@article {pmid42001810,
year = {2026},
author = {Saleem, MS and Khan, SH and Rana, IA and Ahmad, A},
title = {CRISPR/Cas9-mediated editing of the GhJAZ2 gene improves fiber length and lint percentage in Gossypium hirsutum L.},
journal = {GM crops & food},
volume = {17},
number = {1},
pages = {2660546},
doi = {10.1080/21645698.2026.2660546},
pmid = {42001810},
issn = {2164-5701},
mesh = {*Gossypium/genetics/metabolism/growth & development ; *CRISPR-Cas Systems ; *Gene Editing ; *Cotton Fiber/analysis ; Plants, Genetically Modified/genetics ; *Plant Proteins/genetics/metabolism ; },
abstract = {Cotton is regarded as a strategic agricultural commodity owing to its renewable and naturally derived fiber. With the escalating global demand for high-quality fiber, genetic improvement of fiber traits is a critical focus for sustaining and advancing the textile industry standards. The cotton GhJAZ2 gene encodes the Jasmonate ZIM-domain 2 protein, a known repressor in the jasmonic acid signaling pathway and negatively regulates fiber initiation. In this study, we designed a gRNA that simultaneously targets GhJAZ2 homologs and assembled it into the CRISPR vector (pHSE401). Subsequently, the construct (pHSE401-gRNA) was transformed into cotton (Gossypium hirsutum L.) using an Agrobacterium-mediated in planta transformation strategy, targeting the shoot apical meristem as the primary site of transformation. Sanger sequencing analysis revealed consistent single-base pair indels at the targeted site across both A and D sub-genomes, with edited T1 progenies showing both inherited and newly introduced indels at the targeted loci. Fiber analysis of edited lines compared to the control revealed a significant (p < .05) enhancement in lint percentage (≤13.74%) and fiber length (≤16.91%). This study demonstrated the effective application of CRISPR/Cas9 for targeted trait improvement in cotton, offering GhJAZ2-edited lines that can be advanced to develop transgene-free cultivars with improved fiber traits.},
}

*Gossypium/genetics/metabolism/growth & development
*CRISPR-Cas Systems
*Gene Editing
*Cotton Fiber/analysis
Plants, Genetically Modified/genetics
*Plant Proteins/genetics/metabolism

@article {pmid42001897,
year = {2026},
author = {Li, Q and Cao, J and Deng, S and Yu, K},
title = {Precise Multi-Gene Editing Strategies for Xenotransplantation Donor Pigs: Overcoming Immune and Coagulation Barriers.},
journal = {Xenotransplantation},
volume = {33},
number = {2},
pages = {e70128},
doi = {10.1111/xen.70128},
pmid = {42001897},
issn = {1399-3089},
support = {32072722//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Transplantation, Heterologous/methods ; *Gene Editing/methods ; Swine ; Humans ; Graft Rejection/immunology ; CRISPR-Cas Systems/genetics ; *Blood Coagulation/genetics/immunology ; *Heterografts/immunology ; Tissue Donors ; Organ Transplantation ; },
abstract = {Organ transplantation is the preferred treatment for end-stage organ failure, but the severe shortage of donors severely restricts its clinical application. Xenotransplantation, especially using pigs as donors, is considered an ideal source of alternative donors due to the high similarity between their organ structures and those of humans. However, significant differences in immune recognition and coagulation regulation between species can easily induce a series of rejection reactions, including hyperacute rejection, acute humoral rejection, T-cell-mediated rejection, and chronic vascular complications. It also carries risks such as physiological metabolic incompatibility and potential viral transmission. In recent years, with the development of tools such as CRISPR/Cas, precise multi-gene editing technology has become possible, enabling the simultaneous knockout of multiple xenoantigen genes (such as GGTA1, CMAH, and B4GALNT2) and the introduction of human genes regulating complement, coagulation, and immune responses (such as hCD55, hTBM (THBD), and hCD47), significantly improving the immune tolerance and physiological compatibility of donor organs. This article systematically reviews the immune and coagulation barriers in xenotransplantation, focusing on precise multi-gene editing strategies for pigs used in xenotransplantation. It highlights editing pathways such as tandem knock-in at the same site, simultaneous multi-site editing, stepwise modular editing, and homology-directed repair (HDR) enrichment. Combined with representative organ-specific examples (heart, kidney, liver, and lung), including key non-human primate studies and early human exploratory cases where available, it explores the application prospects of these strategies in creating safe clinical-grade donor pigs and promoting the clinical translation of xenotransplantation.},
}

Animals
*Transplantation, Heterologous/methods
*Gene Editing/methods
Swine
Humans
Graft Rejection/immunology
CRISPR-Cas Systems/genetics
*Blood Coagulation/genetics/immunology
*Heterografts/immunology
Tissue Donors
Organ Transplantation

@article {pmid42002179,
year = {2026},
author = {Guha, S and Tharmatt, A and Yadav, S and Siwakoti, P and Kumeria, T and Mittal, A and Chitkara, D},
title = {Lipid-polymer hybrid nanoplex loaded microneedle patches as a corneal delivery platform for CRISPR/Cas expressing plasmid.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {152092},
doi = {10.1016/j.ijbiomac.2026.152092},
pmid = {42002179},
issn = {1879-0003},
abstract = {Lipid-polymer hybrid (LPH) systems have evolved into a promising vehicle for delivering therapeutic agents. This study demonstrates cationic LPH nanoplexes composed of a biodegradable cationic mPEG-polycarbonate based cationic copolymer and cholesterol to enhance delivery efficiency of genetic materials. The optimized blank formulation, BNPX-3, exhibited a particle size of 93.60 nm, polydispersity index (PDI) of 0.116, and a zeta potential (ZP) of 21.9 mV. After complexation of pcDNA3-EGFP plasmid and CRISPRi plasmid at N/P ratios of 10 and 20, respectively, the nanoplexes exhibited particle sizes of 123.4 nm and 131.6 nm, with corresponding PDI values of 0.128 and 0.142 and zeta potentials of 12.2 mV and 10.5 mV, respectively. Cytocompatibility studies demonstrated >80% cell viability in HEK293 and SIRC cells over a wide concentration range. Transfection efficiencies using LPH nanoplexes for pcDNA3-EGFP (6159 bp) and CRISPRi plasmids (11,266 bp) were found to 60.09% and 48.02%, respectively, in HEK293 cells, and 56.04% and 40.34%, respectively, in SIRC cells. These efficiencies were comparable to Lipofectamine 3000 and superior to formulations prepared with cationic polymers alone. To enable efficient delivery of the nanoplexes, the developed nanoplexes were incorporated into dissolvable microneedle (MN) patches. The microneedle patch demonstrated successful loading of pDNA-complexed nanoplexes and efficient delivery ex-vivo.},
}

@article {pmid42002396,
year = {2026},
author = {Chen, L and Luo, J and Zhang, H and Zhao, P},
title = {RPA Combined With CRISPR/Cas12a for Rapid and Ultrasensitive Detection Dual-Gene of Methicillin-Resistant Staphylococcus aureus (MRSA).},
journal = {Journal of molecular recognition : JMR},
volume = {39},
number = {3},
pages = {e70035},
doi = {10.1002/jmr.70035},
pmid = {42002396},
issn = {1099-1352},
support = {2025A1515010579//Natural Science Foundation of Guangdong Province, China/ ; 211102114530659//Shaoguan Municipal Science and Technology Program, China/ ; 220610154531525//Shaoguan Municipal Science and Technology Program, China/ ; 220525096180441//Shaoguan Engineering Research Center for Research and Development of Molecular and Cellular Technology in Rapid Diagnosis of Infectious Diseases and Cancer Program, China/ ; KEYANSHEN (2023) 01//Research Fund for Joint Laboratory for Digital and Precise Detection of Clinical Pathogens, Yuebei People's Hospital Affiliated to Shantou University Medical College, China/ ; RS202001//Research Project for Outstanding Scholar of Yuebei People's Hospital Affiliated to Shantou University Medical College, China/ ; 2023B110008//Research Project for Guangdong Provincial Clinical Research Center for Laboratory Medicine, China/ ; },
mesh = {*Methicillin-Resistant Staphylococcus aureus/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; *Bacterial Proteins/genetics ; Humans ; *Staphylococcal Infections/diagnosis/microbiology ; Penicillin-Binding Proteins/genetics ; Sensitivity and Specificity ; Microbial Sensitivity Tests ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The increasing issue of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) necessitates rapid and reliable diagnostic methods. While existing RPA-CRISPR/Cas12a platforms have demonstrated potential for MRSA detection, most rely on single-gene targets or require multiple Cas enzymes. Here, we have developed a novel dual gene detection strategy that simultaneously detects the S. aureus specific femA gene and the methicillin-resistant mecA gene in a single RPA-CRISPR/Cas12a reaction. This integrated approach enables clear discrimination between MRSA and methicillin-sensitive Staphylococcus aureus (MSSA) in just 30 min, with results visualized via both fluorescence and lateral flow strips. The assay exhibited high specificity (no cross-reactivity with common pathogens) and a sensitivity of 10 copies/μL, comparable to qPCR. Validation with 39 clinical samples showed 100% concordance with antimicrobial susceptibility testing. Our dual-gene RPA-CRISPR/Cas12a platform represents a significant advancement in point-of-care MRSA diagnostics, offering enhanced accuracy and operational simplicity.},
}

*Methicillin-Resistant Staphylococcus aureus/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
*Bacterial Proteins/genetics
Humans
*Staphylococcal Infections/diagnosis/microbiology
Penicillin-Binding Proteins/genetics
Sensitivity and Specificity
Microbial Sensitivity Tests
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid42003550,
year = {2026},
author = {Marsic, T and Gundra, SR and Aouida, M and Masood, M and Salibi, A and Schmidt, F and Alquwayzani, R and Mahfouz, MM},
title = {Precise, specific gene editing via a compact GoCas12m-FokI chimeric nuclease.},
journal = {Nucleic acids research},
volume = {54},
number = {7},
pages = {},
doi = {10.1093/nar/gkag342},
pmid = {42003550},
issn = {1362-4962},
support = {1/1035-01-01//BAS/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; *Deoxyribonucleases, Type II Site-Specific/genetics/metabolism/chemistry ; *CRISPR-Associated Proteins/genetics/metabolism ; HEK293 Cells ; *Endodeoxyribonucleases/genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; Bacterial Proteins ; },
abstract = {CRISPR gene editing technologies have transformed functional genomics and biotechnology. Despite these advances, challenges such as limited delivery capacity and off-target activity continue to hinder their therapeutic translation. We developed a chimeric gene editing platform by fusing the compact, catalytically inactive Cas12m guiding module (GoCas12m) with the FokI nuclease domain. GoCas12m-FokI system integrates the programmable DNA-binding capability of Cas12m with the dimerization-dependent cleavage mechanism of FokI, enabling precise genome editing. Our engineered XTEN-fused GoCas12m-FokI editor exhibits robust activity on both surrogate reporters and endogenous human loci, achieving high-efficiency editing at clinically relevant targets-including CLTA1, HBB, AIFM1, and ABL with no detectable off-target activity at in silico-predicted sites, as confirmed by targeted deep sequencing. Notably, GoCas12m-FokI is nearly half the size of conventional Cas9- or Cas12a-based editors, facilitating delivery via adeno-associated virus and other cargo-limited vectors. This dual-guided editor showed comparable editing efficiency to previously reported FokI-dCas9 systems on endogenous loci, while possessing a different PAM requirement and domain orientation. By combining compact architecture, high specificity, and modular programmability, the GoCas12m-FokI editor offers a powerful alternative for therapeutic genome editing and a promising tool for in vivo gene therapy applications.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems
*Deoxyribonucleases, Type II Site-Specific/genetics/metabolism/chemistry
*CRISPR-Associated Proteins/genetics/metabolism
HEK293 Cells
*Endodeoxyribonucleases/genetics/metabolism
Recombinant Fusion Proteins/genetics/metabolism
Bacterial Proteins

@article {pmid42003552,
year = {2026},
author = {White, N and Hu, YT and Chalk, JA and Kurgan, G and Naseem, A and Schmaljohn, E and Sturgeon, M and Cavazza, A and Thrasher, AJ and Turchiano, G},
title = {DNA-PKcs inhibitor AZD7648 reveals sgRNA cross-contaminants and enhanced sensitivity of genome engineering off-target activity in HSPCs.},
journal = {Nucleic acids research},
volume = {54},
number = {7},
pages = {},
doi = {10.1093/nar/gkag318},
pmid = {42003552},
issn = {1362-4962},
support = {217112/Z/19/Z/WT_/Wellcome Trust/United Kingdom ; 1160024//Great Ormond Street Hospital Children's Charity/ ; //Curing Rare Inherited Diseases Using Innovative Gene Therapies/ ; //European Union's Horizon 2020/ ; },
mesh = {Humans ; *DNA-Activated Protein Kinase/antagonists & inhibitors/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Pyrazoles/pharmacology ; DNA End-Joining Repair/drug effects ; CRISPR-Cas Systems ; Mutation ; },
abstract = {Therapeutic gene editing with designer nucleases can be compromised by undesired repair outcomes. DNA repair inhibitors are used to bias DSB repair toward HDR, but their impact on larger structural rearrangements, including large deletions and translocations, remains unclear. We quantify the mutational burden associated with end-joining inhibitor compounds. With a highly precise Cas9 nuclease, repair inhibition yields modest increases in aberrations, whereas promiscuous single guide RNAs (sgRNAs) amplify aberrant outcomes by orders of magnitude. Donor templates mitigate mutational burden at on-target sites, and in rare cases donor sequences bridge translocations between on- and off-target loci. Because DNA-PKcs inhibition does not itself induce instability over short intervals but increases the likelihood of capturing chromosomal aberrations postediting, we leveraged this to enhance assay performance. Compared to CAST-Seq, high-resolution CAST-Seq achieved a median ~12-fold increase in detected aberrations and, in this higher-sensitivity context, revealed unintended, target-specific sgRNA contaminants in GMP-like batches, underscoring direct genotoxicity risk and the need for stricter guide purity controls. A modified, translocation-quantitative rhAmpSeq reports all translocation combinations between two loci, enabling robust off-target validation beyond indel-only readouts. Finally, we evaluate AZD7648, finding limited aberration increases with precise nucleases and reconciling reports of extensive large deletions by quantifying assay- and design-dependent biases.},
}

Humans
*DNA-Activated Protein Kinase/antagonists & inhibitors/genetics
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
*Pyrazoles/pharmacology
DNA End-Joining Repair/drug effects
CRISPR-Cas Systems
Mutation

@article {pmid41481841,
year = {2026},
author = {Jin, YM and Li, XD and Zhu, JK and Shao, CY and Huang, BB and Huang, HL and Wang, XW and Jiang, HB and Chen, W},
title = {Programmable adenine base editing in cyanobacteria using an engineered TadA-Cas9 fusion.},
journal = {The Plant journal : for cell and molecular biology},
volume = {125},
number = {1},
pages = {e70655},
doi = {10.1111/tpj.70655},
pmid = {41481841},
issn = {1365-313X},
support = {32470092//National Natural Science Foundation of China/ ; 32170108//National Natural Science Foundation of China/ ; 2024QL060//Ningbo Youth Leading Talent Project/ ; //Ningbo University Startup Funding/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Adenine/metabolism ; *Synechocystis/genetics ; *Cyanobacteria/genetics ; Bacterial Proteins/genetics/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Anabaena/genetics ; },
abstract = {Cyanobacteria are photosynthetic prokaryotes with great potential in green biomanufacturing and basic research. Despite decades of pioneering achievements, the application of advanced genome editing tools, particularly CRISPR-based systems, has remained limited in cyanobacteria. In this study, we developed pCyABE, a new adenine base editor for efficient and precise A·T to G·C editing in cyanobacteria. This system utilizes a TadA-Cas9 nickase fusion and functions without double-strand breaks or donor templates. We demonstrated its high editing efficiency in Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, highlighting its broad usability. pCyABE supports multiplex editing and enables start codon disruption for gene functional studies. Furthermore, this tool exhibits low off-target activity and can be effectively removed via sucrose counterselection. In conclusion, pCyABE provides a versatile and efficient genome editing platform that significantly expands the genetic toolbox for cyanobacterial research and biotechnology applications.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Adenine/metabolism
*Synechocystis/genetics
*Cyanobacteria/genetics
Bacterial Proteins/genetics/metabolism
*CRISPR-Associated Protein 9/genetics/metabolism
*Anabaena/genetics

@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 {pmid41483499,
year = {2026},
author = {Jin, G and Yang, C and Deng, Q and Wu, L and Chen, W and Chen, Z},
title = {Establishing a chimeric tRNA-sgRNA scaffold and computational basis for enhanced CRISPR interference.},
journal = {Biochemical and biophysical research communications},
volume = {798},
number = {},
pages = {153222},
doi = {10.1016/j.bbrc.2025.153222},
pmid = {41483499},
issn = {1090-2104},
mesh = {*RNA, Transfer/genetics/chemistry ; *CRISPR-Cas Systems/genetics ; Escherichia coli/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; Humans ; Molecular Docking Simulation ; Gene Editing/methods ; Molecular Dynamics Simulation ; Nucleic Acid Conformation ; },
abstract = {The CRISPR/Cas9 system revolutionizes genome engineering, yet optimizing the stability and expression levels of single-guide RNA (sgRNA) is crucial for achieving more effective gene regulation. Transfer RNAs (tRNA), known for their inherent stability, present a valuable solution. In this study, we developed a chimeric tRNA-sgRNA (tgRNA) by integrating sgRNA into the anticodon stem of a Sephadex aptamer-human HBV ε tRNA (SeptRNA) scaffold, resulting in the formation of SeptgRNA. When applied to target the E. coli ampC and ompA genes, SeptgRNA exhibited significantly increased accumulation compared to conventional sgRNAs. To overcome potential steric hindrance from the tRNA scaffold, we utilized CRISPR interference (CRISPRi) by co-expressing SeptgRNA with deactivated Cas9 (dCas9), which effectively suppressed DNA transcription. This approach demonstrated superior gene expression suppression compared to traditional sgRNA-based CRISPRi. Molecular docking and molecular dynamics simulations revealed that the SeptRNA scaffold stabilizes the sgRNA stem-loop architecture and enhances the stability of the dCas9-tgRNA-DNA ternary complex. Our findings provide proof-of-concept for the use of chimeric tgRNAs in gene knockdown, highlighting their potential for increased expression levels and improved stability. This study advances the CRISPR/Cas9 toolkit and underscores the versatility of tRNA scaffolds in genetic engineering applications.},
}

*RNA, Transfer/genetics/chemistry
*CRISPR-Cas Systems/genetics
Escherichia coli/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry
Humans
Molecular Docking Simulation
Gene Editing/methods
Molecular Dynamics Simulation
Nucleic Acid Conformation

