesis ; *Sequence Deletion ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final step in triacylglycerol (TAG) biosynthesis and is a key determinant of seed oil content and composition. To dissect the functional contribution of the conserved DGAT1 domains, we employed adenine and cytosine base editors and CRISPR/Cas9-mediated in-frame deletion to generate targeted alleles in Arabidopsis thaliana. A total of 25 single guide RNAs were designed to introduce precise nucleotide substitutions across functional domains, and the edited lines were screened using seed fluorescence and Sanger sequencing. Five base-edited (BE) DGAT1 mutants affecting acyl-CoA/CoA allosteric binding site (S124F, S123R/S124L), thiolase acyl-enzyme intermediate signature motif (L229P), diacylglycerol (DAG)-binding motif (W416C/R419Q, V418I), and an in-frame deletion in the intrinsically disordered N-terminal region (Δ49-76H) were characterized. Amino acid substitutions in the conserved domains led to distinct shifts in seed fatty acid profiles. Loss-of-function-like mutants (S123R/S124L, L229P, W416C/R419Q) reduced 18:1 and 20:1 levels and increased 18:3 levels, whereas putative gain-of-function mutants (S124F, V418I) enhanced 20:1 incorporation and elevated total oil content. Overexpression of DGAT1[S124F] and DGAT1[V418I] in the dgat1 mutant background further increased seed oil accumulation beyond that achieved with wild-type DGAT1. Structural modeling of DGAT1 proteins revealed the location of substituted amino acids and their interactions with surrounding residues, as well as the absence of putative N-terminal regulatory segment. These results demonstrate that precise base editing can modulate DGAT1 activity and TAG composition by targeting functional motifs, providing insights into the structure-function relationships of this key enzyme and offering strategies for metabolic engineering of seed oils.},
}

*Arabidopsis/genetics/metabolism
*Diacylglycerol O-Acyltransferase/genetics/metabolism
*Seeds/metabolism/genetics
*Arabidopsis Proteins/genetics/metabolism
*Gene Editing
*Plant Oils/metabolism
Triglycerides/biosynthesis
*Sequence Deletion
Gene Expression Regulation, Plant
CRISPR-Cas Systems

@article {pmid41344457,
year = {2026},
author = {Liu, L and Huang, X and Wan, S and Gao, Z and Liu, H},
title = {Ectodysplasin A regulates the development of scale and intermuscular bone in teleosts.},
journal = {International journal of biological macromolecules},
volume = {337},
number = {Pt 2},
pages = {149465},
doi = {10.1016/j.ijbiomac.2025.149465},
pmid = {41344457},
issn = {1879-0003},
mesh = {Animals ; Zebrafish/genetics/growth & development ; *Ectodysplasins/genetics/metabolism ; *Fishes/genetics/growth & development ; *Bone and Bones/metabolism ; Phylogeny ; *Animal Scales/growth & development/metabolism ; Signal Transduction ; Gene Expression Regulation, Developmental ; *Fish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Most bony fish are extensively covered by scales, which play crucial roles in locomotion, balance, and sensory perception. However, the molecular mechanisms underlying fish scales development remain poorly understood. In this study, comparative genomic analyses were performed between scaled and scaleless fish species, leading to the identification of key genes (eda, wnt3a, gsk3bb, etc.) involved in scale formation. Phylogenetic and sequence analyses of the eda gene revealed that the Eda protein in Monopterus albus lacks a transmembrane domain, disrupting Eda/Edar binding and potentially driving scale degeneration. Using CRISPR/Cas9 technology, we generated zebrafish eda[-/-] mutants, which exhibited a complete absence of scales, fin rays, pharyngeal teeth, and gill rakers. Notably, the intermuscular bones in these mutants showed significantly reduced length, and simplified morphology, indicating impaired growth. Furthermore, quantitative PCR (qPCR) analysis demonstrated that eda deficiency disrupts the Eda/Edar/NF-κB signaling pathway. Our findings provide significant insights into the molecular regulatory mechanisms underlying the development of skin appendages (e.g., scales) and intermuscular bones.},
}

Animals
Zebrafish/genetics/growth & development
*Ectodysplasins/genetics/metabolism
*Fishes/genetics/growth & development
*Bone and Bones/metabolism
Phylogeny
*Animal Scales/growth & development/metabolism
Signal Transduction
Gene Expression Regulation, Developmental
*Fish Proteins/genetics/metabolism
CRISPR-Cas Systems

@article {pmid41298443,
year = {2025},
author = {Wei, T and Yang, X and Jiang, C and Liao, K and Ye, H and Luo, HY and Liu, YR and Wang, PY and Meng, F and Dou, SX and Rong, Z and Li, H},
title = {Osmotic pressure regulates DNA labelling and transcription with dCas9-SunTag system in live cells.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {773},
pmid = {41298443},
issn = {2041-1723},
support = {12122402, 12074043//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Osmotic Pressure/physiology ; *DNA/metabolism/genetics ; Humans ; *Transcription, Genetic ; Cell Nucleus/metabolism/genetics ; CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/metabolism/genetics ; Telomere/metabolism/genetics ; HeLa Cells ; },
abstract = {Extracellular osmotic pressure is a key modulator of intracellular biophysical properties and cellular functions. However, its impact on the cell nucleus remains elusive, largely due to the challenges in real-time measurement of local environmental properties and reaction kinetics at specific loci within the nucleus. Here, we employ the dCas9-SunTag system to investigate the biophysical response at target DNA loci to osmotic pressure alterations. We reveal that variations in extracellular osmotic pressure modulate the efficiency of dCas9-SunTag-mediated fluorescent labelling rapidly and reversibly, with hypoosmotic condition increasing and hyperosmotic condition decreasing the number and fluorescence intensity of foci for telomeres and genes. Strikingly, osmotic pressure also regulates gene transcription with the dCas9-SunTag system, mirroring its effects on fluorescent labelling, as evidenced by changes in mRNA burst frequency. The underlying mechanism is that osmotic pressure shifts the binding-unbinding equilibrium of specific proteins to dCas9-SunTag complex by altering intranuclear crowding. These findings not only highlight the role of mechanical cues in modulating DNA-related processes within the nucleus, but also establish the dCas9-SunTag system as a sensitive probe for intranuclear crowding in response to extracellular cues, notably osmotic pressure.},
}

*Osmotic Pressure/physiology
*DNA/metabolism/genetics
Humans
*Transcription, Genetic
Cell Nucleus/metabolism/genetics
CRISPR-Cas Systems
*CRISPR-Associated Protein 9/metabolism/genetics
Telomere/metabolism/genetics
HeLa Cells

@article {pmid41543170,
year = {2026},
author = {Romanowski, JS and Myles, KM and Adelman, ZN},
title = {Microhomology-mediated end joining is the predominant form of DNA repair in the mosquito Aedes aegypti with implications for gene editing, gene drive, and transgene removal.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41543170},
issn = {1362-4962},
support = {//National Institute of Allergies and Infectious Diseases/ ; AI148787/NH/NIH HHS/United States ; //NIAID/ ; },
mesh = {Animals ; *Aedes/genetics ; *Gene Editing/methods ; *DNA End-Joining Repair ; CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; Transgenes ; Gene Drive Technology ; DNA Repair ; },
abstract = {Programmable site-specific nucleases have revolutionized the field of genetics, and in the field of mosquito vector control, gene editing by these tools has inspired a new wave of population control approaches that aim to prevent disease transmission. Little is known of how DNA repair is prioritized in mosquitoes, which diverged from the nearest model system (Drosophila) by >200 million years, despite site-specific gene editing now being commonplace. Here, we report a scalable, high-throughput platform for studying DNA double-stranded DNA break (DSB) repair in mosquitoes by delivering CRISPR/Cas9, I-SceI, or other nucleases to Aedes aegypti embryos, capable of measuring single-strand annealing (SSA), non-homologous end joining, and microhomology-mediated end-joining (MMEJ) repair outcomes. We find CRISPR/Cas9 can induce deletions of up to 8.6 kb through SSA repair and is tolerant of resection distances of 3.5 kb. Indel events were insensitive to lig4 knockouts, and across 20 synthetic guide RNAs (sgRNAs) representing 5 locations in 2 transgenic strains were almost exclusively attributed to MMEJ repair, establishing MMEJ as the dominant form of repair in A. aegypti at CRISPR/Cas9 DSBs. This information is critical to our understanding of how DNA repair shapes processes required for genetic control strategies involving gene drive action/resistance as well as transgene stability.},
}

Animals
*Aedes/genetics
*Gene Editing/methods
*DNA End-Joining Repair
CRISPR-Cas Systems
DNA Breaks, Double-Stranded
Transgenes
Gene Drive Technology
DNA Repair

@article {pmid41542957,
year = {2026},
author = {Lee, SY and Park, HH},
title = {Structural insights into promoter recognition by Aca7.},
journal = {The FEBS journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/febs.70405},
pmid = {41542957},
issn = {1742-4658},
support = {RS-2025-02316334//NRF/ ; RS-2025-16065724//NRF korea/ ; },
abstract = {CRISPR-Cas systems provide adaptive immunity to bacteria, although bacteriophages counter these defenses with anti-CRISPR (Acr) proteins. Acr expression is frequently regulated by anti-CRISPR associated (Aca) proteins, which repress transcription by binding inverted repeat (IR) sequences in operon promoters. Here, we report the first identification of an IR motif within the AcrIF11-Aca7 operon promoter from Halomonas caseinilytica and present the crystal structure of Aca7 bound to this IR DNA. Biochemical assays demonstrated that Aca7 specifically recognizes the IR element, and structural analysis revealed a symmetric Aca7 dimer engaging both major grooves via helix-turn-helix motifs while stabilizing DNA bending through minor groove contacts. Residue-level interactions, including those mediated by R38, Q42, K46, and K49, establish a detailed basis for sequence-specific recognition. Comparison with Aca2 highlights distinct dimer architectures and DNA deformation strategies among Aca proteins. Our findings uncover the molecular mechanism by which Aca7 represses AcrIF11 expression and broaden the understanding of Aca-mediated transcriptional regulation.},
}

@article {pmid41541975,
year = {2025},
author = {Hummel, L and Carr, C and Biow, S and Asher, K and Sauer, T and Maylin, M},
title = {Re-examining the Diagnostic Criteria for Wilson's Disease: A Case Report and Literature Review.},
journal = {Cureus},
volume = {17},
number = {12},
pages = {e99271},
pmid = {41541975},
issn = {2168-8184},
abstract = {Wilson's disease (WD) is a heterogeneous genetic disorder for which diagnosis is challenging. We present the case of a 54-year-old woman with a complicated medical history, including hepatitis C, cirrhosis, hepatic encephalopathy, and extensive psychiatric disease, who was transferred to our hospital for management of a spinal epidural abscess. Further findings suggested undiagnosed WD, including bilateral rings around Descemet's membrane and modestly low ceruloplasmin but normal urinary copper excretion. Many algorithms have been proposed for Wilson disease diagnosis, including clinical, laboratory, imaging, and genetic findings; however, no single test is diagnostic. The European Association for the Study of the Liver (EASL) guidelines are the most commonly employed algorithm. Updated 2022 guidelines by both the American Association for the Study of Liver Diseases (AASLD) and the British Association for the Study of the Liver (BASL) build upon this algorithm. Our patient scored 5 points on the EASL scale, which is sufficient for diagnosis. Nevertheless, we believe that she met the diagnostic criteria without having the actual disease. Newer guidelines by AASLD and BASL do not provide additional conclusivity. Our case demonstrates the need for re-evaluation of the diagnostic criteria of WD, where uncertainty can mean permanent hepatic and brain damage. Diagnostic guidelines should incorporate new biomarkers, ophthalmological techniques, and advanced technologies such as next-generation sequencing or CRISPR-Cas-based tools.},
}

@article {pmid41499124,
year = {2026},
author = {Wang, K and Wang, W and Hu, Y and Li, X and Yu, H and Xia, X and Kai, T and Wen, M},
title = {A Relay CRISPR/Cas12a System for Extraction-Free and Ultrasensitive Detection of Staphylococcus aureus in Complex Food Samples.},
journal = {Analytical chemistry},
volume = {98},
number = {2},
pages = {1657-1666},
doi = {10.1021/acs.analchem.5c06345},
pmid = {41499124},
issn = {1520-6882},
mesh = {*Staphylococcus aureus/isolation & purification/genetics ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Food Microbiology ; Electrochemical Techniques ; DNA, Bacterial/genetics/analysis ; Limit of Detection ; RNA, Ribosomal, 16S/genetics ; },
abstract = {Staphylococcus aureus (SA) is a predominant foodborne pathogen, frequently associated with nutrient-rich matrices. However, rapid and ultrasensitive detection of SA in complex food samples remains a formidable challenge. In this work, we developed a relay biosensing system for detecting SA-16S rDNA that combines a magnetic bead-anchored APE1 DNA walker with an asymmetric CRISPR/Cas12a system. The magnetic bead-anchored DNA walker enables efficient separation of SA-targets from complex food matrices, while generating abundant activators through APE1-mediated catalytic cleavage. Employing spatially decoupled reactions with differential crRNAs, the asymmetric Cas12a system achieves programmable cascade amplification. Dual-mode fluorescence/electrochemical readout supports both laboratory analysis and on-site detection. The system demonstrates exceptional sensitivity, capable of detecting trace amounts of SA-16S rDNA (7.5 aM by fluorescence, 1.0 fM by electrochemistry) and SA (4 CFU/mL by fluorescence, 34 CFU/mL by electrochemistry). Overall, this work provides a modular and programmable biosensing strategy for bacterial identification, thereby facilitating advanced nucleic acid analysis within complex biological matrices.},
}

*Staphylococcus aureus/isolation & purification/genetics
*CRISPR-Cas Systems
*Biosensing Techniques/methods
*Food Microbiology
Electrochemical Techniques
DNA, Bacterial/genetics/analysis
Limit of Detection
RNA, Ribosomal, 16S/genetics