@article {pmid41484149,
year = {2026},
author = {Kremer, N and Mueller, F and Nguyen, H and Schulz, L and Popp, T and Artes, E and Wolters, J and Renner, M and Vetter, I and Maffini, S and Robles, MS and Musacchio, A and Bange, T},
title = {CUL4A-DDB1-DCAF10 is an N-recognin for N-terminally acetylated Src kinases.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {132},
pmid = {41484149},
issn = {2041-1723},
support = {5041 140321//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Cullin Proteins/metabolism/genetics ; Humans ; Acetylation ; *src-Family Kinases/metabolism/genetics ; HEK293 Cells ; *DNA-Binding Proteins/metabolism/genetics ; Proteolysis ; Protein Processing, Post-Translational ; Ubiquitination ; Ubiquitin-Protein Ligases/metabolism ; CRISPR-Cas Systems ; },
abstract = {Co-translational N-terminal modifications such as methionine excision, acetylation, and myristoylation govern protein stability, localization, and folding. Disruption can expose N-terminal degrons that trigger ubiquitin-mediated degradation, safeguarding the proteome. N-terminal acetylation usually protects proteins from degradation, but can also promote it through the Ac/N-degron pathway. Src-family kinases (SFKs), signaling enzymes implicated in tumorigenesis, require N-terminal myristoylation for function. Using peptide pull-downs, mass spectrometry, and AlphaFold 3 predictions, we identify DCAF10 as the E3 ligase substrate receptor for alternatively N-terminally acetylated SFKs. Combining siRNA-mediated knockdown and CRISPR/Cas9-mediated knockout of endogenous Lyn with inducible Lyn-GFP variants confirms that DCAF10 regulates SFK levels by recognizing an N-terminal acetylated glycine residue. In vitro, a CUL4A-DDB1-DCAF10 complex ubiquitinates N-terminally acetylated SFKs. Thus, we define a novel N-degron pathway that monitors replacement of myristoylation by acetylation and activates degradation of SFKs upon acetylation. This mechanism may extend to other N-terminally myristoylated proteins beyond SFKs.},
}

*Cullin Proteins/metabolism/genetics
Humans
Acetylation
*src-Family Kinases/metabolism/genetics
HEK293 Cells
*DNA-Binding Proteins/metabolism/genetics
Proteolysis
Protein Processing, Post-Translational
Ubiquitination
Ubiquitin-Protein Ligases/metabolism
CRISPR-Cas Systems

@article {pmid41484984,
year = {2026},
author = {Forbes, CA and Shaw, NC and Chen, KG and Hedges, M and Er, TS and Hool, L and Ward, M and Poulton, C and Baynam, G and Lassmann, T and Fear, VS},
title = {A precision medicine approach to interpret a GATA4 genetic variant in a paediatric patient with congenital heart disease.},
journal = {Human genomics},
volume = {20},
number = {1},
pages = {29},
pmid = {41484984},
issn = {1479-7364},
support = {Telethon 7 Ball, Gift of Giving, 2020.//McCusker Charitable Foundation/ ; Perron People//Stan Perron Charitable Foundation/ ; Perron Programs and Partnership, 2025//Stan Perron Charitable Foundation/ ; /WT_/Wellcome Trust/United Kingdom ; Lassmann//Feilman Foundation/ ; UWA-VCCRI Chair in Cardiovascular Research//Wesfarmers/ ; APP2004282//National Health and Medical Research Council/ ; },
mesh = {Humans ; *GATA4 Transcription Factor/genetics ; Induced Pluripotent Stem Cells/metabolism/pathology ; Myocytes, Cardiac/metabolism/pathology ; *Heart Defects, Congenital/genetics/pathology ; *Precision Medicine/methods ; Gene Editing ; *Genetic Variation ; Cell Differentiation/genetics ; CRISPR-Cas Systems ; Male ; Child ; Female ; },
abstract = {BACKGROUND: Patients with congenital heart disease are identified in 1% of live births. Improved surgical intervention means many patients now survive to adulthood, the corollary of which is increased mortality in the over-65-year-old congenital heart disease (CHD) population. In the clinic, genetic sequencing increasingly identifies novel genetic variants in genes related to CHD. Traditional assays for interpreting novel genetic variants are often limited by gene-specificity, whereas animal models are cumbersome and may not accurately reflect human disease. This study investigates CRISPR gene editing in induced pluripotent stem cells and cardiomyocyte-directed differentiation as a human disease model to investigate novel genetic variants identified in association with CHD.

METHODS AND RESULTS: We identified a GATA4 p.Arg284His genetic variant in a paediatric patient. This genetic variant was introduced into induced pluripotent stem cells (iPSCs) using CRISPR gene editing with homology-directed-repair. GATA4 genetic variant and isogenic control iPSCs were selected and differentiated into cardiomyocytes. Expression of the GATA4 p.Arg284His variant resulted in altered calcium transients, indicative of CHD and consistent with the patient's clinical phenotype. Transcriptomics revealed cellular pathway changes in cardiac development, calcium handling, and energy metabolism that contribute to disease aetiology, mechanism and identification of potential treatments.

CONCLUSION: Directed differentiation of iPSCs harbouring the GATA4 p.Arg284His genetic variant recapitulated the CHD phenotype, indicated disease mechanisms, and pointed to potential sites for targeting with therapy. The study highlights the utility of transcriptomics for the functional interpretation of cardiac genetic variants and is an exemplar for precision medicine approaches for the investigation of CHD.},
}

Humans
*GATA4 Transcription Factor/genetics
Induced Pluripotent Stem Cells/metabolism/pathology
Myocytes, Cardiac/metabolism/pathology
*Heart Defects, Congenital/genetics/pathology
*Precision Medicine/methods
Gene Editing
*Genetic Variation
Cell Differentiation/genetics
CRISPR-Cas Systems
Male
Child
Female

@article {pmid41485050,
year = {2026},
author = {Wang, K and Li, X and Li, J and Yu, Z and Li, P and Xie, Y and Liu, J and Huang, H and Zhang, S and Zhang, M and Ma, W and Gao, F and Du, X and Wang, J and Capecchi, MR and Wu, S},
title = {Basic enables selection-free efficient knockin of large DNA in primary human T cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {4},
pages = {2309-2323},
pmid = {41485050},
issn = {1525-0024},
mesh = {Humans ; *T-Lymphocytes/metabolism/immunology ; Genetic Vectors/genetics ; *Receptors, Chimeric Antigen/genetics/metabolism ; Animals ; *Gene Knock-In Techniques/methods ; Dependovirus/genetics ; Mice ; *DNA/genetics ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {Efficient and precise insertion of large DNA fragments into primary human T cells has remained a bottleneck for gene and cell therapy. We present BaEVshort-AAV6 site-specific integration for CAR T (BASIC), a modular platform that combines BaEVshort-pseudotyped virus-like particles for Cas9 RNP delivery with AAV6 donor vectors for homology-directed repair. BASIC achieves >85% knockin efficiency without drug selection or electroporation, preserving cell viability while enabling multiplex genome engineering. Edited chimeric antigen receptor (CAR)-T cells show uniform CAR expression, enhanced cytotoxicity, and complete tumor clearance in vivo. BASIC offers a clinically scalable solution for next-generation cell therapies.},
}

Humans
*T-Lymphocytes/metabolism/immunology
Genetic Vectors/genetics
*Receptors, Chimeric Antigen/genetics/metabolism
Animals
*Gene Knock-In Techniques/methods
Dependovirus/genetics
Mice
*DNA/genetics
Gene Editing
CRISPR-Cas Systems

@article {pmid41485557,
year = {2026},
author = {Li, D},
title = {Rising Star Engineering the Genome for Curative Futures.},
journal = {Journal of molecular biology},
volume = {438},
number = {5},
pages = {169618},
doi = {10.1016/j.jmb.2025.169618},
pmid = {41485557},
issn = {1089-8638},
mesh = {*Gene Editing/methods ; Humans ; Animals ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; *Genetic Engineering/methods ; Mice ; Neoplasms/therapy/genetics ; Disease Models, Animal ; },
abstract = {As a professor of biomedicine in the School of Life Sciences at East China Normal University (ECNU), I am dedicated to developing advanced genome editing technologies for disease modeling and precise gene therapy. My foundational training at Hunan Normal University and Texas A&M University cultivated a deep interest in using engineered cellular and animal models to understand human diseases. Since 2013, my laboratory at ECNU has pioneered the use of TALEN and CRISPR/Cas9 for the rapid generation of knockout mouse and rat models for disease studies. Once stepped in genome editing field, I shifted my focus to advancing editing tools and developing gene therapy strategies for genetic disorders and cancer. My team has developed a suite of high-performance base editors for nuclear DNA, mitochondrial DNA, and RNA, broadening editing capabilities while enhancing precision and safety. Leveraging these technologies, we have designed several therapeutic strategies that have shown efficacy in cellular and animal models of genetic diseases. Through collaborative efforts, we have successfully translated genome editing into clinical applications, contributing to the treatment of patients with β-thalassemia. Additionally, we have developed a non-viral, site-specific CAR-T platform for lymphoma therapy. Looking forward, I aim to develop the next generation of long-fragment, site-specific integration technologies and accelerate clinical translation to bring transformative cures to more patients.},
}

*Gene Editing/methods
Humans
Animals
*Genetic Therapy/methods
CRISPR-Cas Systems
*Genetic Engineering/methods
Mice
Neoplasms/therapy/genetics
Disease Models, Animal

@article {pmid41485979,
year = {2026},
author = {Shimizu, T and Okamoto, M and Kawamoto, K},
title = {Evaluation of a d-octaarginine-linked polymer for CRISPR-Cas9 ribonucleoprotein (RNP) delivery and genome editing in murine dendritic cells.},
journal = {The Journal of veterinary medical science},
volume = {88},
number = {3},
pages = {374-383},
pmid = {41485979},
issn = {1347-7439},
mesh = {Animals ; *Dendritic Cells/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Mice ; *Ribonucleoproteins/genetics ; *Oligopeptides/chemistry ; Cell Line ; RNA, Guide, CRISPR-Cas Systems/genetics ; Transfection ; *Polymers/chemistry ; },
abstract = {We previously reported that poly (N-vinylacetamide-co-acrylic acid) coupled with d-octaarginine (VP-R8) efficiently introduces proteins and nucleic acids into cells. Based on these results, we hypothesized that VP-R8 can introduce a complex composed of guide RNA and Cas9 (RNP complex) into cells and induce genome editing mediated by the CRISPR-Cas9 system. We tested this hypothesis using a mouse dendritic cell line and mouse primary dendritic cells. The RNP complexes formed by guide RNA consisting of CRISPR RNA (crRNA), fluorescently labeled trans-activating crRNA (tracrRNA), and GFP-fused Cas9 were introduced into a mouse dendritic cell line using VP-R8 or control transfection reagents. Cells transfected using VP-R8 exhibited higher fluorescence than those transfected with other transfection reagents, indicating that VP-R8 efficiently introduced the RNP complex into the mouse dendritic cell line. Genome editing of the target DNA was detected in cells transfected with the RNP complex using VP-R8 and dominant relative to other transfection reagents. We also observed that VP-R8 effectively delivered RNP complexes consisting of single-guide RNA and Cas9 and induced genome editing in the dendritic cell line. Additionally, VP-R8 efficiently delivered RNP complexes into mouse primary dendritic cells and induced genome editing of the functional gene without producing early inflammatory cytokines. Thus, VP-R8 shows potential as a transfection tool to generate dendritic cells with specific gene regions deleted by genome editing via the CRISPR-Cas9 system. This approach aims to elucidate the detailed molecular mechanisms of dendritic cell function for its application to vaccines.},
}

Animals
*Dendritic Cells/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems
Mice
*Ribonucleoproteins/genetics
*Oligopeptides/chemistry
Cell Line
RNA, Guide, CRISPR-Cas Systems/genetics
Transfection
*Polymers/chemistry

@article {pmid41486484,
year = {2025},
author = {Boogari, M and Mohebbi, M and Hadidi, N},
title = {Genetically Engineered Probiotics: Design, Therapeutics, and Clinical Translation.},
journal = {Iranian biomedical journal},
volume = {29},
number = {6},
pages = {374-383},
doi = {10.61882/ibj.5197},
pmid = {41486484},
issn = {2008-823X},
mesh = {*Probiotics/therapeutic use ; Humans ; *Genetic Engineering/methods ; *Translational Research, Biomedical ; Animals ; },
abstract = {Genetically engineered probiotics (GEPs) aim to address transient colonization and the intra- and inter-subject variability that limit conventional probiotics. These strains utilize Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas editing, programmable gene circuits, and biosensors in chassis such as E. coli Nissle 1917 and L. lactis. This narrative review summarizes the current engineering toolkits and standards (e.g., SEVA), chassis selection criteria, biocontainment strategies, and translational requirements under CMC/GMP frameworks and discusses regulatory considerations for clinical translation. Representative examples include IL-10-secreting Lactococcus lactis and phenylalanine-metabolizing strains for phenylketonuria (SYNB1618/SYNB1934), which illustrate pharmacodynamic target engagement and short-term preclinical safety. We outline clinical advancements in predefined pharmacodynamics, durability of function, monitoring shedding and horizontal gene transfer, and genomic-microbiome-informed patient stratification. Systems modeling approaches (Genome-Scale Metabolic Model/ Agent-Based Model) are discussed as tools to guide rational design. GEPs offer programmable “sense-and-respond” therapeutics, with successful clinical adoption depending on durable efficacy, long-term safety, and clearly defined regulatory pathways.},
}

*Probiotics/therapeutic use
Humans
*Genetic Engineering/methods
*Translational Research, Biomedical
Animals

@article {pmid41486850,
year = {2026},
author = {Shi, M and Yu, P and Liu, L and Cheng, J and Shao, R and Sun, Y and Lv, J and Li, Y and Zheng, Z and Yu, J and Xu, B and Gan, L and Liang, Y and Zhang, Y and Fang, Y and Shen, W and Huang, J and Zhu, X and Hong, J and Ma, R and Wu, L and Cheng, Y and Zhao, C},
title = {Fluoropolymer-Mediated Delivery of a Dual TSHR/IGF1R-Targeting CRISPR-Cas9 System for Localized Therapy in Thyroid-Associated Ophthalmopathy.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {38},
number = {11},
pages = {e11078},
doi = {10.1002/adma.202511078},
pmid = {41486850},
issn = {1521-4095},
support = {DGF828030-3/005//Clinical Scientist Foundation of Fudan University/ ; pp25014//Clinical Scientist Foundation of Fudan University/ ; U25A20258//National Natural Science Foundation of China/ ; 82271126//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Receptor, IGF Type 1/genetics/metabolism ; *Graves Ophthalmopathy/therapy/genetics/pathology ; *CRISPR-Cas Systems/genetics ; Humans ; Mice ; *Receptors, Thyrotropin/genetics/metabolism ; Gene Editing ; *Polymers/chemistry ; Fibroblasts/metabolism ; Disease Models, Animal ; Adipocytes/metabolism ; Genetic Therapy ; Organoids/metabolism ; Antibodies, Monoclonal, Humanized ; },
abstract = {Thyroid-associated ophthalmopathy (TAO), a vision-threatening and disfiguring autoimmune orbital disorder, remains a therapeutic challenge due to the lack of therapies with orbital specificity, sustained efficacy, and minimal side effects. Herein, we present G4F7-CRISPR, a fluoropolymer-based CRISPR-Cas9 delivery platform engineered for localized and efficient disruption of thyroid-stimulating hormone receptor (TSHR) and insulin-like growth factor 1 receptor (IGF1R), two key mediators of TAO pathogenesis. G4F7-CRISPR achieved high insertion/deletion frequencies in primary orbital fibroblasts (Tshr: 37.2%; Igf1r: 42.8%) and mature adipocytes (Tshr: 22.4%; Igf1r: 24.3%), and maintained robust editing efficiency in orbital adipose tissue of TAO mouse models (Tshr: 30.7%; Igf1r: 32.4%). In both TAO mouse models and 3D human orbital organoids, dual-gene editing of Tshr and Igf1r via G4F7-CRISPR significantly suppressed orbital adipogenesis, inflammation, and fibrosis, demonstrating superior therapeutic efficacy over either single-gene approaches. Comprehensive off-target analyses in both TAO mouse models and orbital organoids revealed minimal off-target activity. Furthermore, G4F7-CRISPR exhibited excellent short- and long-term ocular and systemic safety in TAO mouse models. Notably, it outperformed teprotumumab-the FDA-approved therapy for TAO-in both therapeutic efficacy and safety, highlighting its potential clinical advantages. Collectively, these findings highlight the translational promise of G4F7-CRISPR as a safe, precise, and clinically viable gene therapy for TAO.},
}

Animals
*Receptor, IGF Type 1/genetics/metabolism
*Graves Ophthalmopathy/therapy/genetics/pathology
*CRISPR-Cas Systems/genetics
Humans
Mice
*Receptors, Thyrotropin/genetics/metabolism
Gene Editing
*Polymers/chemistry
Fibroblasts/metabolism
Disease Models, Animal
Adipocytes/metabolism
Genetic Therapy
Organoids/metabolism
Antibodies, Monoclonal, Humanized

@article {pmid41487293,
year = {2025},
author = {},
title = {Erratum: Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing [Corrigendum].},
journal = {Biologics : targets & therapy},
volume = {19},
number = {},
pages = {745-746},
doi = {10.2147/BTT.S585961},
pmid = {41487293},
issn = {1177-5475},
abstract = {[This corrects the article DOI: 10.2147/BTT.S326422.].},
}

@article {pmid41488303,
year = {2025},
author = {Tsolakidou, PJ},
title = {CRISPR-Cas systems against carbapenem resistance: from proof-of-concept to clinical translation.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1725247},
pmid = {41488303},
issn = {1664-302X},
abstract = {Carbapenem-resistant Enterobacterales (CRE) pose a major global threat, driven by plasmid-borne carbapenemase genes such as bla KPC, bla NDM and bla OXA-48. CRISPR-Cas systems offer programmable strategies to selectively eliminate these resistance determinants. This mini-review summarizes recent advances in Cas9-based plasmid curing, RNA-targeting approaches such as Cas13a and Cas13d, and DNA-targeting Cas3-enhanced bacteriophage therapeutics that have entered early clinical evaluation. Particular attention is given to conjugative CRISPR-Cas9 plasmid systems, which enable targeted plasmid eradication without laboratory transformation and broaden the delivery toolbox beyond phage vectors. We further discuss major translational challenges, including delivery efficiency, phage host-range constraints, ecological risks of horizontal CRISPR dissemination, and off-target effects. Finally, we highlight emerging delivery platforms-outer membrane vesicles, lipid and polymeric nanoparticles, conjugative plasmids with containment circuits, and engineered live biotherapeutics-that may complement or overcome current limitations. Collectively, these developments illustrate the potential of CRISPR-based antimicrobials to augment traditional therapies through precise gene-level suppression of carbapenem resistance.},
}

@article {pmid41488985,
year = {2026},
author = {Kim, K and Lee, J and Lee, N and Cho, BK},
title = {CRISPR-Based Approaches to Engineer Nonmodel Bacteria for Bioproduction and Biotherapeutics.},
journal = {Biochemistry},
volume = {65},
number = {2},
pages = {166-194},
doi = {10.1021/acs.biochem.5c00613},
pmid = {41488985},
issn = {1520-4995},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Metabolic Engineering/methods ; *Bacteria/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Engineering/methods ; Streptomyces/genetics/metabolism ; Humans ; },
abstract = {Microbial diversity encompasses vast genetic and functional capacities, with immense potential for biotechnological applications. Yet, most biotechnological advances have been confined to a narrow set of model organisms, leaving the broader repertoire of nonmodel microbes largely untapped due to species-specific barriers that hinder genetic manipulation. Over the past decade, the advent of CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems has transformed microbial engineering by enabling precise, programmable, and scalable control of genomes and gene expression. Importantly, the relative independence of many CRISPR effectors from host cofactors has facilitated their use in microbes previously challenging to engineer, thus expanding opportunities to exploit their unique metabolic and biosynthetic traits. In this review, we summarize the major CRISPR-Cas toolkits and highlight recent innovations, with particular emphasis on translational applications in nonmodel organisms such as C1-gas-fixing acetogens, antibiotic-producing Streptomyces, and gut commensal Bacteroides. We emphasize three areas of emerging impact: engineering microbial cell factories for sustainable biomanufacturing, accelerating natural product discovery, and development of next-generation live biotherapeutics. Finally, we discuss current limitations and future opportunities, underscoring how the integration of genome editing, synthetic biology, and systems-level approaches is reshaping the landscape of microbial biotechnology.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Metabolic Engineering/methods
*Bacteria/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats
Genetic Engineering/methods
Streptomyces/genetics/metabolism
Humans

@article {pmid41489362,
year = {2026},
author = {Shin, J and Barrangou, R},
title = {Occurrence and applications of CRISPR-Cas systems in bifidobacteria.},
journal = {Applied and environmental microbiology},
volume = {92},
number = {1},
pages = {e0170325},
pmid = {41489362},
issn = {1098-5336},
support = {internal grant//North Carolina Agricultural Foundation/ ; internal support//College of Agriculture and Life Sciences, North Carolina State University/ ; },
mesh = {*CRISPR-Cas Systems ; *Bifidobacterium/genetics ; *Gene Editing/methods ; Probiotics ; Genome, Bacterial ; Humans ; },
abstract = {Bifidobacterium is a key member of the human gut microbiota, and many strains are widely used as probiotics due to their health-promoting properties. Despite growing interest, genetic studies in Bifidobacterium have been relatively limited, primarily due to the lack of available genome editing tools. Recent advances in genomics and CRISPR-Cas systems provide opportunities for targeted genome modification in this genus. In this review, we provide an overview of the occurrence, diversity, and distribution of CRISPR-Cas systems across Bifidobacterium species and examine the editing tools developed and implemented to date. We also highlight practical challenges such as strain variability and low transformation efficiency and introduce future avenues of research such as large-payload insertion and in situ editing. Expanding the genetic toolbox for Bifidobacterium will broaden our understanding of this important genus and enable the development of next-generation probiotics.},
}

*CRISPR-Cas Systems
*Bifidobacterium/genetics
*Gene Editing/methods
Probiotics
Genome, Bacterial
Humans

@article {pmid41489552,
year = {2026},
author = {Fan, R and Tong, Y and Luo, S and He, Y and Yang, C and Li, W and Liu, J and Pan, J and Zhu, Y and Zhang, X and Zhu, J and Zhu, Y and Guo, Y and Li, L and Situ, B and Yan, X and Ma, W and Chang, L and Zhang, Y},
title = {Integrated Electroporated-Lysis Electrochemical Platform Enables Sensitive and Rapid EV Protein and miRNA Profiling Based on Multiplex-Responsive CRISPR/Cas12a.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {13},
pages = {e13331},
doi = {10.1002/smll.202513331},
pmid = {41489552},
issn = {1613-6829},
support = {2024YFC2419100//National Key R&D Program of China/ ; 82272424//National Natural Science Foundation of China/ ; 82572682//National Natural Science Foundation of China/ ; 82372343//National Natural Science Foundation of China/ ; 82402753//National Natural Science Foundation of China/ ; 22304006//National Natural Science Foundation of China/ ; 62471021//National Natural Science Foundation of China/ ; 2023A1515010909//Guang Dong Basic and Applied Basic Research Foundation of China/ ; JCYJ20230807142204008//Shenzhen Science and Technology Innovation Committee/ ; 2024J004//Southern Medical University/ ; 2024M750606//China Postdoctoral Science Foundation/ ; 2025T180607//China Postdoctoral Science Foundation/ ; T2425021//National Science Fund for Distinguished Young Scholars/ ; },
mesh = {*MicroRNAs/metabolism/genetics ; Humans ; *Extracellular Vesicles/metabolism ; *CRISPR-Cas Systems/genetics ; *Electrochemical Techniques/methods ; *Electroporation/methods ; },
abstract = {Proteins and miRNAs in extracellular vesicles (EVs) have emerged as crucial biomarkers for tumor diagnosis. While CRISPR/Cas12a-based platforms have shown great promise in nucleic acid and protein detection, their susceptibility to off-target activation and structural instability remains a significant limitation. Here, we have developed an electroporation-lysis electrochemical platform integrated with DNA cube-cage-locked CRISPR/Cas12a (DC-Cas12a), termed EL-DC-Cas12a. This platform utilizes an electric field to rapidly lyse EVs, releasing their internal proteins and miRNAs. These released molecules then activate the DC-Cas12a system, thereby triggering the displacement of two distinct crRNA/Cas12a complexes that correspond to EV proteins and miRNAs, respectively. These complexes then specifically recognize and cleave electrochemical probes, generating quantifiable electrochemical signals that enable synchronous and accurate analysis of the two biomarkers. The integrated workflow for EV lysis and detection can be completed within 40 min, greatly simplifying the overall operation. The detection limits (LOD) of this platform for EV PD-L1 protein and miR-1246 were 5.44 × 10[4] particles/mL and 3.59 × 10[3] particles/mL, respectively. Moreover, by applying machine learning algorithms to analyze the EV-associated proteins and miRNAs profiling, the platform demonstrated a diagnostic accuracy of 98.3% in distinguishing healthy donors from early-stage GC patients, and 99% in differentiating early-stage from advanced-stage GC patients in a clinical gastric cancer cohort. Therefore, the proposed platform offers a promising strategy for multiplexed detection of EV biomarkers and precise discrimination of GC.},
}

*MicroRNAs/metabolism/genetics
Humans
*Extracellular Vesicles/metabolism
*CRISPR-Cas Systems/genetics
*Electrochemical Techniques/methods
*Electroporation/methods

@article {pmid41490267,
year = {2026},
author = {Carturan, A and Angelos, MG and Guruprasad, P and Patel, RP and Pajarillo, R and Lee, A and Espie, D and Zhang, Y and Chiang, YH and Xie, W and Rodriguez, JL and Harris, J and Devi, P and Afolayan-Oloye, OI and Xu, J and Sussman, JH and Elghawy, O and Yang, A and Barsouk, A and Cho, JH and Shaw, CE and Singh, E and Ugwuanyi, O and Paruzzo, L and Stella, F and Liu, S and Nason, S and Imparato, A and Rotolo, A and Lemoine, J and Barrett, DM and Posey, A and Rook, AH and Pillai, V and Bagg, A and Pileri, SA and Liu, D and Tan, K and Schuster, SJ and Teachey, DT and Porazzi, P and Ruella, M},
title = {Harnessing the CD2 axis to broaden and enhance the efficacy of CAR T-cell therapies.},
journal = {Blood},
volume = {147},
number = {16},
pages = {1842-1856},
doi = {10.1182/blood.2025031409},
pmid = {41490267},
issn = {1528-0020},
support = {R01 FD008168/FD/FDA HHS/United States ; R37 CA262362/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Immunotherapy, Adoptive/methods ; *CD2 Antigens/immunology/genetics/metabolism ; *Receptors, Chimeric Antigen/immunology/genetics ; Animals ; Mice ; *T-Lymphocytes/immunology ; *Hematologic Neoplasms/therapy/immunology ; CD58 Antigens/immunology ; CRISPR-Cas Systems ; },
abstract = {Patients with T-cell lymphomas and leukemias have overall poor outcomes because of the lack of targeted and effective treatments, particularly in the relapsed and refractory settings. The development of chimeric antigen receptor (CAR) T cells against T-cell neoplasms is limited by a lack of discriminating T-cell antigens that allow for effective antitumor responses while preventing CAR T-cell fratricide. We hypothesized that targeting CD2, a pan-T-cell antigen, with anti-CD2 CAR T cells engineered to lack CD2 expression (CART2) would improve manufacturability and preclinical efficacy. Optimized CD2-knockout CART2 cells, generated using CRISPR-Cas9, eradicated primary patient-derived CD2+ hematological neoplasms in vitro and in vivo, secreted effector cytokines, and exhibited adequate proliferative capacity. Nevertheless, CD2 has a key costimulatory function, and its deletion could lead to CAR T-cell dysfunction. Therefore, we tested the role of the CD2:CD58 axis in CAR T cells, using the anti-CD19 CART models. We demonstrate that CD2 loss attenuates CART19 efficacy by reducing avidity for tumor antigen, costimulation, and ultimately in vivo activity. Analogously, we show that tumor CD58 loss reduces CART19 efficacy. To overcome this issue, we developed a novel programmed cell death protein 1 (PD-1):CD2 switch receptor that rescues intracellular CD2 signaling, particularly when programmed death-ligand 1 is engaged, thereby improving in vivo outcomes. Collectively, we studied the role of CD2 both as a target for CAR T-cell therapy and as a critical costimulatory protein, whose signaling can be rescued using the PD-1:CD2 switch receptor. This receptor can be incorporated into CAR T cells and provides an effective strategy to overcome CD2-signaling deficiencies.},
}

Humans
*Immunotherapy, Adoptive/methods
*CD2 Antigens/immunology/genetics/metabolism
*Receptors, Chimeric Antigen/immunology/genetics
Animals
Mice
*T-Lymphocytes/immunology
*Hematologic Neoplasms/therapy/immunology
CD58 Antigens/immunology
CRISPR-Cas Systems

@article {pmid41491239,
year = {2026},
author = {Amelan, A and Collins, SC and Damseh, NS and Hamada, N and Salim, A and Dvir, E and Monderer-Rothkoff, G and Harel, T and Nagata, KI and Yalcin, B and Shifman, S},
title = {CRISPR knockout screens reveal genes and pathways essential for neuronal differentiation and implicate PEDS1 in neurodevelopment.},
journal = {Nature neuroscience},
volume = {29},
number = {3},
pages = {592-603},
pmid = {41491239},
issn = {1546-1726},
support = {466/21//Israel Science Foundation (ISF)/ ; 1863/24//Israel Science Foundation (ISF)/ ; ANR-18-CE12-0009//Agence Nationale de la Recherche (French National Research Agency)/ ; },
mesh = {Animals ; Mice ; *Cell Differentiation/genetics ; *Neurons/physiology ; *Neurogenesis/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; *Neurodevelopmental Disorders/genetics ; Gene Knockout Techniques/methods ; Mice, Knockout ; CRISPR-Cas Systems ; Male ; },
abstract = {Neurodevelopmental disorders (NDDs) arise from disruptions in brain development, yet the underlying pathways remain incompletely understood. Here we demonstrate that genome-wide CRISPR knockout screens in mouse embryonic stem cells differentiating into neural lineages identify hundreds of essential genes, only a minority of which are currently implicated in NDDs. Dominant NDD genes were enriched for transcriptional regulators, whereas recessive NDD genes were predominantly involved in metabolic processes. Mouse models for eight genes (Eml1, Dusp26, Dynlrb2, Mta3, Peds1, Sgms1, Slitrk4 and Vamp3) revealed marked neuroanatomical abnormalities, including microcephaly in half of the cases. Focusing on PEDS1, a key enzyme in plasmalogen biosynthesis, we identified a bi-allelic variant in individuals with microcephaly, global developmental delay and congenital cataracts. In mice, Peds1 deficiency led to accelerated cell-cycle exit and impaired neuronal differentiation and migration. These pathways required for neural differentiation provide a genetic framework for discovering additional NDD genes.},
}

Animals
Mice
*Cell Differentiation/genetics
*Neurons/physiology
*Neurogenesis/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Humans
*Neurodevelopmental Disorders/genetics
Gene Knockout Techniques/methods
Mice, Knockout
CRISPR-Cas Systems
Male

@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},
pmid = {41491310},
issn = {1095-564X},
support = {R01 NS124166/NS/NINDS NIH HHS/United States ; R35 NS122191/NS/NINDS NIH HHS/United States ; },
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 {pmid41492065,
year = {2026},
author = {Wang, Z and Wang, Y and Gao, H and Dai, J and Tang, N and Wang, Y and Ji, Q},
title = {Phage-associated Cas12p nucleases require binding to bacterial thioredoxin for activation and cleavage of target DNA.},
journal = {Nature microbiology},
volume = {11},
number = {1},
pages = {81-93},
pmid = {41492065},
issn = {2058-5276},
mesh = {*Thioredoxins/metabolism/genetics ; *CRISPR-Cas Systems ; *Bacteriophages/genetics/enzymology/metabolism ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Cryoelectron Microscopy ; Protein Binding ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; DNA Cleavage ; DNA/metabolism ; Escherichia coli/genetics/virology/metabolism ; },
abstract = {The evolutionary competition within phage-host systems led to the emergence of CRISPR-Cas defence mechanisms in bacteria and anti-CRISPR elements in bacteriophages. Although anti-CRISPR elements are well characterized, the role of bacterial factors that influence CRISPR-Cas efficacy has been comparatively overlooked. Type V CRISPR-Cas12 systems display striking functional and mechanistic diversity for nucleic acid targeting. Here we use a bioinformatic approach to identify Cas12p, a phage-associated nuclease that forms complexes with the bacterial thioredoxin protein TrxA to enable target DNA degradation. This represents an unexpected phage-bacteria interaction, in which the bacteriophage co-opts a bacterial factor to augment its own genome degradation machinery, potentially against competing phages. Biochemical characterization, cryo-EM-based structural analysis of the Cas12p-TrxA-sgRNA-dsDNA complex at 2.67 Å and bacterial defence assays reveal that TrxA directly binds and activates Cas12p, enabling its nuclease activity and subsequent CRISPR immunity. These findings expand our understanding of the multilayered intricacies of phage-bacteria molecular interactions.},
}

*Thioredoxins/metabolism/genetics
*CRISPR-Cas Systems
*Bacteriophages/genetics/enzymology/metabolism
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
Cryoelectron Microscopy
Protein Binding
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics
DNA Cleavage
DNA/metabolism
Escherichia coli/genetics/virology/metabolism

@article {pmid41494531,
year = {2026},
author = {Wan, W and Ji, X and Song, H and Zhang, Z and Kwok, CK and Fang, X and Li, X},
title = {Fluorogenic CRISPR for DNA imaging in live mammalian cells.},
journal = {Cell chemical biology},
volume = {33},
number = {1},
pages = {33-44},
doi = {10.1016/j.chembiol.2025.10.013},
pmid = {41494531},
issn = {2451-9448},
mesh = {Humans ; *DNA/genetics/analysis/metabolism ; *Fluorescent Dyes/chemistry ; Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Optical Imaging ; },
abstract = {Spatiotemporal imaging of genomic DNA dynamics in live mammalian cells is essential for elucidating eukaryotic organization and processes relevant to health and disease. CRISPR systems greatly facilitate the development of live cell DNA imaging tools. However, conventional CRISPR imaging tools typically utilize constitutively fluorescent proteins, resulting in high background noise, nonspecific nucleolar signals, and low signal-to-noise ratios. To address this, fluorogenic CRISPR-based imaging tools have been developed. These tools remain non-fluorescent until they bind to the target DNA, thus significantly reducing the background and enhancing the sensitivity. This review summarizes four fluorogenic CRISPR strategies, each utilizing different fluorogenic reporters, including fluorogenic proteins, fluorogenic RNA aptamers, split fluorescent proteins, and molecular beacons. These fluorogenic CRISPR approaches successfully monitored the subnucleus gene loci localization, dynamics, and DNA breaks and repairs. We anticipate that this review can inspire researchers to expand the fluorogenic CRISPR for cellular DNA imaging and diverse bioapplications.},
}

Humans
*DNA/genetics/analysis/metabolism
*Fluorescent Dyes/chemistry
Animals
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
*Optical Imaging

@article {pmid41494573,
year = {2026},
author = {Pu, Z and Wang, X and Chen, Y and Li, J and He, X and Chen, W and Zhao, C},
title = {Application of CRISPR/Cas9 gene editing system in microalgal metabolic engineering and synthetic strategies of functional food ingredients.},
journal = {Biotechnology advances},
volume = {87},
number = {},
pages = {108796},
doi = {10.1016/j.biotechadv.2026.108796},
pmid = {41494573},
issn = {1873-1899},
mesh = {*Microalgae/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Metabolic Engineering/methods ; *Functional Food ; *Food Ingredients ; },
abstract = {Microalgae are natural and sustainable biological resources rich in high-value nutrients such as lipids, proteins, and functional pigments, which show great potential in the fields of functional foods, dietary supplements, and natural colorants. However, the yields of target components in natural microalgae are often insufficient to meet commercialization demands. The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing system, a revolutionary technology, provides a precise and effective means for targeted improvement of microalgae to enhance their nutritional value and yields. This review first outlines the basic principles of the CRISPR/Cas9 system, including its core components and gene editing mechanism. It then summarizes the application of this technology in microalgae, focusing on successful cases of modifying metabolic pathways to enrich specific nutrients, such as increasing the unsaturated fatty acid content of lipids, increasing the proportion of edible proteins, and enriching natural pigments with antioxidant properties. In addition, this review discusses the main challenges faced when applying this technology to microalgae, including delivery difficulties due to strong cell walls, low efficiency of genetic transformation, and the risk of off-target effects. Finally, the paper describes cutting-edge strategies to address these challenges, such as the development of high-fidelity Cas9 enzymes and the optimization of a single-guide RNA (sgRNA) design. Continued advances in these technologies are propelling microalgae into efficient and sustainable "cell factories", providing the food industry with more natural, healthy, and high-value functional ingredients.},
}

*Microalgae/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Metabolic Engineering/methods
*Functional Food
*Food Ingredients

@article {pmid41494604,
year = {2026},
author = {Zeng, Y and Zhao, G and Wu, S and Hu, B and Forn-Cuní, G and Knol, R and El Ghalbzouri, A and Snaar-Jagalska, E and Kros, A},
title = {CD44-targeted lipid nanoparticles for enhanced CRISPR/Cas9 delivery in cancer gene editing.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {391},
number = {},
pages = {114598},
doi = {10.1016/j.jconrel.2025.114598},
pmid = {41494604},
issn = {1873-4995},
mesh = {*Nanoparticles/administration & dosage/chemistry ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; *Hyaluronan Receptors/metabolism/genetics ; Humans ; *Lipids/chemistry/administration & dosage ; Cell Line, Tumor ; *Melanoma/genetics/therapy/pathology ; Polo-Like Kinase 1 ; *Skin Neoplasms/genetics/therapy/pathology ; Protein Serine-Threonine Kinases/genetics ; RNA, Guide, CRISPR-Cas Systems/administration & dosage/genetics ; Mice ; Cell Cycle Proteins/genetics ; Peptides/chemistry ; Gene Transfer Techniques ; Female ; Liposomes ; },
abstract = {Skin cancer is the third most common malignancy, with melanoma being the most challenging due to its resistance to current therapies. Gene editing technologies like CRISPR/Cas9 offer a promising strategy for targeting cancer-specific genes, but the efficient delivery of these tools to tumor sites remains a significant challenge. Lipid nanoparticles (LNPs) have emerged as the leading platform for gene editing tools due to their ability to protect and transport large payloads. To enhance the precision of gene editing in melanoma, we developed CD44-specific peptide-modified LNPs for targeted delivery of CRISPR/Cas9 mRNA and guide RNA against polo-like kinase 1 (sgPLK1). Our approach led to enhanced targeting and gene editing efficacy by specifically delivering CRISPR/Cas9 and sgPLK1 to melanoma tumor cells, resulting in significant inhibition of tumor growth in both in vitro and in vivo skin melanoma models. Moreover, this platform showed the capacity to reach metastatic melanoma in the brain and resulting in substantial suppression of tumor growth in brain metastasis models. We envision that this peptide-modification strategy could be further employed to improve the targeting capabilities and therapeutic outcomes of LNPs for CRISPR/Cas9-based gene editing, paving the way for more precise and effective cancer treatments.},
}

*Nanoparticles/administration & dosage/chemistry
*CRISPR-Cas Systems
*Gene Editing/methods
Animals
*Hyaluronan Receptors/metabolism/genetics
Humans
*Lipids/chemistry/administration & dosage
Cell Line, Tumor
*Melanoma/genetics/therapy/pathology
Polo-Like Kinase 1
*Skin Neoplasms/genetics/therapy/pathology
Protein Serine-Threonine Kinases/genetics
RNA, Guide, CRISPR-Cas Systems/administration & dosage/genetics
Mice
Cell Cycle Proteins/genetics
Peptides/chemistry
Gene Transfer Techniques
Female
Liposomes