@article {pmid41439322,
year = {2026},
author = {Wang, Z and Yang, F and Zeng, S and Sun, R and Hu, Q and Du, Y},
title = {An integrated valved microfluidic platform for rapid and simultaneous nucleic acid detection.},
journal = {Lab on a chip},
volume = {26},
number = {2},
pages = {507-514},
doi = {10.1039/d5lc01096a},
pmid = {41439322},
issn = {1473-0189},
mesh = {Humans ; *Lab-On-A-Chip Devices ; *Nucleic Acid Amplification Techniques/instrumentation ; Human papillomavirus 16/genetics/isolation & purification ; Human papillomavirus 18/genetics/isolation & purification ; *DNA, Viral/analysis/genetics ; *Nucleic Acids/analysis ; *Microfluidic Analytical Techniques/instrumentation ; CRISPR-Cas Systems ; },
abstract = {Applying CRISPR-based diagnostics to point-of-care pathogen detection remains challenging because of the multi-step and time-consuming sample preparation process. This study presents a low-cost, integrated valved microfluidic device that combines recombinase polymerase amplification (RPA), CRISPR signal amplification, and lateral flow readout for simultaneous nucleic acid detection. The core advantage of the platform lies in its ability to sequentially control the entire multi-step assay through simple valve operation, significantly minimizing user intervention. All key reagents, including the RPA mix, Cas12a/crRNA complex, and proteinase K lysis buffer, are pre-lyophilized, ensuring stability and ready-to-use functionality. The platform demonstrates a sensitivity of 20 copies/reaction for HPV16/18 plasmids and accurately genotypes HPV in lysates of cervical cancer cells within one hour, showing complete concordance with quantitative PCR results. This integrated device, achieving a user-friendly protocol and visual readout, provides a powerful tool for nucleic acid-based point-of-care testing and self-testing in resource-limited settings.},
}

Humans
*Lab-On-A-Chip Devices
*Nucleic Acid Amplification Techniques/instrumentation
Human papillomavirus 16/genetics/isolation & purification
Human papillomavirus 18/genetics/isolation & purification
*DNA, Viral/analysis/genetics
*Nucleic Acids/analysis
*Microfluidic Analytical Techniques/instrumentation
CRISPR-Cas Systems

@article {pmid41422144,
year = {2025},
author = {Felício, D and Osório, H and Pereira, C and Brandão, AF and Freixo, JP and Carvalho, I and Sousa, AP and Castro-Caldas, M and Sequeiros, J and Lemos, C and Santos, M},
title = {Missense variant in TTBK2 kinase domain causes loss of function and impaired protein phosphorylation.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {2501},
pmid = {41422144},
issn = {2045-2322},
support = {UI/BD/154402/2023//Fundação para a Ciência e a Tecnologia/ ; UIDB/00215/2020, UIDP/00215/2020, LA/P/0064/2020 , UID/215/2025//Fundação para a Ciência e a Tecnologia/ ; DL 57/2016 - Norma Transitória//Fundação para a Ciência e a Tecnologia/ ; ROTEIRO/0028/2013; LISBOA-01-0145-FEDER-022125//Rede Nacional de Espectrometria de Massa/ ; ZGRACA//Ataxia UK/ ; },
mesh = {Humans ; Phosphorylation ; *Mutation, Missense ; *Protein Serine-Threonine Kinases/genetics/metabolism/chemistry ; Protein Domains ; CRISPR-Cas Systems ; *Loss of Function Mutation ; HEK293 Cells ; },
abstract = {Tau tubulin kinase 2 (TTBK2) is a ubiquitous serine-threonine protein kinase implicated in diverse cellular processes, including microtubule regulation, ciliogenesis, synaptic signaling, and the phosphorylation of key proteins like TDP-43. Despite its relevance, many aspects of TTBK2 function in both physiological and pathological conditions remain poorly understood. Truncating variants in TTBK2 gene cause spinocerebellar ataxia type 11 (SCA11), a rare form of autosomal dominant cerebellar ataxia. However, the functional consequences and pathogenic potential of missense variants have yet to be elucidated. In this study, we developed a CRISPR/Cas9 knock-in cell model harboring a missense variant in TTBK2 kinase domain (NM_173500.4:c.625 C > T; p.Leu209Phe) to evaluate its impact on TTBK2 expression, associated protein levels, and phosphoproteomic profiles. TTBK2 missense variant (TTBK2-L209F) was associated with reduced TTBK2 protein levels, altered levels of cytoskeleton-related proteins, and impaired kinase activity, namely toward TDP-43. Phosphoproteomic analyses identified dysregulation in pathways linked to gene regulation, protein degradation, cytoskeletal organization, and TGF-β signaling. These findings provide valuable insights into the biological roles of TTBK2 in cellular signaling. Moreover, this study underscores the importance of functional studies to better understand the consequences of TTBK2 missense variants, particularly those affecting the kinase domain, and their potential contribution to disease.},
}

Humans
Phosphorylation
*Mutation, Missense
*Protein Serine-Threonine Kinases/genetics/metabolism/chemistry
Protein Domains
CRISPR-Cas Systems
*Loss of Function Mutation
HEK293 Cells

@article {pmid41388295,
year = {2025},
author = {Ji, T and Zhang, Y and Wang, Y and Yuan, K and Wang, M and Ye, J and Zhang, H and Zhang, N and Zhang, H},
title = {AND logic-gated CRISPR/Cas9 and hybridization chain reaction system for precise ctDNA detection.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {43},
pmid = {41388295},
issn = {1477-3155},
support = {2022YFB3808200//National Key Research and Development Program of China/ ; },
mesh = {*Circulating Tumor DNA/genetics/blood/analysis ; Humans ; *CRISPR-Cas Systems/genetics ; Polymorphism, Single Nucleotide ; Nucleic Acid Amplification Techniques/methods ; Biomarkers, Tumor/genetics/blood ; Nucleic Acid Hybridization ; Mutation ; Proto-Oncogene Proteins p21(ras)/genetics ; Limit of Detection ; },
abstract = {Circulating tumor DNA (ctDNA) is a critical biomarker for liquid biopsies, enabling the non-invasive acquisition of cancer-related information from blood samples. Precise detection of ctDNA, particularly the identification of single-nucleotide variations (SNVs), is crucial for early cancer diagnosis, therapeutic monitoring, and prognostic evaluation. However, current ctDNA detection methods often encounter challenges such as complex procedures, difficult data analysis, and false-positive signals during pre-amplification. In this study, we introduce a novel detection method based on AND logic-gated integration of interspaced short palindromic repeats and associated proteins (CRISPR/Cas9) system with hybridization chain reaction (HCR) isothermal amplification. This strategy enhances the specific and sensitive detection of ctDNA. The incorporation of the AND logic gate effectively minimizes the off-target effects of Cas9 and enables the differentiation of single-nucleotide mutations, such as KRAS G12D, even in complex serum environments. Our system exhibits high sensitivity and specificity, achieving a limit of detection as low as 1 fM and capable of identifying SNVs mutations with allele fractions as low as 0.1% among wild-type sequences. Furthermore, we validated the specificity of our approach by successfully detecting various mutations, including KRAS G12C, KRAS G12D, EGFR T790M and TP53 R273H, in simulated clinical samples. These findings highlight a reliable method for precise ctDNA detection, offering high specificity, selectivity, and accuracy, thus paving the way for potential cancer diagnostic application.},
}

*Circulating Tumor DNA/genetics/blood/analysis
Humans
*CRISPR-Cas Systems/genetics
Polymorphism, Single Nucleotide
Nucleic Acid Amplification Techniques/methods
Biomarkers, Tumor/genetics/blood
Nucleic Acid Hybridization
Mutation
Proto-Oncogene Proteins p21(ras)/genetics
Limit of Detection

@article {pmid41387738,
year = {2025},
author = {Zhang, X and Zhu, T and Zhang, W and Zhang, Y and Zhang, J and Yang, J and Xia, C and Zhao, H and Yu, Y and Wen, C},
title = {Ethylene promotes branch formation but inhibits tendril development in cucumber.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {745},
pmid = {41387738},
issn = {2041-1723},
support = {JKZX202207//Beijing Academy of Agricultural and Forestry Sciences (BAAFS)/ ; },
mesh = {*Ethylenes/pharmacology/metabolism ; *Cucumis sativus/growth & development/genetics/metabolism/drug effects ; Gene Expression Regulation, Plant/drug effects ; Plant Proteins/genetics/metabolism ; Signal Transduction/drug effects ; CRISPR-Cas Systems ; *Plant Growth Regulators/pharmacology/metabolism ; Mutation ; Promoter Regions, Genetic ; },
abstract = {Ethylene coordinates numerous plant growth processes, particularly in cucurbit crops, yet its role in vegetative growth regulation remains largely unexplored. Here, we report the function of ethylene in controlling branch and tendril development in cucumber. We find that ethylene promotes branches formation but inhibits tendrils development in a dose-dependent manner. CRISPR-Cas9-generated gene-edited Csein2 and Csein3/Cseil1 mutants exhibit few branches and more tendrils. Exogenous ethylene can recover the branch/tendril defective phenotypes of the Csein3 and Cseil1 mutants but not those of the Csein2 mutant or the Csein3/Cseil1 double mutant. Transcriptomic and metabolic analyses reveal that CsCYP707A4 and CsTL are the key downstream targets of ethylene signaling. We show that CsEIN3 can bind to its promoters to activate the expression of CsCYP707A4 but inhibit the expression of CsTL, which leads to the opposite effect on branch and tendril development. The study sets the foundation for designing ideal plant architecture to increase production efficiency.},
}

*Ethylenes/pharmacology/metabolism
*Cucumis sativus/growth & development/genetics/metabolism/drug effects
Gene Expression Regulation, Plant/drug effects
Plant Proteins/genetics/metabolism
Signal Transduction/drug effects
CRISPR-Cas Systems
*Plant Growth Regulators/pharmacology/metabolism
Mutation
Promoter Regions, Genetic

@article {pmid41382257,
year = {2025},
author = {Chen, W and Wu, P and Champer, J},
title = {Strategies to improve the efficiency of homing gene drives with multiplexed gRNAs.},
journal = {BMC biology},
volume = {24},
number = {1},
pages = {12},
pmid = {41382257},
issn = {1741-7007},
support = {32270672//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Drosophila melanogaster/genetics ; *Gene Drive Technology/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; },
abstract = {BACKGROUND: CRISPR homing gene drive holds great potential for pest control, but its success is challenged by the generation of resistance alleles through end-joining repair. Using multiple gRNAs to target adjacent sites within a conserved gene can prevent functional resistance by allowing repeated cleavage events, but poor homology during DNA repair may compromise efficiency.

RESULTS: We first assessed the efficiency of single gRNA drives with truncated homology arms in Drosophila melanogaster mimicking a multiplexed system in which only one site is cleaved. Integrating results into a detailed gRNA multiplexing model, we found that efficiency loss was greater than expected. To mitigate this, we evaluated two new strategies: (1) extended homology arms to span all target sites (with mutations in the PAMs to prevent self-cleavage) and (2) a population-level gRNA multiplexing system involving two or more drives, each carrying two gRNAs. Extended homology arms did not result in notable improvement in conversion efficiency, and the extended region could be lost during drive conversion. The population-level multiplexing gRNAs strategy was more promising, though the intentionally mutated PAM also could not be consistently inherited. Simulations of homing suppression drives applying population-level multiplexed gRNAs increased the success rate of population elimination and reduced the time required for suppression.

CONCLUSIONS: Future drive designs requiring a larger number of gRNAs could potentially be improved. The design relying on extended homology arms may not represent an optimal strategy. However, population-level multiplexing gRNAs could serve as a promising alternative, enhancing efficiency while maintaining tolerance to functional resistance.},
}

Animals
*Drosophila melanogaster/genetics
*Gene Drive Technology/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems

@article {pmid41370122,
year = {2026},
author = {Pfisterer, L and Boyle, C and Cole, A and Mitchell, I and Flanagan, M and Gromley, Z and Gromley, A},
title = {Disruption of the centriolin/Cep110 gene (CNTRL) with CRISPR/Cas9 leads to cell cycle arrest and cell death of rhabdomyosarcoma cells in vitro.},
journal = {Molecular biology of the cell},
volume = {37},
number = {2},
pages = {br4},
doi = {10.1091/mbc.E25-08-0365},
pmid = {41370122},
issn = {1939-4586},
mesh = {CRISPR-Cas Systems/genetics ; Humans ; *Rhabdomyosarcoma/genetics/metabolism/pathology ; Cell Line, Tumor ; *Cell Cycle Checkpoints/genetics ; *Cell Cycle Proteins/genetics/metabolism ; Gene Editing/methods ; Centrosome/metabolism ; Cell Death/genetics ; *Microtubule-Associated Proteins/genetics/metabolism ; Cilia/metabolism ; Cell Survival/genetics ; Cell Proliferation/genetics ; Apoptosis/genetics ; },
abstract = {Rhabdomyosarcoma is the most common pediatric soft tissue cancer, thought to arise from primitive mesenchymal cells that differentiate into skeletal muscle. Previous studies suggest that primary cilia may play a role in the development of rhabdomyosarcoma. Primary cilia are cellular structures that arise from the centrosome and serve important functions in sensory signaling, cell migration, and developmental processes. However, most rhabdomyosarcoma cell lines do not have primary cilia. Because primary cilia are derived from centrosomes, the development of rhabdomyosarcoma may, in fact, be due to the function of centrosome proteins rather than the primary cilia itself. Therefore, this study sought to determine if the centrosomal protein centriolin/Cep110, which is localized to both centrosomes and primary cilia, plays a role in rhabdomyosarcoma biology. The gene editing tool CRISPR/Cas9 was used to disrupt the centriolin/Cep110 gene in the rhabdomyosarcoma cell line CCL-136, and the effects on cell viability and cell cycle progression were assayed. Our results show that loss of centriolin/Cep110 leads to cell cycle arrest and apoptotic cell death in rhabdomyosarcoma cells. These findings suggest that centriolin/Cep110 plays a key role in rhabdomyosarcoma cell proliferation and viability and that this centrosome protein may represent a potential target for future rhabdomyosarcoma therapies.},
}