@article {pmid41494883,
year = {2026},
author = {Mahmood, MA and Greenwood, JR and Millar, AA and Susila, H},
title = {Next-generation genome editing: no transgene, no tissue culture.},
journal = {Trends in plant science},
volume = {31},
number = {4},
pages = {383-385},
doi = {10.1016/j.tplants.2025.12.008},
pmid = {41494883},
issn = {1878-4372},
mesh = {*Transgenes/genetics ; *Genome, Plant/genetics ; Plants, Genetically Modified/genetics ; *Gene Editing/methods ; *Genetic Engineering/methods ; Tissue Culture Techniques ; CRISPR-Cas Systems ; },
abstract = {New approaches to engineering plant genomes have the potential to improve agriculture. However, transgenes insertion and tissue culture have become bottlenecks to genome-editing technology becoming widely adopted and achieving the promise of targeted editing. Recent developments in particle bombardment and viral vector-mediated delivery can open doors to overcome these limitations.},
}

*Transgenes/genetics
*Genome, Plant/genetics
Plants, Genetically Modified/genetics
*Gene Editing/methods
*Genetic Engineering/methods
Tissue Culture Techniques
CRISPR-Cas Systems

@article {pmid41494892,
year = {2026},
author = {Wang, MR and Mu, W and Zhen, A and Kitchen, SG},
title = {CRISPR/Cas strategies to enhance CAR T-cell function and persistence via metabolic reprogramming.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.12.001},
pmid = {41494892},
issn = {1879-3096},
abstract = {While chimeric antigen receptor (CAR) T-cell therapy has become a standard of care in various blood cancers, its full curative potential for other diseases has yet to be maximized. One key limiting factor is progressive T-cell exhaustion and differentiation over time, leading to the loss of the CAR-expressing cells. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein) gene manipulation to enhance CAR T-cell therapy has revolutionized the field in recent years. In this review, we will examine the application of CRISPR/Cas aimed at improving CAR T-cell function and persistence to combat the issues of exhaustion and dysfunction, with a focus on metabolic reprogramming. Understanding current preclinical CRISPR/Cas strategies for modulating CAR T-cell metabolism is critical in advancing CAR-T therapies to clinical applications.},
}

@article {pmid41495049,
year = {2026},
author = {Li, X and Shang, X and Liu, J and Zhang, Y and Jia, X and Li, H and Wang, Y and Gao, J and Ma, X and Zhang, X and Rong, X and Gan, W and Zhang, Y and Chen, J and Wang, L and Bao, Z and He, L and Yan, X and Liu, Y and Shao, J and Xiao, Z and Wang, Z and Zhu, H and Wang, Z and Wu, Y and Huang, Y},
title = {Intrathecal CRISPR-edited allogeneic IL-13Rα2 CAR T Cells for recurrent high-grade Glioma: preclinical characterization and phase I trial.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1362},
pmid = {41495049},
issn = {2041-1723},
mesh = {Humans ; Female ; Male ; *Immunotherapy, Adoptive/methods/adverse effects ; *Interleukin-13 Receptor alpha2 Subunit/genetics/immunology ; Middle Aged ; Adult ; *Glioma/therapy/immunology/pathology ; CRISPR-Cas Systems/genetics ; *Receptors, Chimeric Antigen/genetics/immunology ; *T-Lymphocytes/immunology/transplantation/metabolism ; *Brain Neoplasms/therapy/immunology/pathology ; Gene Editing ; Neoplasm Recurrence, Local/therapy ; Animals ; Graft vs Host Disease/prevention & control/immunology ; Glioblastoma/therapy/immunology ; Mice ; Killer Cells, Natural/immunology ; Aged ; Treatment Outcome ; Cell Line, Tumor ; },
abstract = {Patients with recurrent high-grade glioblastoma have a median survival of 6-8 months, with limited therapeutic options. In recent years, interest has grown in applying chimeric antigen receptor T (CAR-T) cells to solid cancers, including advanced gliomas. Here we generated off-the-shelf CRISPR-Cas9-edited IL-13Rα2-specific allogeneic universal CAR-T cells (MT026) by disrupting the endogenous TCR to prevent graft-versus-host disease and knocking out HLA class I molecules to mitigate the host-versus-graft response, and observed minimal NK-cell-mediated rejection in preclinical studies. In a first-in-human, single-center, open-label investigator-initiated trial (ChiCTR2000028801) in patients with high-grade glioma with prior therapy failure and short life expectancy, intrathecal injection of MT026 via lumbar puncture (1.0-3.0×10^7 cells per dose) demonstrated favorable tolerability and safety (primary outcome), pharmacokinetic characteristics, and preliminary clinical activity (secondary outcomes). Among the five patients enrolled, one achieved a complete response and three achieved partial responses. No grade ≥3 adverse events were observed; the predominant treatment-related toxicities were grade 1-2 pyrexia, hypoxia, and vomiting. Trial enrolment was halted after enrolment of the first five patients, however these preliminary clinical data support the potential benefit of locally administered allogeneic universal CAR-T cell therapy for recurrent glioblastoma.},
}

Humans
Female
Male
*Immunotherapy, Adoptive/methods/adverse effects
*Interleukin-13 Receptor alpha2 Subunit/genetics/immunology
Middle Aged
Adult
*Glioma/therapy/immunology/pathology
CRISPR-Cas Systems/genetics
*Receptors, Chimeric Antigen/genetics/immunology
*T-Lymphocytes/immunology/transplantation/metabolism
*Brain Neoplasms/therapy/immunology/pathology
Gene Editing
Neoplasm Recurrence, Local/therapy
Animals
Graft vs Host Disease/prevention & control/immunology
Glioblastoma/therapy/immunology
Mice
Killer Cells, Natural/immunology
Aged
Treatment Outcome
Cell Line, Tumor

@article {pmid41495894,
year = {2026},
author = {Ishihara, K and Matsumoto, S and Gerle, C and Gopalasingam, CC and Shigematsu, H and Shirai, T and Numata, T},
title = {Sequential structural rearrangements at the PAM-distal site of a type I-F3 CRISPR-Cas effector enabling RNA-guided DNA transposition.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41495894},
issn = {1362-4962},
support = {20H02916//Japan Society for the Promotion of Science/ ; 24H00505//Japan Society for the Promotion of Science/ ; //Japan Foundation for Applied Enzymology/ ; //Naito Foundation/ ; //Institute for Fermentation, Osaka/ ; //Noda Institute for Scientific Research/ ; 23KJ1734//JSPS/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; *DNA Transposable Elements/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism ; *Bacterial Proteins/chemistry/genetics/metabolism ; Vibrio parahaemolyticus/genetics ; DNA/chemistry/metabolism/genetics ; Cryoelectron Microscopy ; Models, Molecular ; },
abstract = {Some prokaryotes carry CRISPR-associated transposons (CASTs), Tn7-like elements that incorporate genes encoding CRISPR-Cas effectors. CAST insertion is directed by CRISPR-Cas effectors through RNA-guided DNA binding and interactions with transposition-associated proteins. Although efficient sequence-specific DNA integration requires both precise target DNA recognition and coordinated interactions between effectors and transposition-associated proteins, the underlying mechanism remains elusive. Here, we determined three cryo-EM structures of target DNA-bound type I-F3 TniQ-Cascade from Vibrio parahaemolyticus, revealing how Cas8/5 recognizes the protospacer adjacent motif (PAM) and identifying a key residue responsible for the cytidine preference at position -2 of the PAM. We revealed mismatch tolerance at the PAM-proximal site. Structural analyses showed that correct base pairing at the PAM-distal site correlates with conformational changes in the Cas8/5 helical bundle and TniQ, bending the DNA to guide its downstream region toward the transposition machinery. Together, these dynamic rearrangements at the PAM-distal region provide insights into the licensing mechanism of type I-F3 CAST transposition and highlight its potential for genome engineering applications.},
}

*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/chemistry/genetics/metabolism
*DNA Transposable Elements/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism
*Bacterial Proteins/chemistry/genetics/metabolism
Vibrio parahaemolyticus/genetics
DNA/chemistry/metabolism/genetics
Cryoelectron Microscopy
Models, Molecular

@article {pmid41495909,
year = {2026},
author = {Derollez, E and Roson-Calero, N and Rouzé, P and Dedieu-Berne, A and Ballesté-Delpierre, C and Fraikin, N and Iorga, BI and Huang, TD and Bigot, S and Vila, J and Bogaerts, P and Lesterlin, C},
title = {Specific killing and resensitization of pathogenic Escherichia coli strains carrying blaCTX-M-15 β-lactamase using targeted-antibacterial-plasmids (TAPs).},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41495909},
issn = {1362-4962},
support = {//Joint Programming Initiative on Antimicrobial Resistance/ ; JPIAMR2021-194//JPIAMR/ ; FRM-EQU202103012587//Foundation for Medical Research/ ; ANR-20-PAMR-0010//French Priority Research Program/ ; //Agence Nationale de la Recherches/ ; },
mesh = {*beta-Lactamases/genetics/metabolism ; *Escherichia coli/genetics/drug effects/enzymology ; *Plasmids/genetics ; CRISPR-Cas Systems ; Humans ; Anti-Bacterial Agents/pharmacology ; Conjugation, Genetic ; Drug Resistance, Multiple, Bacterial/genetics ; Escherichia coli Infections/microbiology ; },
abstract = {Targeted-Antibacterial-Plasmids (TAPs) offer a precise approach to combat multidrug-resistant bacteria by selectively removing resistant strains while preserving commensals. Here, we assess TAPs that deliver CRISPR/Cas systems via conjugation to kill or resensitize extended-spectrum β-lactamase (ESBL)-producing Escherichia coli carrying the blaCTX-M-15 gene. We systematically tested multiple variables in the TAP approach, including two commensal E. coli donors, three distinct helper plasmids encoding the transfer machineries, and six recipient strains harbouring the resistance gene either chromosomally or on a plasmid. Cas9-based TAPs induced double-stranded breaks in chromosomal blaCTX-M-15 genes, resulting in immediate bacterial death. When the target gene was plasmid-borne, Cas9 cleavage triggered plasmid loss and partial toxin-antitoxin-mediated killing. In contrast, dCas9-based TAPs inhibited blaCTX-M-15 expression without affecting cell viability, thereby restoring third-generation cephalosporin susceptibility. In mixed-culture experiments, TAPs specifically eliminated only blaCTX-M-15-carrying E. coli while sparing other non-targeted bacterial species. Conjugation assays in human faeces demonstrated substantial suppression of cefotaxime-resistant (CtxR) E. coli by both Cas9- and dCas9-based TAPs, underscoring their efficacy in complex microbial environments. These findings highlight TAPs' decolonization promise, paving the way for future microbiome-editing interventions against multidrug-resistant carriage or infection.},
}

*beta-Lactamases/genetics/metabolism
*Escherichia coli/genetics/drug effects/enzymology
*Plasmids/genetics
CRISPR-Cas Systems
Humans
Anti-Bacterial Agents/pharmacology
Conjugation, Genetic
Drug Resistance, Multiple, Bacterial/genetics
Escherichia coli Infections/microbiology

@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 {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 {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 {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 {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 {pmid41496452,
year = {2026},
author = {Liu, J and Liu, Z and Qin, D and Qi, X and Chen, M and Cheng, D and Guo, S and Zhang, X and Zhang, L and Yan, T and Li, F and He, W and Lu, Z and Wang, H and Li, J and Yang, X and Shi, Y and Zhang, S and Zhao, H and Liu, C and Chen, S and Zhong, Y},
title = {An all-in-one visual selection system for male-sterile line production in maize and rice.},
journal = {Plant communications},
volume = {7},
number = {2},
pages = {101693},
pmid = {41496452},
issn = {2590-3462},
mesh = {*Zea mays/genetics/physiology ; *Oryza/genetics/physiology ; *Plant Infertility/genetics ; Seeds/genetics/growth & development ; *Plant Breeding/methods ; CRISPR-Cas Systems ; Hybrid Vigor/genetics ; Plants, Genetically Modified ; },
abstract = {Heterosis has significantly improved crop yields, yet hybrid seed production remains hindered by labor-intensive manual emasculation. Although current male-sterility systems, such as cytoplasmic male sterility and environment-sensitive genic male sterility, have improved the efficiency of hybrid seed production, their limited genetic adaptability and high environmental dependence remain major challenges. Here, we report an all-in-one seed production technology (ASPT) that integrates CRISPR-Cas9, RUBY, and key seed production technology (SPT) components into a single vector, enabling efficient generation and propagation of male-sterile lines in both maize and rice. The engineered RUBY marker enables visual identification of male-sterile and maintainer lines, with an accuracy of 99.81% in automated seed sorting and 100% in secondary field screening. Notably, ASPT was successfully introduced into 21 genetically diverse elite maize inbred lines, demonstrating broad compatibility. ASPT enables scalable and precise propagation of male-sterile lines in both maize and rice, providing a broadly applicable strategy to advance hybrid seed production in crops.},
}

*Zea mays/genetics/physiology
*Oryza/genetics/physiology
*Plant Infertility/genetics
Seeds/genetics/growth & development
*Plant Breeding/methods
CRISPR-Cas Systems
Hybrid Vigor/genetics
Plants, Genetically Modified

@article {pmid41496629,
year = {2026},
author = {Li, J and Wang, L and Yang, S and Zhou, X and Gou, Q and Cai, J and Yang, H and Wang, Q and Li, S},
title = {Chicken Shank Color Determined by Inhibition of Dermal Melanin (ID) is Mediated by a Structural Variation Regulating CDKN2A Expression.},
journal = {Pigment cell & melanoma research},
volume = {39},
number = {1},
pages = {e70072},
doi = {10.1111/pcmr.70072},
pmid = {41496629},
issn = {1755-148X},
support = {2022hszd006//Major Project of Hubei Hongshan Laboratory/ ; 31772585//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Chickens/genetics ; *Melanins/metabolism ; *Cyclin-Dependent Kinase Inhibitor p16/genetics/metabolism ; *Skin Pigmentation/genetics ; Melanocytes/metabolism ; *Gene Expression Regulation ; Polymorphism, Single Nucleotide/genetics ; Female ; Male ; CRISPR-Cas Systems/genetics ; Genome-Wide Association Study ; *Skin/metabolism ; },
abstract = {Shank color in chickens is a classic quantitative trait governed by four genetic loci. Among these, the Inhibition of dermal melanin (ID) locus, which suppresses dermal melanogenesis in the shank, is the sole sex-linked mutation and its molecular mechanisms remain elusive. To identify the causal mutation, we established a resource population segregating for shank colors. A genome-wide association study utilizing FarmCPU software identified a top-associated SNP on the Z chromosome. Linkage mapping subsequently narrowed the candidate region, within which we discovered a candidate structural variant associated with the yellow shank phenotype. This variant is characterized by a 143 bp deletion coupled with a 2 bp insertion. CDKN2A was the only gene within the same topologically associating domain to exhibit differential expression. Functional validation via CRISPR/Cas9-edited cells demonstrated that this mutation regulates CDKN2A transcription and is responsible for the ID shank color in chickens. We propose that the resulting absence of melanocytes is likely due to apoptosis. This work resolves the molecular basis of the ID locus, thereby completing the genetic puzzle of chicken shank color. This discovery enables the development of molecular markers for auto-sexing of day-old chicks, a tool with significant potential for the poultry industry.},
}

Animals
*Chickens/genetics
*Melanins/metabolism
*Cyclin-Dependent Kinase Inhibitor p16/genetics/metabolism
*Skin Pigmentation/genetics
Melanocytes/metabolism
*Gene Expression Regulation
Polymorphism, Single Nucleotide/genetics
Female
Male
CRISPR-Cas Systems/genetics
Genome-Wide Association Study
*Skin/metabolism

@article {pmid41496894,
year = {2026},
author = {Kantor, B and Duke, L and Bhide, PG},
title = {CRISPR-Cas editing technologies for viral-mediated gene therapies of human diseases: Mechanisms, progress, and challenges.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {1},
pages = {102786},
pmid = {41496894},
issn = {2162-2531},
abstract = {The gene therapy landscape has evolved substantially in recent years, beginning with the approval of the first adeno-associated virus-based gene therapy, Luxterna, in 2017. Since then, the US FDA has approved nearly 30 new viral gene therapy programs, with notable examples including Zolgensma, Spinraza, Hemgenix, Zynteglo, Lyfgenia, Kymriah, Skysona, and Tecelra. Remarkably, all these products rely on delivery via adeno-associated vectors (AAVs) and lentiviral vectors (LVs). Improvements in viral-mediated gene transfer efficiency and clinical-scale manufacturing, together with immense commercial interest, have greatly propelled the clinical adoption of gene therapy products. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR) and its related Cas proteins (CRISPR-Cas) have made significant advances in gene therapy, offering next-generation approaches for curative gene editing to treat genetic diseases and disorders. In this review, we examine the range of these therapeutics and their viral carriers, focusing primarily on LVs and AAVs. We provide a snapshot of the current status of the field and highlight some of the current challenges in the clinical application of gene therapy, with particular emphasis on viral CRISPR-Cas-based technologies and their future potential.},
}

@article {pmid41496934,
year = {2026},
author = {Khizar, M and Aminpoor, H and Zaib, M and Ali, Q and Karimi, H},
title = {Nanoparticle-enhanced CRISPR delivery: paving the path for in vivo tumor gene editing.},
journal = {Annals of medicine and surgery (2012)},
volume = {88},
number = {1},
pages = {1054-1055},
pmid = {41496934},
issn = {2049-0801},
abstract = {Nanoparticle-based delivery systems are redefining how CRISPR/Cas technology can be used in cancer treatment. By encapsulating CRISPR components within lipid, polymeric, or inorganic nanoparticles, researchers have improved their stability, circulation time, and tumor-targeting precision. The NTLA-2001 trial demonstrated the first successful use of lipid nanoparticles for in vivo CRISPR delivery in humans, paving the way for potential applications in oncology. Preclinical studies have shown promising results, with efficient gene knockout and tumor suppression across multiple models. Despite these advances, barriers remain, including limited delivery to solid tumors, potential off-target effects, and inconsistent nanoparticle formulations. Global research efforts spanning the United States, China, Europe, and India are now focused on refining delivery platforms and standardizing protocols. This letter highlights current progress, ongoing challenges, and the need for transparent, globally coordinated development. Nanoparticle-enhanced CRISPR delivery has the potential to bring genetic precision therapy from the laboratory to the clinic, offering a new avenue for durable and accessible cancer care.},
}

@article {pmid41497644,
year = {2025},
author = {Stringer, AM and Wade, JT},
title = {Identification of PAM Requirements for the Vibrio cholerae type I-E CRISPR-Cas System.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {41497644},
issn = {2692-8205},
support = {R01 GM122836/GM/NIGMS NIH HHS/United States ; R35 GM144328/GM/NIGMS NIH HHS/United States ; },
abstract = {CRISPR-Cas systems are prokaryotic adaptive immune systems that use RNA-guided protein complexes to target invading nucleic acid. A surveillance complex consisting of protein and a CRISPR-RNA (crRNA) binds target nucleic acid via base-pairing interactions, typically leading to processing of the target nucleic acid by a nuclease. CRISPR-Cas systems are classified based on their mechanism of action, with type I systems being the most prevalent in nature. Type I CRISPR-Cas systems target DNA, and require extensive complementarity between the crRNA and the target DNA. Moreover, type I systems require the presence of a "Protospacer Adjacent Motif" (PAM) sequence in the target DNA immediately adjacent to the expected region of base-pairing with the crRNA. Classical biotypes of the bacterial pathogen Vibrio cholerae have active type I-E CRISPR-Cas systems. While the optimal PAM sequence for this CRISPR-Cas system is known to be AAY, the activity of other sequences as possible PAMs has not been determined. Here, we quantify the effectiveness of all possible trinucleotide sequences in the PAM position for the V. cholerae type I-E CRISPR-Cas system. Our data indicate a hierarchy of PAM efficacy, with 15 of the 64 trinucleotide sequences functioning as a PAM.},
}

@article {pmid41497669,
year = {2025},
author = {Kumbara, A and Tognon, M and Carone, G and Fontanesi, A and Bombieri, N and Giugno, R and Pinello, L},
title = {CRISPR-HAWK: Haplotype- and Variant-aware guide design toolkit for CRISPR-Cas.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {41497669},
issn = {2692-8205},
support = {R01 HG013618/HG/NHGRI NIH HHS/United States ; UM1 HG012010/HG/NHGRI NIH HHS/United States ; },
abstract = {MOTIVATION: Current CRISPR guide RNA design tools rely on reference genomes, overlooking how genetic variation impacts editing outcomes. As genome editing advances toward clinical applications, incorporating population diversity becomes essential for ensuring therapeutic efficacy across diverse populations.

RESULTS: We present CRISPR-HAWK, a framework integrating individual- and population-scale variants and haplotypes into gRNA design. Analyzing therapeutic targets across 79,648 genomes reveals that genetic variants substantially alter guide performance. For the clinically approved sickle cell disease therapeutic guide targeting BCL11A, we identify haplotypes that completely abolish predicted cutting activity. Across seven therapeutic loci, 82.5% of guides contain variants modifying on-target activity. Variants also create novel protospacer adjacent motif sites generating individual-specific guides invisible to reference-based design. These findings demonstrate that variant-aware selection is critical for equitable genome editing.},
}

@article {pmid41498549,
year = {2026},
author = {Gopalaswamy, R and Subbian, S},
title = {The power of resistance: mechanisms of antimicrobial resistance in Mycobacterium tuberculosis and its impact on tuberculosis management.},
journal = {Clinical microbiology reviews},
volume = {39},
number = {1},
pages = {e0019425},
pmid = {41498549},
issn = {1098-6618},
mesh = {*Mycobacterium tuberculosis/drug effects/genetics ; Humans ; *Antitubercular Agents/pharmacology/therapeutic use ; *Tuberculosis, Multidrug-Resistant/drug therapy/microbiology/diagnosis ; *Drug Resistance, Bacterial ; *Tuberculosis/drug therapy/microbiology ; },
abstract = {SUMMARYThe global resurgence of drug-resistant tuberculosis (DR-TB) presents a formidable challenge to public health, driven by a complex interplay of mycobacterial evolution, dynamics and outcomes of host-pathogen interactions and systemic gaps in diagnosis and treatment strategies. This comprehensive review delineates the multifactorial basis of antimicrobial resistance (AMR) in Mycobacterium tuberculosis (Mtb), integrating molecular, immunological, and pharmacological perspectives to inform next-generation strategies for effective TB control. We reconceptualize TB as a dynamic clinical spectrum-ranging from asymptomatic infection to overt disease-shaped by granuloma biology and bacterial adaptation. This spectrum underpins both diagnostic ambiguity and therapeutic failure, particularly in the context of phenotypic drug tolerance/resistance to current anti-TB drugs. We discuss Mtb's intrinsic and extrinsic resistance mechanisms, including the lipid-rich cell envelope, efflux systems, and enzymatic drug modification, which are compounded by acquired mutations that disrupt drug activation, alter targets, and confer cross-resistance. These adaptations are further potentiated by granuloma-induced pharmacokinetic heterogeneity and host-induced metabolic quiescence. We highlight the emerging role of therapeutic drug monitoring and pharmacokinetic/pharmacodynamic modeling in optimizing individualized therapy, particularly for novel regimens incorporating bedaquiline, pretomanid, and linezolid. Moreover, we underscore the diagnostic limitations in detecting heteroresistance and early-stage disease, advocating for expanded deployment of advanced and targeted molecular diagnostic modalities. Finally, we propose a paradigm shift toward integrated, precision-based TB management, leveraging host-directed therapies, biofilm-disrupting agents, and real-time pharmacokinetics-guided dosing to preempt resistance emergence and improve clinical outcomes. This review provides a translational framework for addressing the biological and operational complexities of DR-TB in the era of AMR.},
}

*Mycobacterium tuberculosis/drug effects/genetics
Humans
*Antitubercular Agents/pharmacology/therapeutic use
*Tuberculosis, Multidrug-Resistant/drug therapy/microbiology/diagnosis
*Drug Resistance, Bacterial
*Tuberculosis/drug therapy/microbiology

@article {pmid41498761,
year = {2026},
author = {Fan, X and Zhang, R and Yang, L and Chen, S and He, M and Wang, Y and Huang, L and Zeng, J and Wu, S and Gao, M and Wang, H},
title = {CRISPR-Cas9-Loaded Theranostic Liposomes for Enhancing Radiosensitization of Prostate Cancer through POLD4 Gene Editing under Real-Time MRI Monitoring.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {16},
pages = {e19704},
pmid = {41498761},
issn = {2198-3844},
support = {SHDC22011310-A//Shanghai Municipal Hospital Development Center/ ; SHDC22025311-B//Shanghai Municipal Hospital Development Center/ ; 25ZR1401440//Science and Technology Commission of Shanghai Municipality/ ; 82271969//National Natural Science Foundation of China/ ; 202101070002E00085//Major Program of Shanghai Municipal Commission of Education/ ; },
mesh = {Male ; *Prostatic Neoplasms/genetics/radiotherapy/therapy/diagnostic imaging ; *CRISPR-Cas Systems/genetics ; Humans ; *Liposomes ; Animals ; *Magnetic Resonance Imaging/methods ; *Gene Editing/methods ; Mice ; Cell Line, Tumor ; Theranostic Nanomedicine/methods ; Genetic Therapy/methods ; },
abstract = {Radiotherapy is a fundamental treatment for prostate cancer; however, its therapeutic efficacy is frequently limited by radioresistance mediated through DNA repair mechanisms and other biological factors. Although gene therapy holds promise for overcoming such resistance, identifying effective radiosensitization targets and developing efficient gene delivery systems remain practically challenging. In this study, transcriptomic analysis of radiotherapy-treated prostate cancer cells revealed a marked upregulation of DNA polymerase delta subunit 4 (POLD4), a target that has not been systematically studied. To evaluate the potential of POLD4 for overcoming radioresistance, CRISPR-Cas9-based plasmids along with ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) were encapsulated within cationic liposomes for achieving an MRI-trackable gene delivery platform (plasmid and iron oxide co-loaded liposomes, termed PIO@Lipo). Comprehensive in vitro and in vivo studies demonstrated that PIO@Lipo enabled efficient POLD4 knockdown. Furthermore, PIO@Lipo synergized with radiotherapy to induce extensive DNA damage, promote tumor cell apoptosis, and remodel the immunosuppressive microenvironment. Notably, PIO@Lipo displayed superior MRI contrast enhancement capability and passive tumor targeting ability. In conclusion, this study has identified POLD4 as a potent target for radiosensitization, capable of disrupting DNA damage-repair homeostasis through MRI-monitored gene editing, thereby offering a promising strategy to enhance the efficacy of radiotherapy in prostate cancer.},
}

Male
*Prostatic Neoplasms/genetics/radiotherapy/therapy/diagnostic imaging
*CRISPR-Cas Systems/genetics
Humans
*Liposomes
Animals
*Magnetic Resonance Imaging/methods
*Gene Editing/methods
Mice
Cell Line, Tumor
Theranostic Nanomedicine/methods
Genetic Therapy/methods

@article {pmid41498982,
year = {2026},
author = {Mangal, H and Mathur, S and Kumar, S and Chaurasia, A and Ranjan, R},
title = {Role of CRISPR in bioremediation of heavy metal(loid): a breakthrough in environmental biotechnology.},
journal = {World journal of microbiology & biotechnology},
volume = {42},
number = {1},
pages = {34},
pmid = {41498982},
issn = {1573-0972},
mesh = {*Metals, Heavy/metabolism ; *Biodegradation, Environmental ; *CRISPR-Cas Systems ; *Biotechnology/methods ; Gene Editing/methods ; Bacteria/genetics/metabolism ; Biosensing Techniques ; Plants/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {A number of technological advancements have made bioremediation an emerging and innovative technology, including its economic viability, increased competence, and natural environment friendliness. The efficiency, scalability, and specificity of conventional physical, chemical, and biological remediation techniques are still limited, despite their partial success. Recent developments in CRISPR-based genome engineering have made it possible to precisely manipulate metal transporters, detoxification enzymes, and stress-response pathways in microorganisms and plants, opening up new possibilities to improve bioremediation. This review offers a thorough and integrated examination of enzyme engineering, biosensing systems, microbial bioremediation, and CRISPR-enabled phytoremediation. This work is novel because it presents a unified roadmap for next-generation bioremediation technologies by integrating CRISPR editing with multi-omics, synthetic biology, and emerging CRISPR-based biosensors. We also go over ecological risks, current difficulties, legal issues, and potential field deployment scenarios in the future. These revelations collectively demonstrate the revolutionary potential of CRISPR in creating highly effective, sustainable, and scalable remedies for heavy metal pollution.},
}

*Metals, Heavy/metabolism
*Biodegradation, Environmental
*CRISPR-Cas Systems
*Biotechnology/methods
Gene Editing/methods
Bacteria/genetics/metabolism
Biosensing Techniques
Plants/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats

@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 {pmid41499404,
year = {2026},
author = {Wan, X and Cohen, SM and Yu, Y and Hoan Le, H and Park, H and Groaz, A and Moreno, R and Tan, M and Schneider, J and Gronquist, MR and Shinya, R and Schroeder, FC and Sternberg, PW},
title = {O-acyltransferase genes involved in the production of volatile sex pheromones in Caenorhabditis elegans.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {2},
pages = {e2524778123},
pmid = {41499404},
issn = {1091-6490},
support = {R24OD023041//HHS | NIH (NIH)/ ; 00000//Chuck Lorre Research Scholars Program/ ; DGE 1745301//NSF | NSF Graduate Research Fellowship Program (GRFP)/ ; R24 OD023041/OD/NIH HHS/United States ; 00000//Tianqiao and Chrissy Chen Institute for Neuroscience/ ; },
mesh = {Animals ; *Sex Attractants/biosynthesis/metabolism/genetics ; *Caenorhabditis elegans/genetics/metabolism/enzymology ; Male ; *Acyltransferases/genetics/metabolism ; Female ; *Caenorhabditis elegans Proteins/genetics/metabolism ; Gas Chromatography-Mass Spectrometry ; CRISPR-Cas Systems ; Sexual Behavior, Animal ; },
abstract = {Gene family expansions are critical for functional diversification, yet the contributions of paralogs to metabolic pathways are often unclear. In Caenorhabditis, the expanded O-acyltransferase (OAC) family-enzymes that transfer acyl groups to hydroxylated substrates-remains poorly characterized despite having been implicated in lipid metabolism. Using CRISPR-Cas9 mutagenesis, behavioral assays, gas chromatographic-mass spectral (GC-MS) analyses, and metabolomics, we systematically analyzed 59 OAC-family protein-coding genes to define their roles in regulating signaling molecules. We found that four adjacent paralogs (oac-13, oac-16, oac-25, and oac-28) on chromosome I are required for synthesizing volatile sex pheromones-airborne signals critical for male mate-searching. Specifically, oac-13 and oac-16 are necessary for producing both major pheromone components, while the identical tandem paralogs oac-25 and oac-28 regulate the production of the later-eluting component in gas chromatography. Disruption of these genes reduced production of key pheromone components and impaired male attraction. Metabolomics revealed that oac-16 and other OACs also modulate the synthesis and secretion of nonvolatile ascaroside pheromones, indicating dual roles in chemical signaling. This work uncovers functional specialization within an expanded gene family, illustrating how redundancy and divergence enable adaptive evolution of communication systems.},
}

Animals
*Sex Attractants/biosynthesis/metabolism/genetics
*Caenorhabditis elegans/genetics/metabolism/enzymology
Male
*Acyltransferases/genetics/metabolism
Female
*Caenorhabditis elegans Proteins/genetics/metabolism
Gas Chromatography-Mass Spectrometry
CRISPR-Cas Systems
Sexual Behavior, Animal

@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 {pmid41500141,
year = {2026},
author = {Zheng, S and Tie, H and Chai, S and Wang, M and Wang, S and Zeng, YY and Wu, G and Zhang, TG},
title = {Molecular mechanisms and biotechnological advances in herbicide resistance: Insights into the development of herbicide-tolerant crops.},
journal = {Journal of plant physiology},
volume = {317},
number = {},
pages = {154690},
doi = {10.1016/j.jplph.2026.154690},
pmid = {41500141},
issn = {1618-1328},
mesh = {*Herbicide Resistance/genetics ; *Crops, Agricultural/genetics/drug effects ; *Herbicides/pharmacology ; *Biotechnology ; Gene Editing ; Plants, Genetically Modified/genetics ; *Plant Weeds/drug effects/genetics ; CRISPR-Cas Systems ; },
abstract = {Herbicides play a pivotal role in modern agriculture by controlling weed populations and safeguarding crop yields. However, the long-term and extensive use of herbicides has accelerated the evolution of herbicide-resistant weeds, thereby diminishing their efficacy and posing a serious threat to global food security. Recent advances in molecular biology and plant biotechnology have greatly expanded our understanding of herbicide resistance mechanisms and enabled the development of crops with enhanced herbicide resistance. Herbicide resistance genes function primarily by encoding detoxifying enzymes, modifying herbicide target sites, or activating specific metabolic pathways that mitigate herbicidal toxicity. Emerging genetic tools, including transgenic approaches and CRISPR/Cas-mediated genome editing, have further facilitated the precise introduction of resistance traits into major crops. It is noteworthy that this review offers novel insights into the latest CRISPR/Cas applications, including base editing and prime editing for developing novel, non-transgenic herbicide-resistant crops. Furthermore, it provides a systematic overview of advanced strategies for engineering multi-gene stacking traits to combat complex or evolving weed resistance. This review integrates recent progress in elucidating the molecular targets of herbicides and the underlying resistance mechanisms, and highlights the potential of modern biotechnological strategies for engineering herbicide-resistant crops to promote sustainable and environmentally responsible weed management.},
}

*Herbicide Resistance/genetics
*Crops, Agricultural/genetics/drug effects
*Herbicides/pharmacology
*Biotechnology
Gene Editing
Plants, Genetically Modified/genetics
*Plant Weeds/drug effects/genetics
CRISPR-Cas Systems

@article {pmid41500180,
year = {2026},
author = {Sheng, C and Wang, J and Tan, M and Zhang, J and Sun, M and Sun, J and Shao, Y and Tu, J and Zhu, L and Song, X},
title = {Establishment of detection method of chicken infectious anemia virus based on CRISPR/Cas12a system.},
journal = {Research in veterinary science},
volume = {201},
number = {},
pages = {106046},
doi = {10.1016/j.rvsc.2025.106046},
pmid = {41500180},
issn = {1532-2661},
mesh = {*Chicken anemia virus/isolation & purification/genetics ; *CRISPR-Cas Systems ; Animals ; Chickens ; *Poultry Diseases/diagnosis/virology ; *Circoviridae Infections/veterinary/diagnosis/virology ; Sensitivity and Specificity ; },
abstract = {Chicken Infectious Anemia Virus (CIAV) causes chicken infectious anemia, characterized by anemia and immune dysfunction. The rapid dissemination of this virus is generating substantial economic consequences for poultry producers. The CRISPR/Cas12a system is widely used for virus detection through crRNA-guided target recognition and the paracrine activity of Cas12a. To enable rapid and highly sensitive detection of Chicken Infectious Anemia Virus (CIAV), a CRISPR-Cas12a-based fluorescence assay was refined. Through optimization of the CRISPR/Cas12a system and integration of enzymatic recombinase amplification (ERA), the assay achieved a detection limit of 1 copy/μL, demonstrating its significant utility for CIAV diagnostics. In addition, a CRISPR/Cas12a lateral flow assay was developed and optimized, achieving a sensitivity of 10^3 copies/μL for the rapid and visual detection of target analytes. This technique exhibits high specificity for CIAV, showing no cross-reactivity with other chicken viruses. Overall, the system enables rapid CIAV detection with cost-effective equipment, making it suitable for virus monitoring.},
}

*Chicken anemia virus/isolation & purification/genetics
*CRISPR-Cas Systems
Animals
Chickens
*Poultry Diseases/diagnosis/virology
*Circoviridae Infections/veterinary/diagnosis/virology
Sensitivity and Specificity

@article {pmid41501085,
year = {2026},
author = {Singh, P and D'Rozario, R and Chakraborty, B and Meher, S and Raychaudhuri, D and Tannir, AJ and Li, Y and Majumdar, A and Hawkins, J and Xiong, Y and Lorenzi, P and Sharma, P and Akdemir, K and Pilie, P and Jain, AK and Lee, BHL and Goswami, S},
title = {Loss of KDM6A-mediated genomic instability and metabolic reprogramming regulates response to therapeutic perturbations in bladder cancer.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1382},
pmid = {41501085},
issn = {2041-1723},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; S10 RR029552/RR/NCRR NIH HHS/United States ; S10 OD012304/OD/NIH HHS/United States ; R37 CA279192/CA/NCI NIH HHS/United States ; R25 CA265800/CA/NCI NIH HHS/United States ; },
mesh = {*Urinary Bladder Neoplasms/genetics/drug therapy/metabolism ; Animals ; *Histone Demethylases/genetics/metabolism ; Humans ; Mice ; *Genomic Instability/genetics ; Cell Line, Tumor ; Cisplatin/therapeutic use/pharmacology ; Drug Resistance, Neoplasm/genetics ; CRISPR-Cas Systems ; DNA Repair/genetics ; Female ; Mice, Knockout ; Loss of Function Mutation ; Histones/metabolism ; Metabolic Reprogramming ; },
abstract = {Mutations in epigenetic regulators are common in bladder cancer, yet their impact on therapeutic responses remains unclear. Here, we identify that loss-of-function mutations in KDM6A, a histone demethylase altered in about 26% of advanced bladder cancers, are associated with poor survival after cisplatin chemotherapy, whereas they correlate with improved outcomes with anti-PD-1 therapy. Using CRISPR-Cas9-engineered murine and human bladder cancer models, we show that KDM6A deficiency increases formation of extrachromosomal circular DNA carrying chemoresistance loci, promoting cisplatin resistance. In parallel, KDM6A loss impairs DNA repair and rewires tumor metabolism, reducing glycolysis and lactate output. This metabolic shift diminishes histone lactylation in regulatory T cells, suppressing immunoregulatory genes and limiting expansion of PD-1[hi] regulatory T cells. Collectively, our findings establish KDM6A mutation as a key regulator of therapeutic responses, providing a foundation for its use in guiding precision therapy in advanced bladder cancer.},
}

*Urinary Bladder Neoplasms/genetics/drug therapy/metabolism
Animals
*Histone Demethylases/genetics/metabolism
Humans
Mice
*Genomic Instability/genetics
Cell Line, Tumor
Cisplatin/therapeutic use/pharmacology
Drug Resistance, Neoplasm/genetics
CRISPR-Cas Systems
DNA Repair/genetics
Female
Mice, Knockout
Loss of Function Mutation
Histones/metabolism
Metabolic Reprogramming