CRISPR-Cas Systems/genetics
Humans
*Rhabdomyosarcoma/genetics/metabolism/pathology
Cell Line, Tumor
*Cell Cycle Checkpoints/genetics
*Cell Cycle Proteins/genetics/metabolism
Gene Editing/methods
Centrosome/metabolism
Cell Death/genetics
*Microtubule-Associated Proteins/genetics/metabolism
Cilia/metabolism
Cell Survival/genetics
Cell Proliferation/genetics
Apoptosis/genetics

@article {pmid41540035,
year = {2026},
author = {Zhang, H and Shang, R and Zhang, Z and Zhou, M and Bigot, A and Cai, Y and Zhao, Y and Wang, Y and Deshmukh, A and Kudryashova, E and Kudryashov, DS and He, C and Mouly, V and Bi, P},
title = {Development of a split-toxin CRISPR screening platform to systematically identify regulators of human myoblast fusion.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {547},
pmid = {41540035},
issn = {2041-1723},
support = {GM147209//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; AR080330//U.S. Department of Health & Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/ ; },
mesh = {Humans ; *Myoblasts/metabolism/cytology ; *Muscle Development/genetics ; Cell Differentiation/genetics ; Cell Fusion ; *CRISPR-Cas Systems/genetics ; Mutation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Muscle, Skeletal/metabolism ; High-Throughput Screening Assays/methods ; Single-Cell Analysis ; Cell Line ; },
abstract = {Muscle defects are common in human developmental disorders and often cause severe functional impairment. These defects arise from intricate tissue crosstalk and rare genetic mutations, underscoring the need to systematically identify cell-autonomous mechanisms regulating human myogenesis. Here we show a rationally designed, high-throughput genetic screening platform that integrates human myoblast models, customized CRISPR libraries, and a split-toxin strategy that enables quantitative selection of fusion-defective myocytes. Leveraging this platform, our initial screen uncovers a large group of hits essential for human myoblast fusion. The majority of these hits converge into 23 protein complexes. Notably, mutations in 41 screen hits are associated with human diseases marked by abnormal skeletal-muscle morphology. Applying a new single-cell CRISPR & RNA-seq approach, we show that majority of these hits control human myoblast fusion as well as influence early-stage myogenic differentiation. This work establishes a scalable approach to identify cell-autonomous regulators of human muscle differentiation and fusion.},
}

Humans
*Myoblasts/metabolism/cytology
*Muscle Development/genetics
Cell Differentiation/genetics
Cell Fusion
*CRISPR-Cas Systems/genetics
Mutation
*Clustered Regularly Interspaced Short Palindromic Repeats
Muscle, Skeletal/metabolism
High-Throughput Screening Assays/methods
Single-Cell Analysis
Cell Line

@article {pmid41538317,
year = {2026},
author = {Ham, DT and Browne, TS and Zhang, CQ and Foo, GW and Uruthirapathy, AS and Gloor, GB and Edgell, DR},
title = {Machine learning reveals sequence and methylation determinants of SaCas9-PAM interactions in bacteria.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41538317},
issn = {1362-4962},
support = {PJT 159708/CAPMC/CIHR/Canada ; PJT 191939/CAPMC/CIHR/Canada ; },
mesh = {*Machine Learning ; Staphylococcus aureus/genetics/enzymology ; *CRISPR-Associated Protein 9/metabolism/genetics ; Escherichia coli/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; CRISPR-Cas Systems ; Nucleotide Motifs ; DNA Methylation ; Methylation ; Gene Editing/methods ; },
abstract = {Cas9 nucleases defend bacteria against invading DNA and can be used with single guide RNAs (sgRNAs) as antimicrobials and genome-editing tools. However, bacterial applications are limited by incomplete knowledge of Cas9-target interactions. Here, we generated large-scale Staphylococcus aureus Cas9 (SaCas9)/sgRNA activity datasets in bacteria and trained a machine learning model (crispr macHine trAnsfer Learning) to predict SaCas9 activity. Incorporating downstream sequences flanking the canonical NNGRRN protospacer adjacent motif (PAM) at positions [+1] and [+2] improved predictive performance, with T-rich dinucleotides at these positions correlating with higher in vivo activity. Crucially, SaCas9 showed $\sim$10-fold reduced activity at sites containing a 5$^{\prime}$
-NNGGAT[C]-3$^\prime$ PAM [+1] sequence in pooled sgRNA experiments in Escherichia coli and Citrobacter rodentium. Plasmid cleavage assays in DNA adenine methyltransferase (DAM)-deficient E. coli confirmed that adenine methylation at GATC motifs inhibited SaCas9 activity. Removal of a DAM site within a PAM sequence enhanced cleavage, while introduction of a site reduced activity, directly linking adenine methylation to SaCas9 activity. These findings demonstrate that machine learning can uncover biologically relevant determinants of Cas9 activity. Avoidance of methylated PAMs may reflect an evolutionary adaptation by SaCas9 to discriminate self from nonself or to counter methylation as a phage and plasmid antirestriction strategy.},
}

*Machine Learning
Staphylococcus aureus/genetics/enzymology
*CRISPR-Associated Protein 9/metabolism/genetics
Escherichia coli/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
CRISPR-Cas Systems
Nucleotide Motifs
DNA Methylation
Methylation
Gene Editing/methods

@article {pmid41538311,
year = {2026},
author = {Xiao, Z and Sun, Y},
title = {Illuminating the genome: emerging approaches in CRISPR-Cas live-cell imaging.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41538311},
issn = {1362-4962},
support = {2022YFA1303103//National Key Research and Development Program of China/ ; 2022YFA3401100//National Key Research and Development Program of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Genome ; Gene Editing/methods ; Animals ; },
abstract = {CRISPR-Cas-based live-cell imaging has rapidly become a central technology for studying genome dynamics with high specificity and flexibility. By coupling nuclease-deactivated Cas (dCas) with programmable guide RNAs, genomic loci can be tracked in living cells, providing direct insights into nuclear organization and chromatin behavior. While repetitive regions such as telomeres and centromeres are readily visualized, labeling non-repetitive loci remains more challenging due to weak signals and high background. Recent advances, including multicolor labeling strategies, innovative amplification systems based on dCas9 and single-guide RNA (sgRNA) engineering, and integration with novel fluorescent reporters, have markedly expanded the applicability of CRISPR imaging across the genome. These developments have expanded the multiplexing capacity of CRISPR imaging, improved signal-to-background ratios, and even enabled the visualization of non-repetitive genomic loci. Nonetheless, key challenges remain, including cellular toxicity, replication stress, and genomic instability associated with prolonged CRISPR expression. In this review, we summarize recent advances in CRISPR live-cell imaging and highlight key design trade-offs and biological constraints.},
}

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

@article {pmid41536960,
year = {2026},
author = {van der Wilt, CN and Veltrop, RJA and Janssens, MH and Bakker, I and Stillitano, F and Sluijter, JPG and van Laake, LW and van der Velden, J and Villard, E and Montag, J and Denning, C and van Tintelen, JP and Te Riele, ASJM and van der Harst, P and Schurgers, LJ and van Steenbeek, FG and Harakalova, M},
title = {Setting the stage for cardiomyopathy gene editing trials: a systematic review of isogenic pair use in human induced pluripotent stem cell-derived cardiomyocyte research.},
journal = {European heart journal open},
volume = {6},
number = {1},
pages = {oeaf161},
pmid = {41536960},
issn = {2752-4191},
abstract = {In vitro gene editing using isogenic pairs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has demonstrated the feasibility of introducing or correcting specific pathogenic variants. These successes represent a key first step towards therapeutic genome editing for cardiomyopathies, showing that precise, variant-specific interventions are achievable. To translate in vitro findings to the clinic, it is essential to develop robust disease models that yield meaningful, translatable data. The next challenge is systematically identifying disease-causing variants amenable to gene editing with strong pre-clinical support. Therefore, we conducted a systematic search of published studies on isogenic hiPSC-CM pairs in cardiomyopathy research with specific criteria, including (likely) pathogenic variants causing cardiomyopathy, correction and/or introduction of variants, differentiation into CMs, and functional follow-up. We systematically assessed 785 papers and highlighted 101 studies meeting our inclusion criteria reporting 69 patients carrying 56 unique variants across 31 genes, most commonly MYH7, MYBPC3, and DMD. This expanded to 91 variants across 38 genes upon inclusion of the introduced variants in a donor line. However, reported clinical data were often incomplete, underscoring the need for standardized phenotypic documentation. We reveal a lack of patient details, which creates an incomplete picture of underlying disease variables that hinder the design of targeted personalized treatments. Omitted key clinical data can lead to misinterpretations or overlooked variables that impact treatment outcomes. This systematic review integrates current evidence from successful in vitro studies using isogenic hiPSC-CM models and proposes a reporting framework for variant prioritization and the rigorous application of isogenic controls in cardiomyopathy research.},
}

@article {pmid41536809,
year = {2026},
author = {Liou, RH and Urrutia-Cabrera, D and Liu, CF and Wu, S and Westin, IM and Golovleva, I and Liu, GS and Kumar, S and McLenachan, S and Chen, FK and Hsu, FT and Huang, CL and Edwards, T and Martin, KR and Cheng, AW and Wong, RCB},
title = {Using RNA-targeting CRISPR-Cas13 and engineered U1 systems to target ABCA4 splice variants in Stargardt disease.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {1},
pages = {102789},
pmid = {41536809},
issn = {2162-2531},
abstract = {Dysregulation of the alternative splicing process results in aberrant mRNA transcripts, leading to dysfunctional proteins or nonsense-mediated decay that cause a wide range of mis-splicing diseases. Development of therapeutic strategies to target the alternative splicing process could potentially shift the mRNA splicing from disease isoforms to a normal isoform and restore functional protein. As a proof of concept, we focus on Stargardt disease (STGD1), an autosomal recessive inherited retinal disease caused by biallelic genetic variants in the ABCA4 gene. The splicing variants c.5461-10T>C and c.4773+3A>G in ABCA4 cause the skipping of exon 39-40 and exon 33-34, respectively. In this study, we compared the efficacy of different RNA-targeting systems to modulate these ABCA4 splicing defects, including four CRISPR-Cas13 systems (CASFx-1, CASFx-3, RBFOX1N-dCas13e-C, and RBFOX1N-dPspCas13b-C) as well as an engineered U1 system (ExSpeU1). Using a minigene system containing ABCA4 variants in the human retinal pigment epithelium ARPE19, our results show that RBFOX1N-dPspCas13b-C is the best performing CRISPR-Cas system, which enabled up to 80% reduction of the mis-spliced ABCA4 c.5461-10T>C variants and up to 78% reduction of the ABCA4 c.4773+3A>G variants. In comparison, delivery of a single ExSpeU1 was able to effectively reduce the mis-spliced ABCA4 c.4773+3A>G variants by up to 84%. We observed that the effectiveness of CRISPR-based and U1 splicing regulation is strongly dependent on the sgRNA/snRNA targeting sequences, highlighting that optimal sgRNA/snRNA designing is crucial for efficient targeting of mis-spliced transcripts. Overall, our study demonstrated the potential of using RNA-targeting CRISPR-Cas technology and engineered U1 to reduce mis-spliced transcripts for ABCA4, providing an important step to advance the development of gene therapy to treat STGD1.},
}

@article {pmid41535388,
year = {2026},
author = {Rolando, JC and Thieme, A and Weckman, NE and Kim, N and de Puig, H and Tan, X and Cotnoir, E and Chaturvedi, V and Collins, JJ and Walt, DR},
title = {Digital CRISPR-based diagnostics for quantification of Candida auris and resistance mutations.},
journal = {Nature biomedical engineering},
volume = {},
number = {},
pages = {},
pmid = {41535388},
issn = {2157-846X},
support = {WC-2019-01//New York State Department of Health - Wadsworth Center (Department of Health, Wadsworth Center)/ ; WC-2019-01//New York State Department of Health - Wadsworth Center (Department of Health, Wadsworth Center)/ ; WC-2019-01//New York State Department of Health - Wadsworth Center (Department of Health, Wadsworth Center)/ ; WC-2019-01//New York State Department of Health - Wadsworth Center (Department of Health, Wadsworth Center)/ ; WC-2019-01//New York State Department of Health - Wadsworth Center (Department of Health, Wadsworth Center)/ ; },
abstract = {Candida auris, an increasingly prevalent fungal pathogen, requires both rapid identification and antifungal susceptibility testing to enable proper treatment. This study introduces digital SHERLOCK (dSHERLOCK), a platform that combines CRISPR/Cas nucleic acid detection, single-template quantification and real-time kinetics monitoring. Assays implemented on this platform display excellent sensitivity to C. auris from major clades 1-4, while maintaining specificity when challenged with common environmental and pathogenic fungi. dSHERLOCK detects C. auris within 20 min in minimally processed swab samples and achieves sensitive quantification (1 c.f.u. µl[-1]) within 40 min. To address antifungal susceptibility testing, we develop assays that detect mutations that are commonly associated with azole and echinocandin multidrug resistance. We use machine learning and real-time monitoring of reaction kinetics to achieve highly accurate simultaneous quantification of mutant and wild-type FKS1 SNP alleles in fungal populations with mixed antifungal susceptibility, which would be misdiagnosed as completely susceptible or resistant under standard reaction conditions. Our platform's use of commercially available materials and common laboratory equipment makes C. auris diagnostics widely deployable in global healthcare settings.},
}