@article {pmid41501459,
year = {2026},
author = {Dmytrenko, O and Yuan, B and Crosby, KT and Krebel, M and Chen, X and Nowak, JS and Chramiec-Głąbik, A and Filani, B and Gribling-Burrer, AS and van der Toorn, W and von Kleist, M and Achmedov, T and Smyth, RP and Glatt, S and Bravo, JPK and Heinz, DW and Jackson, RN and Beisel, CL},
title = {RNA-triggered Cas12a3 cleaves tRNA tails to execute bacterial immunity.},
journal = {Nature},
volume = {649},
number = {8099},
pages = {1312-1321},
pmid = {41501459},
issn = {1476-4687},
support = {R35 GM138080/GM/NIGMS NIH HHS/United States ; },
mesh = {*Bacteriophages/physiology ; Catalytic Domain ; *CRISPR-Associated Proteins/metabolism/chemistry ; CRISPR-Cas Systems ; Cryoelectron Microscopy ; Models, Molecular ; *RNA Cleavage ; *RNA, Bacterial/metabolism/genetics/chemistry/immunology ; *RNA, Transfer/metabolism/chemistry/ultrastructure/genetics/immunology ; Bacteria/immunology/virology ; },
abstract = {In all domains of life, tRNAs mediate the transfer of genetic information from mRNAs to proteins. As their depletion suppresses translation and, consequently, viral replication, tRNAs represent long-standing and increasingly recognized targets of innate immunity[1-5]. Here we report Cas12a3 effector nucleases from type V CRISPR-Cas adaptive immune systems in bacteria that preferentially cleave tRNAs after recognition of target RNA. Cas12a3 orthologues belong to one of two previously unreported nuclease clades that exhibit RNA-mediated cleavage of non-target RNA, and are distinct from all other known type V systems. Through cell-based and biochemical assays and direct RNA sequencing, we demonstrate that recognition of a complementary target RNA by the CRISPR RNA triggers Cas12a3 to cleave the conserved 5'-CCA-3' tail of diverse tRNAs to drive growth arrest and anti-phage defence. Cryogenic electron microscopy structures further revealed a distinct tRNA-loading domain that positions the tRNA tail in the RuvC active site of the nuclease. By designing synthetic reporters that mimic the tRNA acceptor stem and tail, we expanded the capacity of current CRISPR-based diagnostics for multiplexed RNA detection. Overall, these findings reveal widespread tRNA inactivation as a previously unrecognized CRISPR-based immune strategy that broadens the application space of the existing CRISPR toolbox.},
}

*Bacteriophages/physiology
Catalytic Domain
*CRISPR-Associated Proteins/metabolism/chemistry
CRISPR-Cas Systems
Cryoelectron Microscopy
Models, Molecular
*RNA Cleavage
*RNA, Bacterial/metabolism/genetics/chemistry/immunology
*RNA, Transfer/metabolism/chemistry/ultrastructure/genetics/immunology
Bacteria/immunology/virology

@article {pmid41501880,
year = {2026},
author = {Zhu, M and Wu, Y and Ou, H and Liu, X and Wang, Y and Liu, X and Zou, C and Yang, G and Du, M and Yu, D and Zheng, D and He, L and Zhang, K and Zhang, W and Wang, S and Qin, H and Hao, Q and He, Y and Lin, W and Zhang, Y and Gu, J and Li, M and Qin, W and Cao, Z},
title = {Targeting TRIM25 as a therapeutic strategy to enhance ferroptosis in glioblastoma cells.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {20},
pmid = {41501880},
issn = {1477-3155},
support = {01-SWKJYCJJ17//the National Defense Biotechnology Fund for Outstanding Young Talents/ ; 2025GTKP008//the National Key Laboratory of Oncology/ ; },
mesh = {*Ferroptosis/drug effects ; Humans ; *Glioblastoma/metabolism/pathology/drug therapy ; Animals ; Cell Line, Tumor ; *Tripartite Motif Proteins/metabolism/genetics ; *Ubiquitin-Protein Ligases/metabolism/genetics ; Mice ; *Brain Neoplasms/metabolism/pathology ; CRISPR-Cas Systems ; Gene Editing ; Mitochondria/metabolism ; Nanoparticles/chemistry ; Mice, Nude ; Ubiquitination ; },
abstract = {BACKGROUND: Glioblastoma (GBM) is the most common malignant brain tumor with a dismal prognosis (< 7% 5-year survival) under current first-line treatment. While inducing programmed cell death (PCD) is a promising antitumor strategy, its effectiveness in GBM remains controversial. Ferroptosis emerged as the most enriched PCD process and was highly correlated with GBM malignant progression.

METHODS: We performed a CRISPR-Cas9 loss-of-function screen to identify critical ferroptosis contributors. Mechanistic studies involved assessing mitochondrial function and morphology. Protein interaction and degradation pathways were investigated using immunoprecipitation and ubiquitination assays. We developed a blood-brain-barrier-penetrating genome editing delivery system, Angiopep-2-modified nanoparticles with disulfide bonds (ANPSS), loaded with Cas9/sgRNA complexes.

RESULTS: Voltage-dependent anion channel 2 (VDAC2) was identified as a critical contributor to ferroptosis. VDAC2 overexpression induced mitochondrial dysfunction and characteristic ferroptotic mitochondrial morphology. The E3 ubiquitin ligase TRIM25 was identified as a key suppressor of VDAC2, directly interacting with it and inducing its K48-linked polyubiquitination and subsequent proteasomal degradation. In vivo, the ANPSS(sgTRIM25) system effectively targeted GBM cells, significantly promoted ferroptosis, and inhibited GBM progression.

CONCLUSIONS: Our findings demonstrate that TRIM25 is a critical negative regulator of VDAC2-dependent ferroptosis in GBM. Targeting TRIM25 using the ANPSS(sgTRIM25) genome editing system effectively overcomes ferroptosis resistance and suppresses tumor growth, representing a viable therapeutic approach for GBM.},
}

*Ferroptosis/drug effects
Humans
*Glioblastoma/metabolism/pathology/drug therapy
Animals
Cell Line, Tumor
*Tripartite Motif Proteins/metabolism/genetics
*Ubiquitin-Protein Ligases/metabolism/genetics
Mice
*Brain Neoplasms/metabolism/pathology
CRISPR-Cas Systems
Gene Editing
Mitochondria/metabolism
Nanoparticles/chemistry
Mice, Nude
Ubiquitination

@article {pmid41502163,
year = {2026},
author = {Manojkumar, C and Limbola, M and Paul, S and Thangadurai, K and Rajendran, KV and Roy, A and Mandal, B and Jeena, K and Bedekar, MK},
title = {CRISPR-Cas12a-based lateral flow detection of white spot syndrome virus: a dual-target approach for detection of early and latent infection.},
journal = {Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc},
volume = {38},
number = {2},
pages = {192-204},
pmid = {41502163},
issn = {1943-4936},
mesh = {*White spot syndrome virus 1/isolation & purification/genetics ; Animals ; *CRISPR-Cas Systems ; *Penaeidae/virology ; Sensitivity and Specificity ; Viral Envelope Proteins/genetics ; },
abstract = {White spot syndrome virus (WSSV; family Nimaviridae; taxon species White spot syndrome virus) is a major viral pathogen that poses a significant threat to the global shrimp industry, with early detection being the most effective strategy for disease control. We developed a CRISPR-Cas12a-based dual-target detection assay for WSSV, specifically targeting the VP28 gene (gene product is a major envelope protein) and WSSV366 (a latency-associated gene), optimized using Indian WSSV isolates. Our CRISPR RNAs for both targets had high efficiency, and we evaluated the assay using fluorescence-based and lateral flow strip (LFS) endpoint detection. In fluorescence assays, the Cr-WSSV assay (without recombinase polymerase amplification, RPA) detected WSSV at 3 × 10[5] copies/μL; RPA integration significantly enhanced sensitivity, allowing detection at as low as 20 and 200 copies for VP28 and WSSV366, respectively, with 100% specificity. We developed a CRISPR-based LFS assay with optimized FAM-biotin reporter concentrations of 100 nM and 250 nM, yielding robust and reproducible results for improved field applicability. Performance evaluation confirmed lack of cross-reactivity to other WOAH-listed shrimp pathogens, while maintaining detection limits of 20 and 200 copies of VP28 and WSSV366. Clinical validation further demonstrated that the RPA-Cr-WSSV-LFS assay successfully detected WSSV366 even in VP28-negative samples, underscoring the importance of detecting WSSV366 in latent infections. Our rapid, cost-effective, and highly sensitive CRISPR-Cas-based assay enhances WSSV surveillance and biosecurity in shrimp aquaculture by incorporating structural and latency-associated gene markers, making it a promising alternative to conventional molecular testing.},
}

*White spot syndrome virus 1/isolation & purification/genetics
Animals
*CRISPR-Cas Systems
*Penaeidae/virology
Sensitivity and Specificity
Viral Envelope Proteins/genetics

@article {pmid41503690,
year = {2026},
author = {Ma, N and Zhang, M and Ghonaim, AH and Zhou, P and Wang, C and Zhou, J and Guo, G and Lebbink, RJ and Bosch, BJ and Zhu, H and Li, W and He, Q},
title = {The essential role of heparan sulfate in the entry of PDCoV and other porcine coronaviruses.},
journal = {Virulence},
volume = {17},
number = {1},
pages = {2614154},
pmid = {41503690},
issn = {2150-5608},
mesh = {Animals ; *Heparan Sulfate/metabolism ; Swine ; *Deltacoronavirus/physiology ; *Virus Internalization/drug effects ; Swine Diseases/virology ; Humans ; *Porcine epidemic diarrhea virus/physiology ; *Coronavirus Infections/virology/veterinary ; Cell Line ; CRISPR-Cas Systems ; Receptors, Virus/metabolism ; Transmissible gastroenteritis virus/physiology ; Alphacoronavirus ; },
abstract = {Porcine enteric coronaviruses, including porcine deltacoronavirus (PDCoV), porcine epidemic diarrhea virus (PEDV), swine acute diarrhea syndrome coronavirus (SADS-CoV), and transmissible gastroenteritis coronavirus (TGEV), can cause acute diarrhea, vomiting, dehydration, and high mortality in suckling piglets. Recent studies revealing human PDCoV infections and the potential of SADS-CoV to penetrate human cell lines have heightened apprehensions about the zoonotic transmission risks of these viruses. While heparan sulfate (HS) serves as a receptor in PDCoV binding, the key host genes involved in HS biogenesis and the specific molecular mechanisms underlying this process have not been fully examined. Enzymes involved in HS biosynthesis, including SLC35B2, EXT1, and NDST1, were identified as critical host factors via the use of CRISPR-Cas9 knockout cells. Moreover, inhibition assays using heparin sodium, a competitive HS mimic, demonstrated dose-dependent reductions in PDCoV infection in vitro. Additionally, mitoxantrone, an HS-binding drug, reduced PDCoV infection. Furthermore, HS was confirmed to facilitate the entry of other porcine enteric coronaviruses (SeCoVs), including PEDV, SADS-CoV, and TGEV, underscoring the conserved role of HS in CoV pathogenesis. These insights contribute to the understanding of porcine coronavirus-host interactions and support the development of innovative antiviral interventions.},
}

Animals
*Heparan Sulfate/metabolism
Swine
*Deltacoronavirus/physiology
*Virus Internalization/drug effects
Swine Diseases/virology
Humans
*Porcine epidemic diarrhea virus/physiology
*Coronavirus Infections/virology/veterinary
Cell Line
CRISPR-Cas Systems
Receptors, Virus/metabolism
Transmissible gastroenteritis virus/physiology
Alphacoronavirus

@article {pmid41503845,
year = {2026},
author = {Li, MF and Zubair, A and Wdidi, S and He, S},
title = {Disrupting Viral Persistence: CRISPR/Cas9-Based Strategies for Hepatitis B and C Treatment, and Challenges.},
journal = {Journal of cellular and molecular medicine},
volume = {30},
number = {1},
pages = {e70986},
pmid = {41503845},
issn = {1582-4934},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Hepatitis B virus/genetics ; *Hepatitis B/therapy/virology/genetics ; Gene Editing/methods ; *Hepatitis C/therapy/virology/genetics ; *Hepacivirus/genetics ; Animals ; Antiviral Agents/therapeutic use/pharmacology ; Genome, Viral ; DNA, Circular/genetics ; DNA, Viral/genetics ; Genetic Therapy ; Virus Replication/genetics ; },
abstract = {Hepatitis B and C viruses (HBV and HCV) remain among the leading causes of liver disease worldwide. Current antiviral drugs, such as nucleotide analogues (NAs), can reduce the replication of new HBV and HCV infections but cannot completely eliminate chronic infections. This is primarily because a stable form of viral DNA, known as covalently closed circular DNA (cccDNA), persists in liver cells and continues to sustain the infection. In recent years, the CRISPR/Cas9 gene-editing system has emerged as a powerful tool for precisely cutting and inactivating specific DNA sequences. Due to its efficiency and ease of use, researchers have applied CRISPR/Cas9 in numerous studies to directly target and disrupt the HBV genome, demonstrating promising antiviral effects in both cell cultures and animal models. Targeting multiple sites within the HBV genome has been shown to further enhance its effectiveness, paving the way for potential combination therapies aimed at disabling both cccDNA and HBV and HCV DNA integrated into the host genome. Despite its potential, CRISPR/Cas9 still faces significant challenges before clinical application, most notably the risk of off-target effects-unintended cleavage of non-target DNA sequences-and the difficulty of delivering the system efficiently into liver cells in vivo. Future progress will depend on improving the tool's precision, efficiency, flexibility and delivery methods. In this review, we explore recent advances in designing guide RNAs (gRNAs) for targeting HBV and HCV, as well as the delivery systems used to transport CRISPR/Cas9 into cells. We also discuss the remaining challenges and potential strategies for advancing CRISPR/Cas9 from the laboratory toward a viable clinical cure for HBV and HCV.},
}

*CRISPR-Cas Systems/genetics
Humans
*Hepatitis B virus/genetics
*Hepatitis B/therapy/virology/genetics
Gene Editing/methods
*Hepatitis C/therapy/virology/genetics
*Hepacivirus/genetics
Animals
Antiviral Agents/therapeutic use/pharmacology
Genome, Viral
DNA, Circular/genetics
DNA, Viral/genetics
Genetic Therapy
Virus Replication/genetics

@article {pmid41504765,
year = {2026},
author = {Osei, EK and O'Mahony, AK and O'Hea, R and Moriarty, J and O'Doherty, Á and Wilson, M and Garcia Manzanilla, E and Mahony, J and Kenny, JG},
title = {Genomics of Irish swine-derived Streptococcus suis: population structure, prophages and anti-viral defence mechanisms.},
journal = {Microbial genomics},
volume = {12},
number = {1},
pages = {},
pmid = {41504765},
issn = {2057-5858},
mesh = {Animals ; *Streptococcus suis/genetics/virology/classification/isolation & purification ; Swine/microbiology ; *Prophages/genetics ; Ireland ; Phylogeny ; *Streptococcal Infections/microbiology/veterinary ; Whole Genome Sequencing ; Genomics ; *Swine Diseases/microbiology ; Multilocus Sequence Typing ; Genome, Bacterial ; Genetic Variation ; Serogroup ; },
abstract = {Streptococcus suis is a major pig pathogen with zoonotic potential, posing an occupational risk to farmers and meat handlers. We characterized 110 S. suis strains from diseased pigs in Ireland (2005-2022) using whole-genome sequencing to investigate population structure and phage-host dynamics. We identified 15 distinct serotypes, with serotypes 9 and 2 being the most dominant. In silico multi-locus sequence typing revealed high diversity within the collection, identifying several sequence types (STs), including 26 novel STs. Investigation of strain-level genomic clustering using PopPUNK against global S. suis genomes showed that the Irish isolates were phylogenetically dispersed across the broader global S. suis population rather than clustering in a single clonal group. The majority of Irish isolates fall within the ten established pathogenic lineages, including the highly virulent zoonotic lineage 1. A locally persistent clonal lineage was identified among Irish isolates, showing minimal genetic variation over a decade.Prophage analysis revealed novel viral taxa that were interspersed among known streptococcal phages, rather than clustering distinctly. Restriction-modification systems were the predominant anti-viral defence systems identified across genomes. CRISPR-Cas systems were present in limited strains but showed substantial targeting bias toward full-length prophages, indicating ongoing phage pressure. CRISPR spacers matched non-S. suis streptococcal phages, and phylogenomic analysis revealed that Vansinderenvirus phages clustered with S. suis rather than other Streptococcus thermophilus phages, suggesting evolutionary connections between phage lineages infecting different streptococci.This study presents the first comprehensive genomic characterization of S. suis in Ireland, revealing a diverse population with significant implications for animal and human health.},
}

Animals
*Streptococcus suis/genetics/virology/classification/isolation & purification
Swine/microbiology
*Prophages/genetics
Ireland
Phylogeny
*Streptococcal Infections/microbiology/veterinary
Whole Genome Sequencing
Genomics
*Swine Diseases/microbiology
Multilocus Sequence Typing
Genome, Bacterial
Genetic Variation
Serogroup

@article {pmid41505087,
year = {2026},
author = {Di Pede, AC and Bagheri, N and Belforte, E and Palone, A and Rossetti, M and Porchetta, A},
title = {Triplex DNA clamp regulates Cas12a activation for ssDNA and RNA sensing.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41505087},
issn = {1362-4962},
support = {MFAG 2022-ID. 27151//AIRC/ ; //National Recovery and Resilience Plan/ ; //Italian Ministry of University and Research/ ; D53D23009090001//European Union/ ; 2022FPYZ2N//European Union/ ; PNRR M4C2-Investimento 1.4- CN00000041//Italian Ministry of Ministry of University and Research/ ; //Fondazione Umberto Veronesi/ ; },
mesh = {*DNA, Single-Stranded/metabolism/genetics/chemistry ; *CRISPR-Associated Proteins/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *RNA/metabolism/genetics/chemistry ; *DNA/chemistry/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {We present a molecular strategy that enables the programmable activation of the CRISPR-Cas12a system in response to triplex DNA formation triggered by single-stranded DNA (ssDNA) or RNA inputs. Our triplex-controlled Cas12a assay leverages the high specificity of clamp-like triplex structures to control a toehold-based strand displacement reaction within a rationally designed DNA hairpin (PAM-Switch). Upon displacement and protospacer adjacent motif (PAM) complementation, the Cas12a ribonucleoprotein (RNP) is activated, initiating trans-cleavage and producing a concentration-dependent fluorescent signal. By decoupling target recognition (via triplex formation) from direct hybridization with the Cas12a-crRNA complex, the assay eliminates the need for target-specific crRNAs. This design also allows multiple detection of distinct nucleic acid (NA) targets using a single Cas12a reaction mix. Through the use of triplex-based clamps, the proposed platform achieves enhanced specificity for single-nucleotide variants and supports the detection of both ssDNA and RNA targets across a broad range of lengths (10-20 nucleotides), addressing key limitations in current Cas12a-based diagnostics and opening new avenues for NA sensing.},
}

*DNA, Single-Stranded/metabolism/genetics/chemistry
*CRISPR-Associated Proteins/metabolism/genetics
*CRISPR-Cas Systems/genetics
*RNA/metabolism/genetics/chemistry
*DNA/chemistry/metabolism/genetics
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics

@article {pmid41505088,
year = {2026},
author = {Lee-Yow, YC and Valbuena, RC and Richter, CS and Chang, HY and Engreitz, JM},
title = {Junction-targeting designs limit the application of CRISPR-Cas13d in circular RNA perturbation studies.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41505088},
issn = {1362-4962},
support = {//Lucile Packard Children's Hospital at Stanford University/ ; NNF21SA0072102//Novo Nordisk Foundation/ ; R35 HG011324/HG/NHGRI NIH HHS/United States ; DGE-1656518//GRFP/ ; /HHMI/Howard Hughes Medical Institute/United States ; //Stanford Interdisciplinary Graduate Fellowship/ ; T32 GM007790/GM/NIGMS NIH HHS/United States ; R35HG011324/HG/NHGRI NIH HHS/United States ; 5T32GM007790/GF/NIH HHS/United States ; },
mesh = {RNA, Circular/genetics ; Humans ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *RNA/genetics/metabolism ; K562 Cells ; Gene Knockdown Techniques/methods ; Exons ; },
abstract = {Circular RNAs (circRNAs) are RNA molecules formed through the backsplicing of linear exons. Several thousand have been identified, yet relatively few are functionally characterized due to challenges in distinguishing effects of circular from linear RNA targets. Recently, CRISPR-Cas13 systems have been utilized to directly target unique junctions formed through backsplicing, potentially allowing for selective degradation of circular isoforms. Applying this approach in pooled screens has indeed identified circRNAs proposed to affect viability in several cancer cell lines. However, the design limitations of applying Cas13d to study circRNAs are not fully characterized. Here, we assessed the limitations of Cas13d-mediated circRNA knockdowns by performing essentiality screens on 900 highly expressed circRNAs in K562, an ENCODE tier 1 cell line. We observed consistent off-target knockdown of linear isoforms by certain circRNA-targeting single-guide RNAs (sgRNAs). Re-analysis of existing Cas13d screens in other cell types revealed similar off-target effects. Using machine learning models that predict Cas13d sgRNA efficacy, we further found that most circRNA-targeting sgRNAs are unlikely to induce strong knockdown. After accounting for these design constraints, 0 of 346 circRNAs testable in our screens had detectable effects on proliferation. Our findings highlight key limitations of junction-targeting strategies, with implications for future circRNA perturbation studies.},
}

RNA, Circular/genetics
Humans
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*RNA/genetics/metabolism
K562 Cells
Gene Knockdown Techniques/methods
Exons

@article {pmid41505097,
year = {2026},
author = {Wang, L and Xiang, X and Yin, G and Shu, H and Wu, Y and Chen, H and Na, R and Gijzen, M and Hou, Y and Dong, S},
title = {Effector gene silencing coordinated by histone methylation and small RNAs enhances host adaptation in a plant pathogen.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41505097},
issn = {1362-4962},
support = {32400164//National Natural Science Foundation of China/ ; 32488302//National Natural Science Foundation of China/ ; 25ZR1401177//Natural Science Foundation of Shanghai/ ; //Shanghai Jiao Tong University/ ; },
mesh = {*Histones/metabolism/genetics ; *Gene Silencing ; *Phytophthora/genetics/pathogenicity ; Plant Diseases/microbiology/genetics/parasitology ; Methylation ; *Host-Pathogen Interactions/genetics ; Epigenesis, Genetic ; CRISPR-Cas Systems ; Virulence/genetics ; *RNA, Small Untranslated/genetics/metabolism ; },
abstract = {Pathogen adaptability driven by epigenetic processes remains poorly understood and poses a significant challenge to sustainable disease management. Histone 3 lysine 27 trimethylation (H3K27me3) and small RNA (sRNA)-mediated silencing of avirulence (Avr) genes are two major strategies that pathogens employ to evade recognition by host resistance (R) proteins. Here, we demonstrate that these two epigenetic mechanisms operate in a coordinated manner to silence Avr genes in the oomycete Phytophthora sojae. CRISPR/Cas9-mediated editing of PsSu(z)12, a core component of the Polycomb repressive complex 2 (PRC2), abolished H3K27me3 deposition at Avr1b and Avr3a, leading to transcriptional reactivation and loss of avirulence. Complementation with PsSu(z)12 restored H3K27me3 and silencing at Avr1b, but not at Avr3a. This prompted sRNA profiling at both loci, revealing differential co-enrichment of sRNA and H3K27me3. Integrated analysis of H3K27me3-enriched chromatin immunoprecipitation, RNA and sRNA sequencing data uncovered a strong locus-specific co-silencing pattern, with 11 out of 12 H3K27me3-regulated arginine-X-leucine-arginine effectors also targeted by sRNAs. Notably, epigenetic variation among field isolates indicated regulatory heterogeneity and plasticity in effector control. Together, our findings establish PsSu(z)12 as a central hub coordinating H3K27me3 and sRNA-mediated effector gene silencing, revealing a dual-layered epigenetic mechanism that enables immune evasion and promotes pathogen adaptation.},
}

*Histones/metabolism/genetics
*Gene Silencing
*Phytophthora/genetics/pathogenicity
Plant Diseases/microbiology/genetics/parasitology
Methylation
*Host-Pathogen Interactions/genetics
Epigenesis, Genetic
CRISPR-Cas Systems
Virulence/genetics
*RNA, Small Untranslated/genetics/metabolism

@article {pmid41505099,
year = {2026},
author = {Xu, B and Li, S and Li, Y and Zhao, S and Li, X and Han, J and Wu, D and Li, S and Chen, L and Xie, S and Han, X and Zhao, C},
title = {Enhanced SfaTnpB enables single-base-specific, one-pot nucleic acid detection for high-sensitivity diagnostics.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41505099},
issn = {1362-4962},
support = {2023YFF1001000//National Key Research & Developmental Program of China/ ; SH23YCKY01//Basic Research Project of Yazhouwan National Laboratory/ ; 32202634//Natural Science Foundation of China/ ; //Yazhouwan National Laboratory/ ; },
mesh = {Humans ; Polymorphism, Single Nucleotide ; CRISPR-Cas Systems ; *Transposases/genetics/metabolism ; *Nucleic Acid Amplification Techniques/methods ; Papillomavirus Infections/diagnosis/virology ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR/Cas12-based nucleic acid detection has revolutionized molecular diagnostics but shows limited single-nucleotide specificity, limited high-fidelity subtype discrimination, and limited compatibility with one-pot assays, restricting its broader clinical application. Here, we report a transposon-associated transposase B (TnpB) ortholog, SfaTnpB, with high trans-cleavage activity, robust single-base mismatch discrimination, and broad temperature tolerance. By stepwise engineering of its guide RNA (ωRNA), we developed an enhanced SfaTnpB (enSfaTnpB) system with markedly improved trans-cleavage efficiency. In combination with a TAM-independent split-activator strategy, this system enables precise detection of single-nucleotide polymorphisms. We further developed TOPIC (TnpB-based One-Pot nucleIC acid detection), a one-pot detection platform coupling enSfaTnpB with recombinase-aided amplification (RAA) or loop-mediated isothermal amplification that enables ultrasensitive detection of human papillomavirus (HPV) subtypes 16 and 18 (∼4 copies/μl) and African swine fever virus DNA (∼3 copies/μl). Finally, RAA-TOPIC accurately detected and genotyped 14 high-risk HPV subtypes with high-fidelity subtype discrimination, showing complete concordance with quantitative real-time PCR-based clinical diagnostics. These findings establish TOPIC as a compact, programmable, and scalable molecular detection tool with broad potential for precision diagnostics and point-of-care testing, particularly in resource-limited settings.},
}

Humans
Polymorphism, Single Nucleotide
CRISPR-Cas Systems
*Transposases/genetics/metabolism
*Nucleic Acid Amplification Techniques/methods
Papillomavirus Infections/diagnosis/virology
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41505207,
year = {2026},
author = {Gao, JL and Li, Z and Calderon-Perez, R and Pavek, A and Kim, L and McDermott, DH and Murphy, PM},
title = {Gene therapy via CRISPR/Cas9-mediated Cxcr4 disease allele inactivation reverses leukopenia in WHIM mice.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {5},
pages = {},
pmid = {41505207},
issn = {1558-8238},
mesh = {Animals ; *Receptors, CXCR4/genetics ; Mice ; *CRISPR-Cas Systems ; *Genetic Therapy ; *Leukopenia/therapy/genetics/pathology ; *Warts/therapy/genetics/pathology ; *Alleles ; *Immunologic Deficiency Syndromes/therapy/genetics/pathology ; Primary Immunodeficiency Diseases/therapy/genetics ; Hematopoietic Stem Cell Transplantation ; Humans ; Gene Editing ; },
abstract = {Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is an immunodeficiency caused by autosomal dominant hyperfunctional mutations in chemokine receptor CXCR4 that promote panleukopenia due to BM retention. We previously reported a preclinical gene therapy protocol involving allele-nonspecific Cxcr4 CRISPR/Cas9 inactivation, leveraging the known in vivo dominance of Cxcr4+/o (+, WT; o, inactivated) hematopoietic stem cells (HSCs) for autologous BM engraftment and leukocyte reconstitution over HSCs with other Cxcr4 genotypes. Here, we show that without BM conditioning, this approach is not able to correct leukopenia in WHIM mice. We therefore modified the protocol by adding conditioning with a nongenotoxic CD117-targeted immunotoxin, CD117-antibody-saporin-conjugate. With this change, donor-derived blood cells rapidly reached ~95% chimerism after transplantation, which was stable without adverse events. Mice receiving edited HSCs showed rapid normalization of absolute myeloid cell counts, the key blood subset responsible for WHIM syndrome. In competitive transplants using equal numbers of edited and unedited donor HSCs, over 80% of blood cells originated from the edited population, predominantly with the Cxcr4+/o genotype. These results provide proof of principle that CRISPR/Cas9-mediated inactivation of the Cxcr4 disease allele, combined with nongenotoxic HSC-targeted conditioning, may offer a safe and effective gene therapy strategy generalizable to all WHIM-causing mutations.},
}

Animals
*Receptors, CXCR4/genetics
Mice
*CRISPR-Cas Systems
*Genetic Therapy
*Leukopenia/therapy/genetics/pathology
*Warts/therapy/genetics/pathology
*Alleles
*Immunologic Deficiency Syndromes/therapy/genetics/pathology
Primary Immunodeficiency Diseases/therapy/genetics
Hematopoietic Stem Cell Transplantation
Humans
Gene Editing

@article {pmid41505468,
year = {2026},
author = {Ma, J and Zhang, Y and Li, Z and Liu, L and A, J and Liang, R and Cao, C and Zhou, J and Cheng, P and Li, Y and Li, Z and Ma, L and Jiang, L and A, X},
title = {CRISPR/Cas12a-RCA enables ultrasensitive detection of circulating free DNA for noninvasive diagnosis of echinococcosis.},
journal = {PLoS neglected tropical diseases},
volume = {20},
number = {1},
pages = {e0013069},
pmid = {41505468},
issn = {1935-2735},
mesh = {Humans ; *CRISPR-Cas Systems ; *Echinococcosis/diagnosis/blood/parasitology ; *Echinococcus/genetics/isolation & purification ; *Cell-Free Nucleic Acids/blood/genetics ; Sensitivity and Specificity ; *DNA, Helminth/blood/genetics ; *Molecular Diagnostic Techniques/methods ; Animals ; Male ; Female ; Limit of Detection ; Middle Aged ; High-Throughput Nucleotide Sequencing ; Adult ; },
abstract = {OBJECTIVE: To develop a novel non-invasive CRISPR/Cas12a-RCA assay for the primary screening of human echinococcosis via detection of circulating Echinococcus cell-free DNA (cfDNA) in peripheral blood.

METHODS: Plasma cfDNA from 20 AE patients was analyzed via high-throughput sequencing to identify conserved repetitive Echinococcus fragments.A one-pot RCA system coupled with CRISPR/Cas12a was optimized for Echinococcus-cfDNA detection. The limit of detection (LOD) was determined using serially diluted synthetic standards, while specificity was validated through mismatch probes and cross-reactivity testing. Clinical validation included 50 AE cases, 22 cystic echinococcosis (CE) cases, 43 non-Echinococcus hepatic disease (HD) cases, and 53 healthy controls (CON).

RESULTS: A conserved repetitive 28S rDNA fragment (pan-Echinococcus-28S) was identified as a biomarker. The CRISPR/Cas12a-RCA assay achieved amplification within 30 minutes at 37 °C, with a linear range of 1 aM to 100 pM and an LOD of 1.41 aM. Visual detection limits were 10 aM (UV light) and 1 aM (blue light). The assay demonstrated high sensitivity (87.5%) and specificity (96.9%, AUC = 0.933) in distinguishing Echinococcus infection (AE/CE) from HD and CON.

CONCLUSION: The optimized one-pot CRISPR/Cas12a-RCA system enables rapid and ultrasensitive detection of pan-Echinococcus cfDNA, providing a cost-effective and highly accurate solution for the primary screening of echinococcosis.},
}

Humans
*CRISPR-Cas Systems
*Echinococcosis/diagnosis/blood/parasitology
*Echinococcus/genetics/isolation & purification
*Cell-Free Nucleic Acids/blood/genetics
Sensitivity and Specificity
*DNA, Helminth/blood/genetics
*Molecular Diagnostic Techniques/methods
Animals
Male
Female
Limit of Detection
Middle Aged
High-Throughput Nucleotide Sequencing
Adult

@article {pmid41506042,
year = {2026},
author = {Jiang, H and Yang, J and Qian, C and Li, A and Liu, Y and Zhang, F and Deng, Y and Duan, J and Lv, X},
title = {Logic-gated CRISPR-Cas12a assay with engineered signal amplification for sensitive multiplexed detection of HCC miRNAs.},
journal = {Biosensors & bioelectronics},
volume = {297},
number = {},
pages = {118376},
doi = {10.1016/j.bios.2026.118376},
pmid = {41506042},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/isolation & purification ; Humans ; *Liver Neoplasms/genetics/diagnosis/blood ; *Carcinoma, Hepatocellular/genetics/diagnosis/blood ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Biomarkers, Tumor/genetics ; },
abstract = {Rapid, sensitive, and accurate multi-target analysis is of great significance in biomedical detection. Traditional methods, however, often function as a mere "one-pot" collection of individual assays, ignoring the intrinsic relationships among biomarkers. To address this, we established a novel biosensing platform that integrates circle-to-circle AND logic gate circuit with an engineered CRISPR-Cas system for the early screening of hepatocellular carcinoma. In this design, the logic gate processes multiple miRNA inputs (e.g., miRNA 122 and miRNA 223), and only upon co-recognition, generates a unified DNA output. This output then directly activates a CRISPR-Cas12a system, which has been enhanced by a multi-legged crRNA assembled on a DNA tetrahedra and a cube-based framework probe to enhance the trans-cleavage activity and reaction kinetics. This integration converts complex multi-target recognition into a single, amplified detection signal, minimizing systemic interference. Under optimal conditions, the method achieved detection limits as low as 78.88 fM for miRNA 122 and 65.26 fM for miRNA 223, with serum recovery rates of 89.66 %-108.08 %. Clinical validation using 36 samples showed that excellent correlation with RT-qPCR (all R[2] > 0.98) and areas under the ROC curves of 0.8514 and 0.9244, effectively distinguishing liver cancer patients from healthy individuals. Combining high sensitivity, specificity, and clinical applicability, this strategy provides a universal platform for logic-operated multiple biomarkers analysis. Looking forward, integration with microfluidic systems could enable automated, high-throughput testing, further enhancing its utility in point-of-care diagnostics. This approach holds great promise not only for early hepatocellular carcinoma screening but also, with adaptation of the input logic, for the detection of a broad spectrum of cancers and other diseases.},
}

*MicroRNAs/genetics/isolation & purification
Humans
*Liver Neoplasms/genetics/diagnosis/blood
*Carcinoma, Hepatocellular/genetics/diagnosis/blood
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Limit of Detection
Biomarkers, Tumor/genetics

@article {pmid41506049,
year = {2026},
author = {Yaşar, S and Gehrke, F and Capdeville, N and Puchta, H},
title = {Recent progress in plant genome engineering: from large insertions to chromosome number changes.},
journal = {Current opinion in biotechnology},
volume = {97},
number = {},
pages = {103426},
doi = {10.1016/j.copbio.2025.103426},
pmid = {41506049},
issn = {1879-0429},
mesh = {*Genome, Plant/genetics ; *Genetic Engineering/methods ; *Chromosomes, Plant/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Plants/genetics ; },
abstract = {The adaptation of the CRISPR/Cas system as a biotechnological tool has enabled a wide spectrum of targeted genome modifications. Whereas earlier approaches focused on small sequence changes, recent years have seen a shift toward larger-scale alterations. Advances in homology-directed gene targeting now enable efficient, scar-free kilobase insertions, while combining nuclease-deficient Cas effectors with recombinases or transposases allows the integration of much larger sequences. Prime editing further expands this scope, enabling inversions, replacements, and deletions spanning hundreds of kilobases to several megabases. More recently, genome engineering has reached a new stage with chromosome fission and fusion, demonstrating the feasibility of controlled karyotype restructuring. Together, these advances open new opportunities for crop improvement, from establishing reproductive barriers and mimicking evolutionary processes to trait stacking on Plant Artificial Chromosomes.},
}

*Genome, Plant/genetics
*Genetic Engineering/methods
*Chromosomes, Plant/genetics
CRISPR-Cas Systems/genetics
*Gene Editing
Plants/genetics

@article {pmid41506374,
year = {2026},
author = {Marschhofer, M and Chen, S and Molbay, M and Winkeljann, B and Villano, E and Giancaspro, C and Kourou, A and Berninghausen, O and Rieder, S and Ungewickell, C and Beckmann, R and Popper, B and Torres, AM and Vidal, A and Merkel, OM and Carneiro, SP},
title = {Optimized lipid nanoparticles for pulmonary delivery of CRISPR/Cas9 targeting KRAS G12S in lung cancer.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {391},
number = {},
pages = {114607},
doi = {10.1016/j.jconrel.2026.114607},
pmid = {41506374},
issn = {1873-4995},
mesh = {Animals ; *Lung Neoplasms/genetics/therapy ; *CRISPR-Cas Systems ; Humans ; *Nanoparticles/administration & dosage/chemistry ; *Proto-Oncogene Proteins p21(ras)/genetics ; *Lipids/chemistry/administration & dosage ; Gene Editing ; A549 Cells ; *Carcinoma, Non-Small-Cell Lung/genetics/therapy ; Mice ; *CRISPR-Associated Protein 9/genetics ; Mice, Nude ; RNA, Guide, CRISPR-Cas Systems/administration & dosage/genetics ; Female ; RNA, Messenger/administration & dosage/genetics ; Apoptosis ; Cell Line, Tumor ; Liposomes ; },
abstract = {KRAS G12S mutations in non-small cell lung cancer (NSCLC) remain refractory to current targeted therapies, with few clinical options and frequent resistance. While CRISPR/Cas9 enables mutation-specific gene disruption, its pulmonary application is limited by systemic clearance, hepatic tropism, and airway mucus barriers. Here, we present lipid nanoparticles (LNPs) specifically engineered for pulmonary delivery of Cas9 mRNA and KRAS G12S-targeting sgRNA, optimized through mRNA surrogate screening and orthogonal mixture design to guide lipid composition and Cas9:sgRNA weight-to-weight ratios. Two lead LNP formulations, A6 3:1 and A8 1:1, exhibited robust critical quality attributes, including particle sizes below 120 nm, low polydispersity, near-neutral zeta potential, and over 80 % encapsulation efficiency. Cryo-TEM revealed distinct morphologies correlated with enhanced transfection. In vitro, A8 1:1 achieved up to 90 % on-target gene editing in A549 cells and a 3.6-fold increase in apoptosis, while A6 3:1 induced a 3.7-fold apoptotic response. Both formulations efficiently traversed airway mucus in air-liquid interface cultures and preserved over 80 % cell viability across doses. In vivo, repeated pulmonary administration was well tolerated, with no signs of systemic toxicity or cytokine elevation in healthy or tumor-bearing mice. In an orthotopic A549-luc lung tumor model, intratracheal delivery of A6 3:1 and A8 1:1 modestly suppressed tumor growth, with histological evidence of tumor cell apoptosis for A8 1:1. Quantification confirmed a statistically significant increase of apoptosis in the A8 1:1 group, consistent with effective KRAS disruption in vivo. Overall, lead LNPs, particularly A8 1:1, enabled efficient and localized RNA-based gene editing that induced downstream apoptotic signaling, demonstrating a preliminary, yet promising, proof-of-concept for CRISPR/Cas9 therapy in NSCLC.},
}