@article {pmid41526513,
year = {2026},
author = {Ngo, W and Wu, JLY and Wasko, KM and Doudna, JA},
title = {Targeted delivery of genome editors in vivo.},
journal = {Nature biotechnology},
volume = {44},
number = {1},
pages = {49-59},
pmid = {41526513},
issn = {1546-1696},
support = {DE-AC52-07NA27344//DOE | LDRD | Lawrence Livermore National Laboratory (LLNL)/ ; },
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems/genetics ; Animals ; *Genetic Therapy/methods ; *Gene Transfer Techniques ; *Drug Delivery Systems ; },
abstract = {Genome editing has revolutionized the treatment of genetic diseases, yet the difficulty of tissue-specific delivery currently limits applications of editing technology. In this Review, we discuss preclinical and clinical advances in delivering genome editors with both established and emerging delivery mechanisms. Targeted delivery promises to considerably expand the therapeutic applicability of genome editing, moving closer to the ideal of a precise 'magic bullet' that safely and effectively treats diverse genetic disorders.},
}

*Gene Editing/methods
Humans
CRISPR-Cas Systems/genetics
Animals
*Genetic Therapy/methods
*Gene Transfer Techniques
*Drug Delivery Systems

@article {pmid41468873,
year = {2026},
author = {Zhang, J and Zhang, X and Xie, X and Han, B and Zhao, F and Yang, X and Zhang, W and Jiang, Y and Zhang, X},
title = {CRISPR/Cas12a-mediated marker-free fluorescent biosensing platform based on a lightful copper nanocluster for highly sensitive detection of mycotoxin.},
journal = {Talanta},
volume = {301},
number = {},
pages = {129326},
doi = {10.1016/j.talanta.2025.129326},
pmid = {41468873},
issn = {1873-3573},
mesh = {*Copper/chemistry ; *Biosensing Techniques/methods ; *Aflatoxin B1/analysis ; *CRISPR-Cas Systems ; *Metal Nanoparticles/chemistry ; Limit of Detection ; Food Contamination/analysis ; Fluorescence ; *Mycotoxins/analysis ; },
abstract = {The problem of mycotoxin contamination in foodstuffs has attracted widespread attention and posed a great threat to human health. Therefore, the sensitive and effective detection of mycotoxins is of great importance for preserving public health worldwide. In this study, a CRISPR/Cas12a-mediated marker-free fluorescent biosensing platform was constructed for highly sensitive and fast detection of aflatoxin B1. The copper nanoclusters were synthesized with marker-free DNA single strands within 5 min, showing the outstanding fluorescence properties. With the existence of aflatoxin B1, the released complementary DNA (cDNA) triggered multiple isothermal amplification reaction. Subsequently, the obtained amplification products triggered the trans-cleavage activity of CRISPR/Cas12a system, which degraded the DNA single strands for synthesis of copper nanoclusters, leading to a decreased fluorescent signal. Benefiting from good fluorescence properties of copper nanoclusters, the established biosensing platform for aflatoxin B1 detection had a high specificity and a limit of detection of 47.51 pg/mL in the linear range of 0.05-10 ng/mL. The proposed platform provided a new insight for the detection of non-nucleic acid targets.},
}

*Copper/chemistry
*Biosensing Techniques/methods
*Aflatoxin B1/analysis
*CRISPR-Cas Systems
*Metal Nanoparticles/chemistry
Limit of Detection
Food Contamination/analysis
Fluorescence
*Mycotoxins/analysis

@article {pmid41454886,
year = {2026},
author = {Yang, H and Gao, X and Jin, ZC and Zhang, R and Ning, B and Yan, X},
title = {Simple and Versatile Toolkit for Genetic Manipulation of Bacillus licheniformis.},
journal = {ACS synthetic biology},
volume = {15},
number = {1},
pages = {262-270},
doi = {10.1021/acssynbio.5c00699},
pmid = {41454886},
issn = {2161-5063},
mesh = {*Bacillus licheniformis/genetics ; Plasmids/genetics ; *Genetic Engineering/methods ; DNA Transposable Elements/genetics ; Mutagenesis ; CRISPR-Cas Systems/genetics ; },
abstract = {Bacillus licheniformis is a spore-forming bacterium with probiotic, environmental, and industrial applications. Many wild strains with diverse functions have been described in recent years. Nevertheless, the lack of efficient and universal genetic manipulation tools hinders the study and engineering of these strains. Here, a versatile and simple genetic manipulation toolkit is established for B. licheniformis. The cornerstone of this toolkit is a conjugative DNA transfer system. This system could effectively transfer temperature-sensitive plasmid pTSMK into all ten tested B. licheniformis strains, with efficiencies ranging from 10[-5] to 10[-3]. Based on this DNA transfer system, the tools for maker-free knockout and knock-in, CRISPRi, as well as transposon mutagenesis, were built. A transposition frequency of 7.68 × 10[-3] was observed. The toolkit developed in this study fulfills most tasks in the engineering of this species and will promote the basic and applied research of B. licheniformis.},
}

*Bacillus licheniformis/genetics
Plasmids/genetics
*Genetic Engineering/methods
DNA Transposable Elements/genetics
Mutagenesis
CRISPR-Cas Systems/genetics

@article {pmid41418786,
year = {2026},
author = {Sherman, A and Benvenisty, N},
title = {Genetic screening of long non-coding RNAs in human embryonic stem cells reveals novel regulators of pluripotency.},
journal = {Stem cell reports},
volume = {21},
number = {1},
pages = {102743},
doi = {10.1016/j.stemcr.2025.102743},
pmid = {41418786},
issn = {2213-6711},
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Human Embryonic Stem Cells/metabolism/cytology ; Cell Differentiation/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; Octamer Transcription Factor-3/genetics/metabolism ; *Genetic Testing ; Cell Line ; CRISPR-Cas Systems ; Apoptosis/genetics ; },
abstract = {The human genome encodes thousands of long non-coding RNAs (lncRNAs), transcripts of over 200 nucleotides that lack protein-coding potential. lncRNAs are emerging as key players in diverse cellular processes, particularly in tissue-specific contexts, yet their functionality remained poorly understood. Here, we performed a CRISPR interference (CRISPRi) screen in human embryonic stem cells (hESCs), identifying over 100 essential and about 150 growth-restricting lncRNAs. We show that growth-modifying lncRNAs display distinctive properties, including unique expression signatures, genomic structure, evolutionary conservation, chromosomal distribution, and potential involvement in teratoma formation. Notably, we uncovered two primate-conserved, uncharacterized, essential lncRNAs that regulate neighboring pluripotency transcription factors: lncOCT4, which positively regulates OCT4 and induces p53-mediated apoptosis upon knockdown, and lncVRTN, which acts as a putative negative regulator of VRTN, affecting cell fate determination. These findings shed light on the contribution of lncRNAs to the human-specific pluripotency network and provide insights into lncRNA-mediated regulation of hESC growth and differentiation.},
}

Humans
*RNA, Long Noncoding/genetics/metabolism
*Human Embryonic Stem Cells/metabolism/cytology
Cell Differentiation/genetics
*Pluripotent Stem Cells/metabolism/cytology
Octamer Transcription Factor-3/genetics/metabolism
*Genetic Testing
Cell Line
CRISPR-Cas Systems
Apoptosis/genetics

@article {pmid41391726,
year = {2026},
author = {Liao, XR and Han, D and Qi, LJ and Huang, QY and Gao, QY and He, XY and Guo, T and Lei, JJ and Cheng, SX},
title = {Aptamer-functionalized nanoparticles for CRISPR-Cas9 delivery to circulating malignant cells for therapeutic efficacy evaluation.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {390},
number = {},
pages = {114542},
doi = {10.1016/j.jconrel.2025.114542},
pmid = {41391726},
issn = {1873-4995},
mesh = {Humans ; *CRISPR-Cas Systems ; *Nanoparticles/chemistry/administration & dosage ; *Aptamers, Nucleotide/administration & dosage/chemistry ; Cell Line, Tumor ; Hyaluronic Acid/chemistry ; *Neoplastic Cells, Circulating/metabolism ; Gene Editing/methods ; *Neoplasms/therapy/genetics ; Hyaluronan Receptors/metabolism ; Epithelial Cell Adhesion Molecule/genetics ; Proto-Oncogene Proteins c-met/genetics ; *Gene Transfer Techniques ; ErbB Receptors/genetics ; Plasmids ; },
abstract = {Genome editing therapies targeting oncogenic pathways represent a promising alternative to small-molecule inhibitors, enabling durable therapeutic responses without inducing drug resistance. However, their success hinges on overcoming tumor heterogeneity, as malignant cells of cancer patients exhibit significant phenotypic variability. To advance personalized research on genome editing efficacy, tailored delivery systems capable of precisely targeting heterogeneous cancer cell populations are essential. Herein, we developed a facile modification strategy to construct a multiplexed surface-functionalized gene delivery system targeting heterogeneous cancer cells for personalized therapeutic studies. The system integrates the EGFR-targeting TuTu22 aptamer with SYL3C-conjugated hyaluronic acid (SYL3C-HA) for EpCAM and CD44 recognition. This triple-targeting platform enables efficient delivery of genome editing plasmid for c-Met knockout in both cancer cell lines and circulating malignant cells (CMCs) from cancer patients. The c-Met knockout not only reduces tumor malignancy but also reverses immune suppression, evidenced by PD-L1 downregulation and restored immune surveillance. By combining gene delivery with an ex vivo patient-derived evaluation platform, this system provides a robust tool for personalized research on the therapeutic strategies for tumor progression inhibition and immunity restoration.},
}

Humans
*CRISPR-Cas Systems
*Nanoparticles/chemistry/administration & dosage
*Aptamers, Nucleotide/administration & dosage/chemistry
Cell Line, Tumor
Hyaluronic Acid/chemistry
*Neoplastic Cells, Circulating/metabolism
Gene Editing/methods
*Neoplasms/therapy/genetics
Hyaluronan Receptors/metabolism
Epithelial Cell Adhesion Molecule/genetics
Proto-Oncogene Proteins c-met/genetics
*Gene Transfer Techniques
ErbB Receptors/genetics
Plasmids

@article {pmid41387457,
year = {2025},
author = {Cheng, F and Soleimani Samarkhazan, H and Khazaei, Y},
title = {CRISPR-engineered microbiome: living therapeutics revolutionize blood cancer immunotherapy.},
journal = {NPJ biofilms and microbiomes},
volume = {12},
number = {1},
pages = {17},
pmid = {41387457},
issn = {2055-5008},
mesh = {Humans ; *Immunotherapy/methods ; Animals ; *Hematologic Neoplasms/therapy/immunology/microbiology ; *CRISPR-Cas Systems ; *Gastrointestinal Microbiome/genetics ; *Microbiota ; },
abstract = {Blood cancers such as leukemia, lymphoma, and myeloma remain refractory in many patients due to immune escape, antigen heterogeneity, and therapy‑related toxicities. To address these challenges, we review recent strategies that harness CRISPR‑engineered gut commensals as precision "living therapeutics" to modulate host immunity and directly target malignant clones. We frame this review around three principal themes: (1) mechanistic strategies whereby CRISPR-engineered commensals modulate host immunity and directly antagonize malignant clones; (2) the enabling technologies and delivery/containment platforms, CRISPR variants, phage/LNP delivery, genetic circuits and biocontainment, that make living therapeutics feasible; and (3) translational progress, outstanding technical and safety barriers, and ethical/regulatory challenges that must be addressed for clinical deployment. To illustrate these themes, we discuss three concrete therapeutic modalities: engineered microbial secretion of immunomodulators, targeted delivery of tumor-lytic payloads, and engineered production of anticancer metabolites, and how these are enabled by contemporary CRISPR and synthetic-biology toolkits. Selected preclinical models report substantial antitumor effects, often >60% tumor reduction in rodent studies, and restoration of CAR-T cell function in controlled settings; however, effect sizes vary across models, and human translation remains unproven. We also analyze key technical barriers, strain stability, biocontainment, off‑target effects, and propose solutions, including auxotrophic kill-switches and AI‑guided strain optimization. Finally, we outline future directions, from in situ phage delivery to multi‑omics-driven patient stratification. CRISPR‑microbiome editing represents a paradigm shift in hematologic oncology, offering localized, sustained therapy with reduced systemic toxicity.},
}

Humans
*Immunotherapy/methods
Animals
*Hematologic Neoplasms/therapy/immunology/microbiology
*CRISPR-Cas Systems
*Gastrointestinal Microbiome/genetics
*Microbiota

@article {pmid41319963,
year = {2026},
author = {Kong, H and Wang, S and Zhuo, C and Zhong, Q and Xu, Y and Lao, YH and Lv, S and Xie, X and Yuan, Q and Li, K and Tao, Y and Li, M},
title = {Nanovesicles integrating PD-1-mediated targeting and CRISPR/Cas9-based CD47 editing for dual immune checkpoint blockade.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {390},
number = {},
pages = {114480},
doi = {10.1016/j.jconrel.2025.114480},
pmid = {41319963},
issn = {1873-4995},
mesh = {*CD47 Antigen/genetics ; CRISPR-Cas Systems ; Animals ; Gene Editing ; *Immune Checkpoint Inhibitors/administration & dosage ; *Programmed Cell Death 1 Receptor/genetics/immunology ; Humans ; B7-H1 Antigen/immunology ; Mice ; Cell Line, Tumor ; *Neoplasms/therapy/immunology/genetics ; Mice, Inbred C57BL ; Female ; Immunotherapy/methods ; },
abstract = {Immunotherapy with immune checkpoint inhibitors has revolutionized cancer treatment, yet many tumors evade immune surveillance through multiple suppressive mechanisms. In particular, the adaptive immune checkpoint programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) and the innate "don't eat me" signal CD47/signal-regulatory protein alpha (SIRPα) represent two distinct pathways that cancers exploit to avoid T-cell attack and macrophage phagocytosis, respectively. Herein, we present BITE (Biomimetic Immune Targeting and Editing), a genetically engineered biomimetic nanoplatform designed to concurrently blockade both pathways by combining PD-1-mediated tumor targeting with CRISPR/Cas9 gene editing of CD47. BITE nanovesicles display PD-1 on their surface, enabling selective binding to PD-L1-expressing tumor cells and local disruption of PD-1/PD-L1 signaling. Simultaneously, they deliver a CRISPR/Cas9 payload that knocks out the CD47 gene in tumor cells, abolishing the anti-phagocytic signal and thus activating innate immune clearance. We demonstrate that BITE efficiently homes to PD-L1-positive tumors in vitro and in vivo, achieves significant CD47 gene disruption in tumor cells, and triggers robust phagocytosis by macrophages. In a mouse tumor model, dual checkpoint blockade by BITE reshapes the tumor microenvironment, yielding increased infiltration of CD4[+] T cells, CD8[+] T cells, and M1 macrophages; treatment with BITE induces pronounced tumor regression and extended survival, outperforming single-target controls. Our results establish a proof-of-concept for this dual-function nanovesicle approach, highlighting its potential to engage both adaptive and innate immunity synergistically. The BITE platform offers a versatile and targeted strategy to overcome immune resistance in cancer, representing a promising therapeutic avenue in biomedical engineering and nanomedicine.},
}

*CD47 Antigen/genetics
CRISPR-Cas Systems
Animals
Gene Editing
*Immune Checkpoint Inhibitors/administration & dosage
*Programmed Cell Death 1 Receptor/genetics/immunology
Humans
B7-H1 Antigen/immunology
Mice
Cell Line, Tumor
*Neoplasms/therapy/immunology/genetics
Mice, Inbred C57BL
Female
Immunotherapy/methods

@article {pmid41147788,
year = {2026},
author = {Saika, H and Hara, N and Yasumoto, S and Muranaka, T and Yoshimi, K and Mashimo, T and Toki, S},
title = {Versatile genome editing using Type I-E CRISPR-Cas3 in rice.},
journal = {Plant & cell physiology},
volume = {67},
number = {1},
pages = {82-92},
doi = {10.1093/pcp/pcaf138},
pmid = {41147788},
issn = {1471-9053},
support = {JPJ008000//Ministry of Agriculture, Fisheries and Food/ ; //Cross-ministerial Strategic Innovation Promotion Program/ ; JPJ012287//Bio-oriented Technology Research Advancement Institution/ ; },
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; Plants, Genetically Modified ; },
abstract = {The Type I-E CRISPR-Cas3 derived from Escherichia coli (Eco CRISPR-Cas3) can introduce large deletions in target sites and is available for mammalian genome editing. The use of Eco CRISPR-Cas3 in plants is challenging because seven CRISPR-Cas3 components (six Cas proteins and CRISPR RNA) must be expressed simultaneously in plant cells. To date, application has been limited to maize protoplasts, and no mutant plants have been produced. In this study, we developed a genome editing system in rice using Eco CRISPR-Cas3 via Agrobacterium-mediated transformation. Deletions in the target gene were detected in 39%-71% of transformed calli by polymerase chain reaction (PCR) analysis, and the frequency of alleles lacking a region 7.0 kb upstream of the protospacer adjacent motif sequence was estimated as 21%-61% by quantifying copy number by droplet digital PCR, suggesting that mutant plants could be obtained with reasonably high frequency. Deletions were determined in plants regenerated from transformed calli, and stably inherited to the progenies. Sequencing analysis showed that deletions of 0.1-7.2 kb were obtained, as reported previously in mammals. Interestingly, deletions separated by intervening fragments or with short insertion and inversion were also determined, suggesting the creation of novel alleles. Moreover, we demonstrated C to T base editing based on Type I-E CRISPR-Cas3 in rice, whereas base editing based on Type I-C and Type I-F2 CRISPR-Cas3 has been reported previously only in human cells. Overall, Eco CRISPR-Cas3 could be a promising genome editing tool for gene knockout, gene deletion, base editing, and genome rearrangement in plants.},
}

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

@article {pmid40971496,
year = {2026},
author = {V Pillai, V and Koganti, PP and Gurung, S and Cheong, SH and Selvaraj, V},
title = {Transformed bovine trophoblast stem cell lines, characterization, gene editing and secretion†.},
journal = {Biology of reproduction},
volume = {114},
number = {1},
pages = {273-286},
doi = {10.1093/biolre/ioaf212},
pmid = {40971496},
issn = {1529-7268},
support = {USDA-NIFA 2023-08329//United States Department of Agriculture/ ; NE-2227//USDA-Multistate Program/ ; },
mesh = {Animals ; *Trophoblasts/cytology/physiology/metabolism ; Cattle ; *Gene Editing/veterinary ; Female ; *Stem Cells/cytology/physiology/metabolism ; Pregnancy ; Cell Differentiation ; Cell Line, Transformed ; CRISPR-Cas Systems ; },
abstract = {Trophoblast stem cells (TSCs) serve as a critical model for understanding placental development, early embryo-maternal interactions, and pregnancy establishment in mammals. In cattle, the developing trophectoderm plays an essential role in conceptus elongation and secretion of factors necessary for maternal recognition of pregnancy. Building on previous work identifying signaling pathways regulating bovine TSC self-renewal and differentiation, we report the generation and characterization of transformed bovine TSC (bTSC) lines derived from blastocysts via lentiviral transduction of simian vacuolating virus 40 large T antigen. These rapidly proliferating TSC cell lines, maintained in the presence of Rho-associated protein kinase (ROCK) inhibition, retain key morphological and transcriptional characteristics of bovine TSCs. Upon transforming growth factor β-induced differentiation, they exhibit morphological and molecular changes consistent with trophoblast maturation. To evaluate their utility for functional studies, we demonstrated stable gene introduction of tdTomato and EGFP using lentiviral vectors and employed CRISPR/Cas9-mediated gene editing to target lentiviral EGFP integration sites, confirming efficient gene deletion. Additionally, proteomic analysis of conditioned medium identified secreted proteins with potential roles in embryo-uterine interactions, aligning with factors previously reported in bovine conceptus secretomes. These findings establish transformed bTSC lines as a valuable model for investigating bovine trophoblast biology, functional gene studies, and trophoblast-endometrial signaling. By providing a renewable in vitro system with stable proliferative capacity, these cell lines enable further exploration of the molecular mechanisms governing early pregnancy in cattle.},
}

Animals
*Trophoblasts/cytology/physiology/metabolism
Cattle
*Gene Editing/veterinary
Female
*Stem Cells/cytology/physiology/metabolism
Pregnancy
Cell Differentiation
Cell Line, Transformed
CRISPR-Cas Systems

@article {pmid41535270,
year = {2026},
author = {Taguchi, J and Kikuchi, M and Jeon, H and Shimizu, R and Mori, H and Ikawa, M and Yamada, Y and Sato, K and Ikeda, T and Yamazaki, S and Ozawa, M},
title = {A scalable two-step genome editing strategy for generating full-length gene-humanized mice at diverse genomic loci.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {356},
pmid = {41535270},
issn = {2041-1723},
support = {23K27084//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 25K18393//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 21H05033//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 21H05033//Japan Society for the Promotion of Science London (JSPS London)/ ; },
mesh = {Animals ; *Gene Editing/methods ; Humans ; Mice ; CRISPR-Cas Systems/genetics ; Gene Knock-In Techniques/methods ; Male ; Mice, Transgenic ; Alleles ; Homologous Recombination ; Embryonic Stem Cells/metabolism ; Genetic Loci ; Hematopoiesis/genetics ; Female ; },
abstract = {Full-length gene-humanized mice engineered by completely replacing mouse loci with human counterparts, including untranslated and regulatory regions, provide a robust in vivo platform for human gene function studies. However, reliably humanizing large genomic regions remains challenging due to limited DNA insert sizes, complex protocols, and specialized material requirements. This study introduces a streamlined approach that enables full-length gene humanization through two sequential CRISPR-assisted homologous recombination steps in embryonic stem cells. This method supports targeted knock-in of genomic fragments (> 200 kbp) and is applicable across multiple mouse strains. Humanized alleles generated using the developed method recapitulate human-like splicing isoforms and organ-specific gene expression while restoring essential functions in hematopoiesis, spermatogenesis, and survival. Furthermore, disease-associated mutations can be engineered into humanized alleles to model human genetic disorders in vivo. This versatile platform enables the creation of physiologically relevant, fully gene-humanized mouse models for broad applications in biomedical research.},
}

Animals
*Gene Editing/methods
Humans
Mice
CRISPR-Cas Systems/genetics
Gene Knock-In Techniques/methods
Male
Mice, Transgenic
Alleles
Homologous Recombination
Embryonic Stem Cells/metabolism
Genetic Loci
Hematopoiesis/genetics
Female

@article {pmid41533983,
year = {2026},
author = {Zhang, RR and Wang, YH and Peng, XF and Sun, YJ and Xu, ZS and Liu, H and Xiong, AS},
title = {Expansion protein DcEXP22 regulates taproot enlargement via mediating root cell extension in carrot.},
journal = {The Plant journal : for cell and molecular biology},
volume = {125},
number = {1},
pages = {e70677},
doi = {10.1111/tpj.70677},
pmid = {41533983},
issn = {1365-313X},
support = {KYLH2025002//Fundamental Research Funds for the Central Universities/ ; 2023-SYS-02//Open Research Project of Key Laboratory of Biotechnology of Qinghai-Tibet Plateau Biotechnology of the Ministry of Education/ ; //Priority Academic Program Development of Jiangsu Higher Education Institutions Project PAPD/ ; //Bioinformatics Center of Nanjing Agricultural University/ ; },
mesh = {*Daucus carota/genetics/growth & development/metabolism ; *Plant Roots/growth & development/genetics/cytology/metabolism ; *Plant Proteins/genetics/metabolism/physiology ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; CRISPR-Cas Systems ; },
abstract = {Carrot (Daucus carota L.) is an important root vegetable crop of the Daucus genus in the Apiaceae. As the main product organ of carrot, the taproot has high nutritional and economic value. Expansins, a class of proteins involved in plant cell wall relaxation and cell extension, are mainly found in growing tissues and organs. Expansins play an important role in plant root development. Here, the DcEXP22 gene with a length of 789 bp was cloned from the carrot cultivar 'Kurodagosun'. Based on the stable genetic transformation system and CRISPR/Cas9 gene-editing technology, the DcEXP22 gene was overexpressed and knocked out in carrots. The results indicated that overexpression of the DcEXP22 gene increased carrot root fresh weight, root diameter, and root-shoot ratio, and enlarged the perimeter and area of taproot phloem cells. In contrast, knockout of the DcEXP22 gene inhibited the development of carrot taproots and the extension of phloem cells, suggesting that the DcEXP22 gene might promote the enlargement of carrot taproots by regulating the size of phloem cells. RNA-seq analysis identified several genes that were co-expressed with DcEXP22, including DcCYP734A1, DcERF1, DcMAP2K1, and DcSAD9. It was hypothesized that the DcEXP22 gene might influence the enlargement of carrot taproot by participating in the signal transduction of phytohormones such as brassinosteroids, cell wall synthesis and modification, and fatty acid metabolisms. These findings will advance our knowledge of the molecular mechanisms of carrot taproot enlargement.},
}

*Daucus carota/genetics/growth & development/metabolism
*Plant Roots/growth & development/genetics/cytology/metabolism
*Plant Proteins/genetics/metabolism/physiology
Gene Expression Regulation, Plant
Plants, Genetically Modified
CRISPR-Cas Systems

@article {pmid41500123,
year = {2026},
author = {Zang, Z and Chen, J and Dong, Y and Chen, L and Yang, M and Mu, M and Zhou, L and Zhang, W and Zou, G and Liu, C},
title = {CRISPR-Cas12a biosensing technology advances and applications in precision diagnostics and cancer research.},
journal = {Talanta},
volume = {301},
number = {},
pages = {129298},
doi = {10.1016/j.talanta.2025.129298},
pmid = {41500123},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/diagnosis/genetics ; *Precision Medicine/methods ; Biomarkers, Tumor/genetics ; Gene Editing ; },
abstract = {CRISPR-Cas12a has become a versatile biotechnology platform with important applications in biosensing, diagnostics, and precision genome editing. This system is activated by a single crRNA, exhibits target-responsive trans-cleavage activity, and recognizes programmable PAM sequences. These features provide a robust basis for accurate detection of diverse biomarkers. Its detection capabilities include nucleic acid targets such as viral RNA and cancer mutations, as well as non-nucleic acid molecules like exosomes and proteins. Recent advancements have shown significant advantages, including multi-temperature adaptability, rapid kinetics, and compatibility with both DNA and RNA targets. Technical improvements include machine learning-assisted crRNA design for enhanced prediction accuracy and engineered EnAsCas12a variants that overcome conventional PAM restrictions. Notable achievements involve entropy-driven circuits that achieve attomolar-level sensitivity, smartphone-compatible four-channel quantitative detection systems, and streamlined integrated workflows completed within 30 min. Advances in sensor design, such as metal-organic framework encapsulation and high-performance aptamer-based sensors, have further expanded detection capabilities. In oncology research, CRISPR-Cas12a technology provides powerful tools to comprehensively analyze complex molecular networks within the tumor microenvironment (TME) and facilitate ultrasensitive detection of early-stage cancer biomarkers. Additionally, in genome editing, CRISPR-Cas12a enables precise genomic modifications due to distinct repair pathways, versatile delivery methods, and efficient creation of transgenic models. Thus, it expands its functional scope beyond diagnostics. With ongoing development, this technology is expected to evolve into an integrated platform combining TME research, point-of-care cancer diagnostics, and programmable genome engineering, offering innovative solutions for both biomedical research and clinical translation.},
}

*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
*Neoplasms/diagnosis/genetics
*Precision Medicine/methods
Biomarkers, Tumor/genetics
Gene Editing

@article {pmid41478231,
year = {2026},
author = {Liu, Y and Mei, H and Gao, C and Yang, Y},
title = {Rapid detection of respiratory syncytial virus using RT-LAMP-CRISPR/Cas12b on a gravity-driven microfluidic chip.},
journal = {Diagnostic microbiology and infectious disease},
volume = {114},
number = {3},
pages = {117244},
doi = {10.1016/j.diagmicrobio.2025.117244},
pmid = {41478231},
issn = {1879-0070},
mesh = {Humans ; *Respiratory Syncytial Virus Infections/diagnosis/virology ; *Molecular Diagnostic Techniques/methods/instrumentation ; *CRISPR-Cas Systems ; *Respiratory Syncytial Virus, Human/isolation & purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Lab-On-A-Chip Devices ; RNA, Viral/genetics ; Limit of Detection ; Point-of-Care Systems ; },
abstract = {AIMS: Respiratory syncytial virus (RSV) is the most significant pathogen causing acute lower respiratory tract infections in children. Early detection of RSV can help control disease progression and reduce complications. However, RT-qPCR-based detection methods cannot provide accurate results within one hour and are not suitable for use in resource-limited settings. Therefore, there is a pressing need to develop a rapid and precise bedside RSV detection assay to meet clinical demands.

METHODS AND RESULTS: Firstly, we developed a RT-LAMP-assisted CRISPR/Cas12b method for detecting RSV M gene, capable of identifying target RNA with a limit of detection as low as 100 copies/μL within 40 min. Secondly, we created and tested a sampling lysis reagent, demonstrating its effectiveness in enabling direct detection without the need for nucleic acid extraction, thus improving bedside detection efficiency. Lastly, to facilitate use in resource-limited areas, we designed and developed a gravity-driven microfluidic chip that simplifies the stepwise process of RT-LAMP amplification and CRISPR/Cas12b detection. This chip allows for visual recognition of results without the need for an external power source when used in point-of-care (POC) settings. This assay showed 99% agreement with RT-qPCR, highlighting its potential for practical application. Additionally, no cross-reactivity was observed with other respiratory pathogens infection, demonstrating good clinical specificity.

CONCLUSIONS: In summary, the platform we developed is faster and more user-friendly than RT-qPCR, while achieving comparable sensitivity.

IMPACT STATEMENT: Our finding fills the gap in the inability to detect RSV in POC setting, safeguarding the health of children, and offers new insights for the innovation of CRISPR diagnostics.},
}

Humans
*Respiratory Syncytial Virus Infections/diagnosis/virology
*Molecular Diagnostic Techniques/methods/instrumentation
*CRISPR-Cas Systems
*Respiratory Syncytial Virus, Human/isolation & purification/genetics
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
*Lab-On-A-Chip Devices
RNA, Viral/genetics
Limit of Detection
Point-of-Care Systems

@article {pmid41476394,
year = {2026},
author = {Zhuang, M and Song, J and Hu, X and Wang, X},
title = {Metabolic Engineering of Escherichia coli Nissle 1917 for Efficient Production of p-Coumaric Acid.},
journal = {ACS synthetic biology},
volume = {15},
number = {1},
pages = {210-222},
doi = {10.1021/acssynbio.5c00639},
pmid = {41476394},
issn = {2161-5063},
mesh = {*Escherichia coli/metabolism/genetics ; *Metabolic Engineering/methods ; *Coumaric Acids/metabolism ; *Propionates/metabolism ; Ammonia-Lyases/genetics/metabolism ; Tyrosine/biosynthesis/metabolism ; CRISPR-Cas Systems/genetics ; Plasmids/genetics/metabolism ; Escherichia coli Proteins/genetics/metabolism ; Quorum Sensing/genetics ; Phenylalanine/biosynthesis ; },
abstract = {p-Coumaric acid is a valuable phytochemical with significant roles in anticancer cell proliferation, antianxiety, and neuroprotection and as a key precursor for various flavonoids. However, the production of p-coumaric acid in microorganisms is often limited by enzyme compatibility and its antimicrobial effects. In this study, a p-coumaric acid producing Escherichia coli strain was constructed. First, the cryptic plasmids pMUT1 and pMUT2 were eliminated from E. coli Nissle 1917 by using the CRISPR/Cas9 method to mitigate their interference with heterologous gene expression, and the resulting strain WEN01 was used to screen for the genes encoding for tyrosine ammonia-lyase with superior host compatibility. Next, the gene tyrR encoding a global regulator was knocked out to alleviate the repression of l-tyrosine production. The key genes pheL and pheA involved in phenylalanine biosynthesis were knocked out to reduce byproduct formation, resulting in the strain WEN06. Finally, the quorum sensing system was used to overexpress the key genes aroG[fbr] and tyrA[fbr] in the l-tyrosine biosynthetic pathway, and the resulting strain WEN06/pWT101-AT, pWT104F could produce 462.6 mg/L p-coumaric acid in shake flask fermentation. In fed-batch fermentation, the engineered strain WEN06/pWT101-AT, pWT104F could produce 10.3 g/L p-coumaric acid with a glucose conversion yield of 0.13 g/g and a productivity of 0.14 g/L/h. This work provides a novel strategy for the efficient production of p-coumaric acid and lays a foundation for the efficient production of antimicrobial natural products in bacteria.},
}

*Escherichia coli/metabolism/genetics
*Metabolic Engineering/methods
*Coumaric Acids/metabolism
*Propionates/metabolism
Ammonia-Lyases/genetics/metabolism
Tyrosine/biosynthesis/metabolism
CRISPR-Cas Systems/genetics
Plasmids/genetics/metabolism
Escherichia coli Proteins/genetics/metabolism
Quorum Sensing/genetics
Phenylalanine/biosynthesis

@article {pmid41455263,
year = {2026},
author = {Liu, F and Cheng, AX and Zhang, C and Li, J and Huang, YF and Zhang, YP and Li, CP and Zhao, H},
title = {Amplification-free, CRISPR-Cas12a-mediated fluorescence biosensor using mesoporous silica nanomaterials for ultrasensitive detection of nucleic acid biomarkers.},
journal = {Talanta},
volume = {301},
number = {},
pages = {129289},
doi = {10.1016/j.talanta.2025.129289},
pmid = {41455263},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; *Silicon Dioxide/chemistry ; *CRISPR-Cas Systems ; Humans ; *Nanostructures/chemistry ; Porosity ; Limit of Detection ; Fluorescent Dyes/chemistry ; Rhodamines/chemistry ; Fluorescence ; ErbB Receptors/genetics ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Sensitive detection of nucleic acid biomarkers is crucial in many fields, including biomedical diagnosis, veterinary medicine, and food safety. Thus, developing an accurate and cost-effective detection method for nucleic acid biomarkers is essential. Here, we developed a sensitive CRISPR-Cas12a-based fluorescence biosensor using mesoporous silica nanomaterials (MSNs). A large quantity of rhodamine B (RB) was enriched on the MSNs to synthesize RB@MSN nanocomposites, which served as fluorescent probe materials, and Au NPs acted as fluorescence quenching materials. Combined with the high specific recognition capability of the CRISPR-Cas12a system, we detected three important nucleic acids without requiring amplification: the EGFR exon 19 deletion mutation (EGFR 19Del, found in circulating tumor DNA), African swine fever virus (ASFV), and human papilloma virus (HPV). Under optimal conditions and using quantitative analysis, there were strong linear correlations between the concentrations of the targets and their respective fluorescence intensities. The lowest detection limits were 55 aM for EGFR 19Del, 51 aM for ASFV, and 24 aM for HPV. By enriching and encapsulating MSNs with RB, our method avoided the problems of fluorescence modifications in typical CRISPR-Cas12a systems, such as professional outsourcing requirements and easily quenched fluorescence. Moreover, the results exhibited good repeatability and stability. This method provides a novel approach to nucleic acid fluorescence detection using the CRISPR-Cas12a system.},
}

*Biosensing Techniques/methods
*Silicon Dioxide/chemistry
*CRISPR-Cas Systems
Humans
*Nanostructures/chemistry
Porosity
Limit of Detection
Fluorescent Dyes/chemistry
Rhodamines/chemistry
Fluorescence
ErbB Receptors/genetics
Gold/chemistry
Metal Nanoparticles/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41448060,
year = {2026},
author = {Long, Y and Sun, S and Mei, H and Zhou, D and Zhou, H and Fang, Z and Li, X and Li, N and Zhuang, T and Guo, C},
title = {RT-LAMP-CRISPR/Cas12b-based hand-pressure-actuated microfluidic chip for rapid and portable detection of severe fever with thrombocytopenia syndrome virus.},
journal = {Talanta},
volume = {301},
number = {},
pages = {129277},
doi = {10.1016/j.talanta.2025.129277},
pmid = {41448060},
issn = {1873-3573},
mesh = {*Phlebovirus/genetics/isolation & purification ; *Severe Fever with Thrombocytopenia Syndrome/diagnosis/virology ; *Nucleic Acid Amplification Techniques/instrumentation/methods ; Humans ; *Lab-On-A-Chip Devices ; *Molecular Diagnostic Techniques/instrumentation/methods ; *CRISPR-Cas Systems ; Pressure ; RNA, Viral/genetics/blood ; },
abstract = {Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging, highly pathogenic tick-borne virus causing severe viral hemorrhagic fever, posing a significant public health threat. Rapid and accurate detection of SFTSV in resource-limited settings is critical for early diagnosis and effective control of severe fever with thrombocytopenia syndrome (SFTS). Here, we developed the RT-LAMP-CRISPR/Cas12b-based Hand-Pressure-Actuated Microfluidic Chip for Rapid and Portable Detection of Severe Fever with Thrombocytopenia Syndrome Virus (HARD). The hand-pressure-actuated microfluidic chip integrates reverse transcription loop-mediated isothermal amplification (RT-LAMP), clustered regularly interspaced short palindromic repeats (CRISPR) and its associated proteins (CRISPR associated proteins, Cas) 12b in order to achieve rapid, low-cost, and contamination-free point-of-care testing. The HARD system achieves a detection limit of 5 copies per reaction, utilizing direct RNA lysis from blood samples and a hand warmer as a heat source, enabling electricity-free operation. Clinical validation with blood samples from vector-borne infectious diseases demonstrated high concordance with laboratory RT-qPCR, with 88.9 % sensitivity, 100 % specificity, and 95 % accuracy. Thus, the HARD platform offers a rapid, portable, and efficient solution for the early diagnosis of SFTSV in resource-limited settings, with potential for broader application in POCT for infectious diseases.},
}

*Phlebovirus/genetics/isolation & purification
*Severe Fever with Thrombocytopenia Syndrome/diagnosis/virology
*Nucleic Acid Amplification Techniques/instrumentation/methods
Humans
*Lab-On-A-Chip Devices
*Molecular Diagnostic Techniques/instrumentation/methods
*CRISPR-Cas Systems
Pressure
RNA, Viral/genetics/blood

@article {pmid41439415,
year = {2026},
author = {Zhao, R and Wan, P and Huang, H and Li, Q and Zeng, Z and Xiong, W},
title = {Harnessing CRISPR-Cas9 and CRISPRi systems to reverse antibiotic resistance in a clinical multidrug-resistant Escherichia coli isolate.},
journal = {The Journal of antimicrobial chemotherapy},
volume = {81},
number = {1},
pages = {},
doi = {10.1093/jac/dkaf442},
pmid = {41439415},
issn = {1460-2091},
support = {2022YFD1800400//National Key R&D Program/ ; },
mesh = {*Escherichia coli/drug effects/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Drug Resistance, Multiple, Bacterial/genetics ; Microbial Sensitivity Tests ; *Anti-Bacterial Agents/pharmacology ; Escherichia coli Proteins/genetics ; Escherichia coli Infections/microbiology ; Humans ; Multidrug Resistance-Associated Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Deletion ; },
abstract = {OBJECTIVES: To evaluate the CRISPR-Cas9 and CRISPR interference (CRISPRi) systems as an antibiotic re-sensitization strategy for reversing multidrug resistance in a clinical Escherichia coli isolate.

MATERIALS AND METHODS: The CRISPR-Cas9 system was applied for precise deletion of the acrB gene from clinical E. coli isolate GP53 and homologous recombination (HR) was provided for accurate repairs of double-strand breaks. An arabinose-inducible CRISPRi system was developed and optimized using fluorescent reporter strain GH01. Multiple guide RNAs (gRNAs) targeting acrB were designed, and the most effective gRNA was selected based on its transcriptional suppression of gene acrB. The minimum inhibitory concentrations (MICs) of selected antibiotics in GP53ΔacrB, CRISPRi strains, WT and WT combined with efflux pump inhibitor PAβN were evaluated.

RESULTS: The CRISPR-Cas9 system precisely deleted the acrB gene in clinical E. coli isolate GP53 with 11.46% knockout efficiency. The constructed arabinose-inducible CRISPRi system effectively repressed fluorescent protein expression in strain GH01. Although dCas9 expression increased with L-arabinose concentration, the transcriptional repression efficiency of the target gene under 1 mM induction reached a significant inhibitory level. The CRISPRi system targeting gene acrB exhibited 44.9%, 5.4% and 23.5% inhibition rates on the transcriptional levels with 1 mM L-arabinose for three distinct gRNAs. Both the knockout and CRISPRi strains successfully restored susceptibility of the multidrug-resistant E. coli GP53 to quinolones and tetracyclines, outperforming the effect of PAβN combination therapy.

CONCLUSIONS: In this study, CRISPR-based systems effectively reversed multidrug resistance in a clinical E. coli isolate, advancing the applications of CRISPR systems in controlling bacterial multidrug resistance.},
}

*Escherichia coli/drug effects/genetics/isolation & purification
*CRISPR-Cas Systems
*Drug Resistance, Multiple, Bacterial/genetics
Microbial Sensitivity Tests
*Anti-Bacterial Agents/pharmacology
Escherichia coli Proteins/genetics
Escherichia coli Infections/microbiology
Humans
Multidrug Resistance-Associated Proteins/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Deletion

@article {pmid41433963,
year = {2026},
author = {Schneider, O and Zehl, M and Miele, M and Pace, V and Brungs, C and Cheng, JF and Hummelbrunner, S and Dirsch, VM and Zotchev, SB},
title = {Heterologous Expression and CRISPR/Cas9-Assisted Manipulation of the Hybrid Gene Cluster Specifying the Biosynthesis of Meroterpenoids and Phenazines.},
journal = {ACS synthetic biology},
volume = {15},
number = {1},
pages = {137-148},
doi = {10.1021/acssynbio.5c00531},
pmid = {41433963},
issn = {2161-5063},
mesh = {*Multigene Family/genetics ; *CRISPR-Cas Systems/genetics ; *Phenazines/metabolism/chemistry ; *Terpenes/metabolism/chemistry ; Streptomyces/genetics/metabolism ; Streptomyces coelicolor/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Anti-Bacterial Agents/biosynthesis ; },
abstract = {A hybrid gene cluster, mfq, predicted to govern the biosynthesis of both meroterpenoids and phenaziterpenes, was cloned from the genome of Streptomyces sp. S4.7 and introduced into the heterologous host Streptomyces coelicolor M1154. The biosynthesis of the meroterpenoids marfuraquinocins C and D, previously isolated from Streptomyces niveus SCSIO 3406, as well as a new congener, marfuraquinocin E, which exhibited antibacterial activity, was activated upon overexpression of the regulatory protein MfqF. However, production of neither phenaziterpenes nor phenazines was detected. The structure of marfuraquinocin E was elucidated, revealing the attachment of a terpene moiety at C-2, in contrast to C-6 as seen in the known congeners A-D. Using the CRISPR/Cas9 system, several genes in the mfq cluster were inactivated, confirming the role of MfqW as a prenyltransferase specific to the meroterpenoid pathway. Both gene overexpression and further knockouts provided the first insights into the complex regulation of this hybrid gene cluster. To restore the presumably deficient phenazine biosynthetic pathway, a gene encoding a PhzF homologue from another gene cluster in S4.7 was heterologously expressed alongside the mfq cluster, leading to the production of 1,6-phenazine dicarboxylic acid upon MfqF overexpression. This work lays the foundation for elucidating the complete biosynthetic pathway of marfuraquinocins and its potential coregulation with that of phenazines.},
}

*Multigene Family/genetics
*CRISPR-Cas Systems/genetics
*Phenazines/metabolism/chemistry
*Terpenes/metabolism/chemistry
Streptomyces/genetics/metabolism
Streptomyces coelicolor/genetics/metabolism
Bacterial Proteins/genetics/metabolism
Anti-Bacterial Agents/biosynthesis

@article {pmid41396964,
year = {2026},
author = {Jiang, C and Liu, Y and Han, W and Zou, D and Chen, K and Jiang, X and Ma, A and Wei, X},
title = {Regulation of Single and Multiple Genes in Bacillus amyloliquefaciens by an Evolution System In Vivo.},
journal = {ACS synthetic biology},
volume = {15},
number = {1},
pages = {88-98},
doi = {10.1021/acssynbio.5c00480},
pmid = {41396964},
issn = {2161-5063},
mesh = {*Bacillus amyloliquefaciens/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Directed Molecular Evolution/methods ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Cytidine Deaminase/genetics/metabolism ; Mutation ; },
abstract = {With the development of synthetic biology, an evolution system in vivo has been applied to accelerate the construction of cell factories. In this study, an efficient in vivo evolution system was developed for regulation of single and multiple genes in Bacillus amyloliquefaciens. First, the CRISPR/Cas9n-AID base editor was constructed through integration expression of the fused Cas9n protein and activation-induced cytidine deaminase (AID), and the base conversion efficiency from C to T was as high as 90% in single-gene editing. Subsequently, the evolution template (XP43) with an editable RBS sequence (GGGGGGGG) was designed for in vivo evolution through two strategies. By next-generation sequencing of RBS mutation libraries, the extended sgRNA strategy was confirmed to be the optimal evolution scheme. Using the alkaline protease gene (aprE) as the single gene target, the evolution program was initiated to successfully obtain a series of mutant strains with gradient AprE activities. Furthermore, multiple key genes (dhemA, SAM2, and hemEHY) were evolved simultaneously to balance the heme metabolic network, and the optimal mutant strain (HZHA-C2) produced 14.02 mg/L heme, 93% higher than the control strain. Finally, the overexpression of the hemH gene further increased the heme titer by 49%. By a fed-batch fermentation strategy, the heme titer of the optimal engineered strain (HZHA2/pHY-hemH) was improved by 64%, achieving 32.61 mg/L.},
}

*Bacillus amyloliquefaciens/genetics/metabolism
CRISPR-Cas Systems/genetics
Gene Editing/methods
*Directed Molecular Evolution/methods
Bacterial Proteins/genetics/metabolism
Gene Expression Regulation, Bacterial
Cytidine Deaminase/genetics/metabolism
Mutation

@article {pmid41352420,
year = {2026},
author = {Tang, M and Liang, R and Wu, Z and Chen, C and He, B and Zhou, N and Wang, S and Xiao, X and Li, G and Jiang, Y and Gong, G and Zhou, Y},
title = {Deciphering OCT4A-dose-dependent transcriptional profiles associated with tumorigenic potential in somatic cancer cells.},
journal = {SLAS technology},
volume = {36},
number = {},
pages = {100381},
doi = {10.1016/j.slast.2025.100381},
pmid = {41352420},
issn = {2472-6311},
mesh = {*Octamer Transcription Factor-3/genetics/metabolism ; Humans ; *Neoplasms/genetics/pathology ; Cell Line, Tumor ; *Carcinogenesis/genetics ; Gene Expression Regulation, Neoplastic ; Gene Expression Profiling ; *Transcriptome ; CRISPR-Cas Systems ; Gene Regulatory Networks ; Prognosis ; },
abstract = {AIMS: The transcription factor OCT4A, a well-established master pluripotency factor, exerts regulatory effects on cell fate determination that are closely associated with its protein levels. This study aims to uncover the downstream gene profile features relevant to tumorigenic potential mediated by OCT4A under varying protein abundance in somatic cancer cells (SCCs).

MATERIALS AND METHODS: CRISPR-Cas9-mediated knockout and doxycycline-inducible OCT4A expression systems were established in cervical (HeLa) and hepatocellular (HepG2, Huh7) cancer cells. Single-cell sequencing, spatial transcriptomic and survival analysis data were used to elucidate the expression pattern of OCT4 in somatic cancer tissues and its prognostic relevance. The plate colony formation assay was performed to assess the tumorigenic capacity of SCCs, and Bulk RNA sequencing coupled with weighted gene co-expression network analysis (WGCNA) identified dose-relevant downstream pathways. Functional enrichment, survival modeling, and RT-qPCR validation were used to construct OCT4A-dose-dependent transcriptional regulatory networks.

KEY FINDINGS: OCT4 transcript, is heterogeneously present and confined to a small subset of tumor cells within somatic cancer tissues, with a significantly higher proportion of OCT4-positive cells in tumor tissues compared to paired paraneoplastic tissues and is significantly correlated with poor prognosis in SCCs. Endogenous low-level OCT4A positively regulates tumorigenic capacity predominantly through targeting non-coding genes, whereas high-level OCT4A suppresses tumorigenic capacity primarily via protein-coding genes in SCCs. A prognostic model based on high-level OCT4A-regulated protein-coding genes was associated with favorable clinical outcomes, aligning with in vitro phenotypic results.

SIGNIFICANCE: Our findings further confirm in SCCs that the functional pleiotropy of OCT4A is closely linked to its protein abundance, and further systematically elucidate the molecular signatures of OCT4A-regulated downstream gene networks associated with tumorigenic phenotypes at differential protein levels, providing novel insights for its translational exploitation in both oncological intervention and regenerative medicine strategies.},
}

*Octamer Transcription Factor-3/genetics/metabolism
Humans
*Neoplasms/genetics/pathology
Cell Line, Tumor
*Carcinogenesis/genetics
Gene Expression Regulation, Neoplastic
Gene Expression Profiling
*Transcriptome
CRISPR-Cas Systems
Gene Regulatory Networks
Prognosis

@article {pmid41313840,
year = {2026},
author = {Mohammad, SI and Kareem, AK and Vasudevan, A and Rekha, MM and Jabir, MS and Nayak, P and AlKhafaje, Z and Arora, V and Kadhum, W and Chennakesavulu, K},
title = {Genome editing of immune checkpoints: CRISPR-mediated PD-1 inhibition in cancer.},
journal = {Seminars in oncology},
volume = {53},
number = {1},
pages = {152438},
doi = {10.1016/j.seminoncol.2025.152438},
pmid = {41313840},
issn = {1532-8708},
mesh = {Humans ; *Gene Editing/methods ; *Neoplasms/genetics/therapy/immunology ; *Programmed Cell Death 1 Receptor/antagonists & inhibitors/genetics ; Animals ; *CRISPR-Cas Systems ; *Immune Checkpoint Inhibitors/therapeutic use/pharmacology ; B7-H1 Antigen/antagonists & inhibitors/genetics ; Immunotherapy, Adoptive/methods ; },
abstract = {The programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) immune checkpoint is a primary mechanism by which tumors evade immune surveillance, limiting the efficacy of cytotoxic T lymphocytes (CTLs) and tumor-infiltrating lymphocytes (TILs). Although immune checkpoint blockade therapies have revolutionized cancer treatment, their efficacy is restricted by acquired resistance, T-cell exhaustion, and tumor heterogeneity. The advent of CRISPR-Cas9 genome editing provides a precise and versatile approach to disrupt PD-1 or PD-L1, directly enhancing anti-tumor immune responses. Preclinical studies demonstrate that ex vivo PD-1 knockout in primary human T cells or TILs enhances proliferation, cytokine production, and cytotoxicity, resulting in improved tumor clearance in xenograft and humanized mouse models. In chimeric antigen receptor (CAR) T cell therapy, CRISPR-mediated disruption of PD-1 improves effector function, persistence, and resistance to exhaustion, with universal and allogeneic CAR-T platforms benefiting from multiplex genome editing. Direct PD-L1 knockout in tumor cells, often facilitated via nanoparticle- or biomaterial-assisted delivery, reshapes the immunosuppressive tumor microenvironment, promotes T cell infiltration, and enhances the efficacy of adoptive cellular therapy. Combination approaches integrating PD-1 editing with viral antigen targeting, long noncoding RNA (lncRNA) modulation, or conventional checkpoint blockade demonstrate synergistic anti-tumor effects. Clinically, early-phase trials in non-small cell lung cancer, mesothelin-positive solid tumors, and hematological malignancies establish the feasibility, safety, and preliminary efficacy of PD-1-deficient T cells. Despite these promising outcomes, challenges such as off-target effects, delivery efficiency, immunogenicity, long-term persistence, and regulatory considerations remain. This review aims to comprehensively evaluate preclinical and clinical studies investigating CRISPR-mediated PD-1/PD-L1 inhibition across various cancers, summarize mechanistic insights, and highlight translational opportunities and challenges for clinical implementation.},
}

Humans
*Gene Editing/methods
*Neoplasms/genetics/therapy/immunology
*Programmed Cell Death 1 Receptor/antagonists & inhibitors/genetics
Animals
*CRISPR-Cas Systems
*Immune Checkpoint Inhibitors/therapeutic use/pharmacology
B7-H1 Antigen/antagonists & inhibitors/genetics
Immunotherapy, Adoptive/methods

@article {pmid41286106,
year = {2026},
author = {Angelini Stewart, A and Ahrens-Nicklas, RC and Tsai, SQ and Musunuru, K and Giannikopoulos, P and Clelland, CD},
title = {Measurement and clinical interpretation of CRISPR off-targets.},
journal = {Nature genetics},
volume = {58},
number = {1},
pages = {20-27},
pmid = {41286106},
issn = {1546-1718},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Therapy/methods/adverse effects ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Animals ; },
abstract = {CRISPR genetic therapies are revolutionizing the landscape of preclinical research and clinical studies, providing new potential routes for curative intervention for a range of previously untreatable diseases. As with any therapy, the therapeutic benefits and risks must be weighed against consideration of the disease threat. Genome-related adverse events are an inherent risk of CRISPR genetic therapies, including off-target edits. The perception that CRISPR therapies ought to have near-zero off-targets belies clinical medicine, therapy development and biology, which demonstrate that 'perfect' therapeutics do not exist. Given that not all genomic off-target events are equal, we provide a practical framework to evaluate and assess off-target safety based on the tools available today and ones that will be developed in the future. With the comprehensive information and assessment gathered using these guidelines, we aim to streamline the transition of CRISPR therapeutics from bench to bedside.},
}

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

@article {pmid41270550,
year = {2026},
author = {Hu, Q and Liu, J and Zhang, W and Liao, X and Guo, Y and Lu, Q and Zhang, R and Yang, B and Zhang, T and Zhai, X and Luo, Q},
title = {Rapid nucleic acid detection of Mycoplasma synoviae using dual-mode RAA-CRISPR/Cas12a system.},
journal = {Poultry science},
volume = {105},
number = {1},
pages = {106126},
pmid = {41270550},
issn = {1525-3171},
mesh = {Animals ; *Mycoplasma synoviae/isolation & purification ; *Mycoplasma Infections/veterinary/diagnosis/microbiology ; *Poultry Diseases/diagnosis/microbiology ; *Chickens ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Sensitivity and Specificity ; Ducks ; Columbidae ; },
abstract = {Mycoplasma synoviae is a significant avian pathogen implicated in diverse clinical manifestations, including air sacculitis, infectious synovitis with arthritis, and eggshell apex abnormalities, which significantly threaten the poultry industry. To enable the rapid and accurate detection of M. synoviae, we developed a dual-mode diagnostic platform that combines recombinant enzyme-assisted amplification (RAA) with CRISPR/Cas12a-based fluorescence and lateral flow dipsticks (LFD). The Qsep100 automatic nucleic acid analysis system was used to screen for RAA primers by evaluating the amplicon size and concentration. Fluorescence monitoring revealed that the optimal reaction time for the RAA-CRISPR/Cas12a system was 28 min. After optimizing the concentrations of LbaCas12a, crRNA, and the ssDNA reporter, the assay achieved a sensitivity of 5.2 copies/µL. Cross-reactivity testing with seven common avian pathogens confirmed the high specificity of the established method for detecting M. synoviae. In the clinical validation, the method perfectly matched the results of quantitative real time polymerase chain reaction (qPCR). Furthermore, an epidemiological investigation revealed that chickens had the highest positivity rate for M. synoviae among the chickens, ducks, and pigeons. In summary, we developed a rapid, accurate, and portable diagnostic platform for M. synoviae detection, which provides a valuable tool for disease prevention and control in resource-limited settings.},
}

Animals
*Mycoplasma synoviae/isolation & purification
*Mycoplasma Infections/veterinary/diagnosis/microbiology
*Poultry Diseases/diagnosis/microbiology
*Chickens
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/veterinary/methods
Sensitivity and Specificity
Ducks
Columbidae

@article {pmid41265135,
year = {2026},
author = {Wright, M and Choi, W and Deng, Z and Kim, D and Lee, K},
title = {CRISPR/Cas9 Gene Editing of Turkey Cells Using Adenoviral Delivery Running Head: RESEARCH NOTE.},
journal = {Poultry science},
volume = {105},
number = {1},
pages = {106096},
pmid = {41265135},
issn = {1525-3171},
mesh = {Animals ; *Gene Editing/veterinary/methods ; *CRISPR-Cas Systems ; *Turkeys/genetics ; *Adenoviridae/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Line ; Receptors, Leptin/genetics ; Genetic Vectors ; },
abstract = {Poultry, including birds such as chicken, duck, turkey, geese, and other game birds is the most widely consumed animal protein globally, with per capita poultry consumption steadily rising in the United States over the past several decades. To meet the demand for poultry, many avenues of improving poultry production have been explored including genetic modification solutions. While genetic modification in many poultry species has been well explored, gene editing in turkey has no research into its application. This study investigated the feasibility of using CRISPR/Cas9 mediated gene editing using adenoviral delivery in vitro, a method that has been proven successful for other species of poultry but never turkey. Three guide RNAs (gRNAs) were designed, targeting the leptin receptor (LEPR) gene matching the gene sequences of both chicken and turkey. Following the assessment of each gRNA's editing efficiency in turkey embryonic fibroblast (TEF) cells and commercially available chicken DF-1 cells via liposome-mediated transfection of the CRISPR vector, the most effective gRNA was chosen for recombinant adenovirus type 5 production. From there, both DF-1 and TEF cells were transduced using the adenovirus and the genome editing efficiency was assessed. The results show that turkey cells can be genetically modified in vitro with indel frequencies comparable to those observed in chicken cells, using both CRISPR plasmids and adenoviral vectors. These findings can lay the groundwork for future experiments and adaptation to in vivo modification of turkey in the future.},
}

Animals
*Gene Editing/veterinary/methods
*CRISPR-Cas Systems
*Turkeys/genetics
*Adenoviridae/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Cell Line
Receptors, Leptin/genetics
Genetic Vectors

@article {pmid41236017,
year = {2026},
author = {Zhang, C and Goldsmith, SR and Htut, M and Rhee, JW and Vyas, V and Clark, MC and Armenian, SH and Forman, SJ and Wang, X},
title = {CRISPR Knockdown of CHIP Genes in Macrophages Drives Increased Production of Inflammatory Cytokines in CAR-T Cell Therapy.},
journal = {Transplantation and cellular therapy},
volume = {32},
number = {1},
pages = {10.e1-10.e5},
doi = {10.1016/j.jtct.2025.08.022},
pmid = {41236017},
issn = {2666-6367},
mesh = {Humans ; *Macrophages/metabolism/immunology ; *Cytokines/metabolism ; DNA Methyltransferase 3A ; *Immunotherapy, Adoptive/methods/adverse effects ; *CRISPR-Cas Systems ; Dioxygenases/genetics ; Receptors, Chimeric Antigen ; Gene Knockdown Techniques ; Proto-Oncogene Proteins/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA-Binding Proteins/genetics ; Repressor Proteins/genetics ; DNA (Cytosine-5-)-Methyltransferases/genetics ; Gene Editing ; },
abstract = {Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by somatic mutations in leukemogenic genes (e.g., DNMT3A, TET2, and ASXL1) in hematopoietic stem cells and is linked to age-related clonal expansion and inflammation. We previously showed that patients with CHIP receiving chimeric antigen receptor (CAR)-T cell therapy have a higher risk of developing clinically significant cytokine release syndrome (CRS) compared to those without CHIP. Here, we investigated the mechanisms through which CHIP mutations can contribute to CAR-T-related toxicities. To model the interaction of CAR-T cell-mediated inflammatory response and macrophages harboring CHIP gene mutations, we employed CRISPR-based gene editing to knock down three key CHIP-associated genes (DNMT3A, TET2, and ASXL1) in human macrophages, using multiple guide RNAs for each. We co-cultured gene-edited macrophages with autologous CS1 and BCMA CAR-T cells and multiple myeloma (MM) tumor cells. Compared to nonedited macrophages, DNMT3A-, TET2-, and ASXL1-edited macrophages secreted significantly higher levels of pro-inflammatory cytokines characteristic of CRS, including IL-6, MCP-1, and IL-1β (P < .05 to .01). These results suggest that the presence of CHIP mutations in human macrophages may exacerbate the inflammatory response during CAR-T therapy. This study highlights CHIP mutations as potential biomarkers for identifying patients at high risk of developing CRS and for guiding personalized prophylactic interventions in MM CAR-T therapy.},
}

Humans
*Macrophages/metabolism/immunology
*Cytokines/metabolism
DNA Methyltransferase 3A
*Immunotherapy, Adoptive/methods/adverse effects
*CRISPR-Cas Systems
Dioxygenases/genetics
Receptors, Chimeric Antigen
Gene Knockdown Techniques
Proto-Oncogene Proteins/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
DNA-Binding Proteins/genetics
Repressor Proteins/genetics
DNA (Cytosine-5-)-Methyltransferases/genetics
Gene Editing

@article {pmid41150843,
year = {2026},
author = {Butt, H and Sathish, S and London, E and Le, A and Li, Q and Gudmundsdottir, B and Lee, DY and Burke, EV and Yates, BP and Liu, DR and Hsieh, M and Leonard, A and Eaton, WA and Uchida, N and Pierciey, FJ and Newby, GA and Tisdale, JF and Demirci, S},
title = {Comparative analysis of CRISPR-Cas9, lentiviral transduction, and base editing for sickle cell disease in a murine model.},
journal = {Blood advances},
volume = {10},
number = {2},
pages = {289-300},
doi = {10.1182/bloodadvances.2025017321},
pmid = {41150843},
issn = {2473-9537},
mesh = {Animals ; *Anemia, Sickle Cell/therapy/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Mice ; *Lentivirus/genetics ; Disease Models, Animal ; Humans ; *Transduction, Genetic ; Hematopoietic Stem Cells/metabolism ; Hematopoietic Stem Cell Transplantation ; },
abstract = {Sickle cell disease (SCD) is a red blood cell disorder caused by a mutation in the β-globin gene, leading to sickle hemoglobin polymerization under low oxygen conditions. Both CRISPR-Cas9 editing and lentiviral transduction have shown promising clinical outcomes, but it remains unclear which approach is superior. Alternatively, new editing tools such as base editing may also be promising and reduce risks of genotoxicity. To compare these approaches, we studied them in an immunocompromised mouse model. We optimized ex vivo conditions in CD34+ hematopoietic stem and progenitor cells (HSPC) and infused edited SCD HSPC into busulfan-conditioned nonirradiated NOD,B6.SCID Il2rγ-/-KitW41/W41 (NBSGW) mice. Ex vivo analysis confirmed successful editing and transduction. At 16 weeks, bone marrow analysis showed similar human CD45+ cell engraftment across all groups (75%-90%). In the competitive transplantation group, there was a lower amount of B-cell lymphoma/leukemia 11A enhancer editing than base editing and lentiviral transduction. A secondary transplantation model yielded similar results. An antisickling assay showed significantly higher red blood cell sickling reduction in the base editing, transduction, and competitive transplantation groups compared to CRISPR-Cas9. In conclusion, although all methods showed therapeutic potential, base editing and lentiviral transduction provided superior outcomes over CRISPR-Cas9-mediated editing in a competitive murine transplantation model.},
}

Animals
*Anemia, Sickle Cell/therapy/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
Mice
*Lentivirus/genetics
Disease Models, Animal
Humans
*Transduction, Genetic
Hematopoietic Stem Cells/metabolism
Hematopoietic Stem Cell Transplantation

@article {pmid40971839,
year = {2026},
author = {Lv, X and Wei, Q and Zhi, Q and Liu, X and Li, F and Niu, Y and Sun, H and Jin, K and Chen, GH and Li, B and Zuo, Q},
title = {CRISPR/Cas9-mediated TOP1 knockout in chicken DF-1 cells reveals its critical role in apoptosis regulation and genomic stability.},
journal = {Journal of animal science},
volume = {104},
number = {},
pages = {},
doi = {10.1093/jas/skaf315},
pmid = {40971839},
issn = {1525-3163},
mesh = {Animals ; *Apoptosis/genetics/physiology ; *Chickens/genetics ; *CRISPR-Cas Systems ; *Genomic Instability/genetics ; *DNA Topoisomerases, Type I/genetics/metabolism ; Cell Line ; Gene Knockout Techniques/veterinary ; DNA Damage ; },
abstract = {The role of topoisomerase I (encoded by TOP1) in avian cell survival and apoptosis regulation remains unclear, limiting its potential application in poultry biotechnology. This study aimed to establish a CRISPR/Cas9-mediated TOP1 knockout platform in chicken DF-1 cells and evaluate its functional impact on apoptosis. Three sgRNAs targeting TOP1 were designed and delivered via liposome vectors, achieving knockout efficiencies up to 50% as confirmed by T7 Endonuclease I (T7E1) assay and Sanger sequencing, with no detectable off-target effects. Functional analysis revealed that TOP1 knockout significantly increased apoptosis rates and upregulated DNA damage markers (γH2AX) and apoptotic genes (Caspase 8 and BRCA1). These results demonstrate that TOP1 is essential for maintaining genomic stability in avian somatic cells, and its depletion triggers apoptosis through DNA damage accumulation. Although synthetic lethality-based sex control was not directly tested here, our findings provide critical evidence that TOP1 dysfunction could theoretically enable selective elimination of specific cell populations (e.g., primary germ cells) via CRISPR editing. Notably, developing targeted delivery systems for PGCs-a focus of future research not addressed in this study-will be essential to achieve such selectivity in vivo, highlighting a significant technological hurdle to overcome.},
}

Animals
*Apoptosis/genetics/physiology
*Chickens/genetics
*CRISPR-Cas Systems
*Genomic Instability/genetics
*DNA Topoisomerases, Type I/genetics/metabolism
Cell Line
Gene Knockout Techniques/veterinary
DNA Damage

@article {pmid39962280,
year = {2026},
author = {Zheng, C and Zhang, G and Dean, LJ and Sontheimer, EJ and Xue, W},
title = {The reverse transcriptase domain of prime editors contributes to DNA repair in mammalian cells.},
journal = {Nature biotechnology},
volume = {44},
number = {1},
pages = {146-153},
pmid = {39962280},
issn = {1546-1696},
support = {R01 CA275945/CA/NCI NIH HHS/United States ; UH3HL147367//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; P01HL158506//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R01 GM150273/GM/NIGMS NIH HHS/United States ; UH3 HL147367/HL/NHLBI NIH HHS/United States ; R01GM150279//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; },
mesh = {Humans ; *DNA Repair/genetics ; *RNA-Directed DNA Polymerase/metabolism/genetics ; *Gene Editing/methods ; HEK293 Cells ; Animals ; DNA Damage ; Protein Domains ; DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; Cell Line ; },
abstract = {Reverse transcriptase (RT) has been shown to play a role in double-strand break repair in bacteria, yet the impact of the RT component of prime editors (PEs) on normal mammalian cellular functions is unclear. Here we show that overexpressed RT or PE increases short insertions and diminishes homology-directed repair following Cas9 cleavage at multiple loci in multiple cell lines. Live-cell imaging shows that RT and PEs are rapidly recruited to laser-induced DNA damage sites and promote endogenous repair, independent of known DNA damage sensors. Interestingly, RT-mCherry partially impairs green fluorescent protein-PARP1 recruitment. A compact PE without an RNase H domain shows reduced DNA repair activity and may therefore be more suitable for clinical application. These data reveal a role for untethered RT or the RT domain of PEs in the repair of chromosomal breaks, calling for evaluation of the long-term effect of PEs and retroviral RT in mammalian cells.},
}

Humans
*DNA Repair/genetics
*RNA-Directed DNA Polymerase/metabolism/genetics
*Gene Editing/methods
HEK293 Cells
Animals
DNA Damage
Protein Domains
DNA Breaks, Double-Stranded
CRISPR-Cas Systems
Cell Line

@article {pmid39934271,
year = {2026},
author = {Hart, SK and Müller, S and Wessels, HH and Méndez-Mancilla, A and Drabavicius, G and Choi, O and Sanjana, NE},
title = {Precise RNA targeting with CRISPR-Cas13d.},
journal = {Nature biotechnology},
volume = {44},
number = {1},
pages = {64-69},
pmid = {39934271},
issn = {1546-1696},
support = {DP2HG010099//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *RNA/genetics ; *Gene Editing/methods ; HEK293 Cells ; Transcriptome/genetics ; },
abstract = {The possibility of collateral RNA degradation poses a concern for transcriptome perturbations and therapeutic applications using CRISPR-Cas13. We show that collateral activity only occurs with high RfxCas13d expression. Using low-copy RfxCas13d in transcriptome-scale and combinatorial pooled screens, we achieve high on-target knockdown without extensive collateral activity. Furthermore, analysis of a high-fidelity Cas13 variant suggests that its reduced collateral activity may be due to overall diminished nuclease capability.},
}

*CRISPR-Cas Systems/genetics
Humans
*RNA/genetics
*Gene Editing/methods
HEK293 Cells
Transcriptome/genetics