Animals
*Lung Neoplasms/genetics/therapy
*CRISPR-Cas Systems
Humans
*Nanoparticles/administration & dosage/chemistry
*Proto-Oncogene Proteins p21(ras)/genetics
*Lipids/chemistry/administration & dosage
Gene Editing
A549 Cells
*Carcinoma, Non-Small-Cell Lung/genetics/therapy
Mice
*CRISPR-Associated Protein 9/genetics
Mice, Nude
RNA, Guide, CRISPR-Cas Systems/administration & dosage/genetics
Female
RNA, Messenger/administration & dosage/genetics
Apoptosis
Cell Line, Tumor
Liposomes

@article {pmid41506441,
year = {2026},
author = {Mokhles, F and Moosavi, MA and Gutierrez-Uzquiza, A and Velasco, G and Li, M and Cordani, M},
title = {Unraveling stress-adaptation pathways in cancer: Functional dissection through CRISPR-based genetic screens.},
journal = {Cancer letters},
volume = {644},
number = {},
pages = {218246},
doi = {10.1016/j.canlet.2026.218246},
pmid = {41506441},
issn = {1872-7980},
mesh = {Humans ; *Neoplasms/genetics/pathology/metabolism ; *CRISPR-Cas Systems ; Animals ; Endoplasmic Reticulum Stress/genetics ; *Genetic Testing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Signal Transduction ; Tumor Microenvironment ; Unfolded Protein Response/genetics ; *Stress, Physiological ; Adaptation, Physiological/genetics ; },
abstract = {Cancer cells face a hostile microenvironment characterized by hypoxia, nutrient deprivation, endoplasmic reticulum (ER) stress, and oxidative imbalance. To cope with these challenges, they activate an interconnected network of adaptive pathways including autophagy, the unfolded protein response, metabolic reprogramming, and the integrated stress response., which promote cell survival, therapy resistance, immune evasion, and metastasis. CRISPR-based functional genomics has emerged as a powerful strategy to systematically dissect these stress-adaptive networks, enabling the identification of key regulators and vulnerabilities across diverse contexts. In this review, we first summarize tumor progression in major stress conditions and then highlight how CRISPR screening strategies ranging from genome-wide loss-of-function studies to single-cell and combinatorial platforms, are unraveling critical stress regulators. We further discuss emerging tools, model systems, and translational perspectives, underscoring how the integration of CRISPR technologies with multi-omics, artificial intelligence, and advanced preclinical models is reshaping our understanding of cancer stress biology and guiding the development of novel therapeutic strategies. Finally, we addressed how these novel dissection technologies influence translational opportunities, specifically in the context of combining stress-pathway modulators with immunotherapy and targeted therapy drugs.},
}

Humans
*Neoplasms/genetics/pathology/metabolism
*CRISPR-Cas Systems
Animals
Endoplasmic Reticulum Stress/genetics
*Genetic Testing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
Signal Transduction
Tumor Microenvironment
Unfolded Protein Response/genetics
*Stress, Physiological
Adaptation, Physiological/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 {pmid41507205,
year = {2026},
author = {Wan, H and Kong, D and Yan, T and Zhou, Y and Liu, M and Ma, X and Zhao, T and Zhou, W and Liu, X and Yin, J and Guan, N and Ye, H},
title = {A compact and inducible dCas12f-based CRISPRa platform for programmable in vivo gene activation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1447},
pmid = {41507205},
issn = {2041-1723},
support = {25J22800100//Science and Technology Commission of Shanghai Municipality (Shanghai Municipal Science and Technology Commission)/ ; 32250010//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; Dependovirus/genetics ; Mice ; *CRISPR-Cas Systems/genetics ; Humans ; *Transcriptional Activation/genetics ; HEK293 Cells ; *Gene Editing/methods ; Genetic Therapy/methods ; Mice, Inbred C57BL ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Precise activation of endogenous genes is a powerful strategy for functional genomics and therapeutic development, but current CRISPR-based transcriptional activation (CRISPRa) systems are limited by the large size of Cas proteins for adeno-associated virus (AAV) delivery. Here, we present a high-efficiency dCas12f-based transcriptional activation system (HEAL), which recruits transactivators through MS2 coat protein binding to MS2 aptamers embedded within the sgRNA scaffold. Engineered to enhance DNA binding, nuclear localization, and transactivator recruitment, HEAL induces over 100,000-fold activation of endogenous genes and outperforms existing CRISPRa systems in vitro and in vivo. We further develop red-light-inducible OptoHEAL and small-molecule-inducible ChemHEAL for remote and precise transcriptional control. AAV-delivered HEAL targeting interleukin 10 alleviates acute kidney injury in mice, while ChemHEAL-mediated activation of thymic stromal lymphopoietin reduces body weight in obese mice. HEAL provides a modular, compact, and controllable platform for endogenous gene activation with strong potential for fundamental research and gene therapy.},
}

Animals
Dependovirus/genetics
Mice
*CRISPR-Cas Systems/genetics
Humans
*Transcriptional Activation/genetics
HEK293 Cells
*Gene Editing/methods
Genetic Therapy/methods
Mice, Inbred C57BL
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41507381,
year = {2026},
author = {Gonzales, F and Schneider, C and Alexe, G and Lin, S and Khalid, D and Alvarez, M and Basanthakumar, A and Ellegast, JM and Merickel, L and Salhotra, S and Taillon, A and Giaimo, M and Wunderlich, M and Ansari, M and Perry, JA and Degar, B and Pikman, Y and Stegmaier, K},
title = {Identifying targeted therapies for CBFA2T3::GLIS2 acute myeloid leukemia.},
journal = {Leukemia},
volume = {40},
number = {2},
pages = {383-396},
pmid = {41507381},
issn = {1476-5551},
support = {R00 CA263161/CA/NCI NIH HHS/United States ; R35 CA283977/CA/NCI NIH HHS/United States ; R50 CA211404/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Leukemia, Myeloid, Acute/drug therapy/genetics/pathology/metabolism ; Animals ; Mice ; CRISPR-Cas Systems ; Janus Kinase 2/antagonists & inhibitors/genetics ; Nitriles ; *Molecular Targeted Therapy ; Pyrazoles/pharmacology ; Protein Kinase Inhibitors/pharmacology ; Xenograft Model Antitumor Assays ; Cell Proliferation ; Pyrimidines ; Cell Line, Tumor ; Apoptosis ; Drug Resistance, Neoplasm ; },
abstract = {CBFA2T3::GLIS2-positive pediatric acute myeloid leukemia (AML) remains one of the worst prognostic AML subgroups. To uncover innovative targeted therapy approaches in this disease subtype we performed genome-scale CRISPR-Cas9 screening that highlighted a strong, selective dependency on JAK2 compared to other types of cancer. Using a doxycycline-inducible JAK2 knockout (KO) system, we validated JAK2 dependency in CBFA2T3::GLIS2 cell lines, observing impaired proliferation in vitro and in vivo and apoptosis induction in vitro. Both type I (ruxolitinib) and type II (CHZ868) JAK2 inhibitors showed selective in vitro activity in CBFA2T3::GLIS2-positive AML models. To identify resistance and sensitizer mechanisms to JAK2 inhibitors, we used CRISPR-Cas9 ruxolitinib anchor screening in CBFA2T3::GLIS2 AML. sgRNAs targeting negative regulators of the MAPK pathway were enriched in the ruxolitinib-treated cells. Similarly, CBFA2T3::GLIS2 AML sublines grown to resistance under chronic ruxolitinib treatment expressed pathogenic NRAS mutations. Both approaches converged on MAPK pathway activation as a resistance mechanism to ruxolitinib treatment. Combining ruxolitinib with MEK inhibitors showed a synergistic effect in cell lines and patient-derived xenograft (PDX) cells expressing the fusion and in vivo activity in a CBFA2T3::GLIS2 AML PDX, suggesting a potential approach to target this signaling circuitry in this poor outcome AML subtype.},
}

Humans
*Leukemia, Myeloid, Acute/drug therapy/genetics/pathology/metabolism
Animals
Mice
CRISPR-Cas Systems
Janus Kinase 2/antagonists & inhibitors/genetics
Nitriles
*Molecular Targeted Therapy
Pyrazoles/pharmacology
Protein Kinase Inhibitors/pharmacology
Xenograft Model Antitumor Assays
Cell Proliferation
Pyrimidines
Cell Line, Tumor
Apoptosis
Drug Resistance, Neoplasm

@article {pmid41508115,
year = {2026},
author = {Xiao, Y and Li, X and Jiang, L and Zhao, Y and Wang, L and Feng, Y},
title = {Construction of Escherichia coli L-isoleucine cell factories based on propionate pathway.},
journal = {Journal of biological engineering},
volume = {20},
number = {1},
pages = {28},
pmid = {41508115},
issn = {1754-1611},
abstract = {UNLABELLED: Isoleucine, an essential branched-chain amino acid with broad applications in food, pharmaceuticals, and feed, is predominantly produced via the microbial threonine pathway, which suffers from catalytic complexity and regulatory inefficiency. The propionate pathway offers a streamlined alternative but remains unexplored for L-isoleucine biosynthesis. Here, we engineered Escherichia coli to establish the first propionate pathway-based L-isoleucine cell factory. Critical enzymes—propionyl-CoA synthase (PCS), propionyl-CoA transferase (PCT), and α-ketobutyrate synthase (OBS)—were identified for converting propionate to α-ketobutyrate. Key genes (prpE from Salmonella, pctcP from Gibberella, pctcN from Clostridium propionicum, and nifJ from Moorella thermoacetica) were integrated with the propionate transporter (prpP) and carbonic anhydrase (can) to enhance substrate utilization. ILE-5a, derived from E. coli BW25113 with deletions in brnQ, livJ, and livK, and containing specific insertions, was further modified to create ILE-5b with an additional deletion in yjiP. Plasmid-based expression in these strains, ILE-5a and ILE-5b, yielded top producers ILE-5a-P10 and ILE-5b-P10, which achieved L-isoleucine titers of 304 mg/L and 235 mg/L, respectively, in shake-flask fermentation using glucose and propionate as carbon sources. To stabilize production, the optimal gene set (pctN, nifJ, prpP, can) was genomically integrated via a transposon-encoded CRISPR-Cas system, generating mutants ILE-5a-P11 and ILE-5b-P11. Response surface methodology-optimized medium and 3-L fed-batch fermentation further elevated titers to 1.13 g/L (ILE-5a-P11) and 11.33 g/L (ILE-5b-P11). This study pioneers the propionate pathway for efficient L-isoleucine production, demonstrating its industrial potential through systematic metabolic engineering and process optimization.To our knowledge, this is the first study to design and demonstrate an E. coli platform for isoleucine production that simultaneously leverages the glutamate and propionate precursors.

SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13036-025-00609-6.},
}

@article {pmid41508607,
year = {2026},
author = {Wang, F and Lu, S and Zhu, C and Yang, L},
title = {PCdb: A comprehensive plant genome-editing database integrating sgRNA efficiency, off-target predictions, and epigenomic landscapes.},
journal = {Plant communications},
volume = {7},
number = {4},
pages = {101708},
pmid = {41508607},
issn = {2590-3462},
mesh = {*Genome, Plant/genetics ; *Gene Editing ; Epigenomics ; *Databases, Genetic ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/genetics ; *Plants/genetics ; },
abstract = {CRISPR-Cas-based genome editing has transformed plant biotechnology by enabling precise genomic modifications for crop improvement and functional genomics. The success of these applications hinges on the design of single guide RNAs (sgRNAs) that maximize on-target efficiency while minimizing off-target effects. However, existing resources for sgRNA design and performance evaluation in plants remain fragmented and lack integration with genomic and epigenomic contexts that influence both editing efficacy and specificity. Here, we present PCdb (Plant CRISPR Database; https://gmo.sjtu.edu.cn/pcdb), a comprehensive plant-focused database that integrates experimentally validated sgRNAs, annotated genomic contexts, genome-wide off-target predictions, and multilayer epigenomic annotations. PCdb encompasses 6172 manually curated editing records from 2132 publications, covering 4320 unique sgRNAs and 6 117 424 predicted off-target sites across nine major plant species. Notably, PCdb contextualizes potential editing outcomes-both on target and off target-within the chromatin landscape by incorporating DNA methylation profiles, chromatin accessibility data, and histone modification patterns. The database features an intuitive web interface that supports flexible queries, interactive visualization tools, and comprehensive analytical modules for sgRNA efficiency assessment and off-target analysis. A case study reanalysis of Oryza sativa yield-related genes demonstrates PCdb's ability to generate a detailed performance profile by evaluating both on-target characteristics and off-target risks within their native epigenomic context. Systematic analysis of the database further identifies key sequence and chromatin features that influence editing outcomes, providing novel insights to improve gene-editing efficacy and specificity.},
}

*Genome, Plant/genetics
*Gene Editing
Epigenomics
*Databases, Genetic
*RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems/genetics
*Plants/genetics

@article {pmid41508907,
year = {2026},
author = {Liu, CF and Leon, S and Herrig, I and Wessely, O and Tang, WHW},
title = {Generation of Mice Harboring Bicc1 Conditional Null Alleles.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {64},
number = {1},
pages = {e70038},
pmid = {41508907},
issn = {1526-968X},
support = {R01 DK080745/DK/NIDDK NIH HHS/United States ; MF-2203-02413//G. Harold and Leila Y. Mathers Charitable Foundation/ ; R01DK080745/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Mice ; Alleles ; Mice, Knockout ; CRISPR-Cas Systems ; *Polycystic Kidney Diseases/genetics ; *RNA-Binding Proteins/genetics ; Gene Editing ; Exons ; Female ; },
abstract = {Bicaudal C1 (Bicc1) encodes an RNA-binding protein critical for many organ development and epithelial tissue homeostasis. Bicc1 null mutations have been shown to lead to the development of polycystic kidney disease (PKD) and death at an early prenatal stage. To elucidate the tissue-specific functions of Bicc1, we engineered two independent conditional knockout (cKO) mouse lines targeting distinct exonic regions of the gene. The first line was generated using a traditional embryonic stem (ES) cell-based approach, wherein loxP sites were inserted flanking exon 4 (E4), enabling Cre-mediated excision of a functionally essential coding region. The second line was created using CRISPR/Cas9 genome editing, introducing loxP sites around both exon 4 and exon 5 (E4-5) in a double-step zygote injection strategy. Both alleles were validated by PCR genotyping, sequencing, and functional recombination was confirmed via a tissue-specific Cre driver. These independent cKO models provide a robust platform for dissecting the role of Bicc1 in specific tissues and developmental stages, and offer new avenues for studying the mechanistic basis of PKD and other Bicc1-related pathologies.},
}

Animals
Mice
Alleles
Mice, Knockout
CRISPR-Cas Systems
*Polycystic Kidney Diseases/genetics
*RNA-Binding Proteins/genetics
Gene Editing
Exons
Female

@article {pmid41510162,
year = {2026},
author = {Pan, J and Li, B and Wang, Y and Han, Y and Liu, G and Sun, SK},
title = {Physical stimuli-responsive CRISPR-Cas9 systems for spatiotemporally precise control of genome engineering.},
journal = {Theranostics},
volume = {16},
number = {6},
pages = {2984-3010},
pmid = {41510162},
issn = {1838-7640},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; Animals ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9 (CRISPR-Cas9) endonuclease system has revolutionized biology research by enabling precise, efficient, and versatile genome editing. However, achieving spatiotemporally controlled gene editing within specific organs, tissues, or cells remains a major challenge, as unregulated CRISPR-Cas9 activity can lead to severe off-target effects, hindering its clinical translation. To enhance the on-target precision and reduce the unwanted consequences of aberrant or premature CRISPR-Cas9 activation, various strategies have been developed to regulate its function at translational or post-translational stages using diverse external physicochemical stimuli. While chemical molecule-inducible CRISPR-Cas9 systems have demonstrated significant progress, most of them still suffer from inherent deficiencies, such as unsatisfactory spatiotemporal precision, irreversibility, pharmacokinetic dependence, internal disturbance, and safety concerns related to chemical inducers. By contrast, externally applied physical stimuli provide distinct advantages for triggering CRISPR-Cas9 activity, offering superior spatiotemporal precision, reversibility, and biocompatibility. These features significantly enhance the controllability, target specificity, and practical applicability of CRISPR-Cas9 systems across diverse biological settings. This review systematically explores recent advances in physical stimuli-responsive CRISPR-Cas9 platforms, detailing their design strategies, activation mechanisms, and proof-of-concept applications. Furthermore, we provide a comparative analysis of different stimulation strategies, highlighting their respective characteristics, current limitations, and future prospects. A discussion on the persistent bench-to-bedside gap is also included, aiming to guide future development toward clinically viable solutions.},
}

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

@article {pmid41510594,
year = {2026},
author = {Wei, J and Duan, Y and Xue, C and Zheng, L and Wei, Q and Wu, Z and Xin, H and Zeng, T and Deng, H and Fan, S and Xiong, W and Zeng, Z and Li, M and Zhou, M},
title = {Targeted demethylation of the BRD7 promoter based on CRISPR/dCas9 system inhibits the malignant progression of nasopharyngeal carcinoma.},
journal = {Clinical and translational medicine},
volume = {16},
number = {1},
pages = {e70583},
pmid = {41510594},
issn = {2001-1326},
support = {82473262//the National Natural Science Foundation of China/ ; 82403005//the National Natural Science Foundation of China/ ; 2023SK2008//the Human Provincial key Research and Development Program/ ; 2024ZZTS0160//the Free Exploration Program of Central South University/ ; 20256508//the scientific research plan project of Hunan Provincial Health Commission/ ; 111-2-12//the program of Introducing Talents of Discipline to Universities/ ; },
mesh = {Humans ; *Nasopharyngeal Carcinoma/genetics/pathology ; Promoter Regions, Genetic/genetics ; Mice ; Animals ; *Nasopharyngeal Neoplasms/genetics/pathology ; *Chromosomal Proteins, Non-Histone/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Disease Progression ; Cell Line, Tumor ; DNA Methylation/genetics ; Cell Proliferation/genetics ; Female ; Bromodomain Containing Proteins ; },
abstract = {BACKGROUND: BRD7 has been confirmed to be lowly expressed in nasopharyngeal carcinoma (NPC) tissues and exerts tumour suppressive roles. However, the molecular mechanism of the downregulation of BRD7 expression and whether the strategy of activating BRD7 expression plays anti-tumour effects still needs to be clarified.

METHODS: Methylation-specific polymerase chain reaction (PCR) was used to identify the methylation levels of BRD7 promoter. In vitro experiments were used to evaluate the effects of BRD7-targeted demethylation system on the malignant progression of NPC cells. Chromatin immunoprecipitation (ChIP)-qPCR experiment was employed to examine the regulatory mechanisms underlying the demethylation system. Xenograft tumour models were used to assess impact of this demethylation system on tumour growth in vivo and the anti-tumour effects of the lentivirus-mediated demethylation system in NPC.

RESULTS: There was hypermethylation modification in BRD7 promoter, which was negatively correlated with BRD7 expression. Next, we constructed a LentiCRISPRv2/dCas9-TET1CD-sgRNAs system targeting specific methylation sites of BRD7 promoter based on five sgRNAs, and confirmed that all five sgRNA-guided CRISPR/dCas9 systems could activate BRD7 and inhibit cell proliferation to varying degrees, among which sgRNA2&sgRNA5 were the most significant. Further, we constructed NPC cell lines stably transfected with LentiCRISPRv2/dCas9-TET1CD-sgRNA2&5, and confirmed that both sgRNA2&sgRNA5 could promote the transcriptional activation by reducing its methylation, and inhibit the cell proliferation, migration, invasion and tumour growth in vivo of NPC, and the combination of them has a more significant demethylation, transcriptional activation and anti-tumour effect. In addition, BRD7 had hypermethylation modification in its promoter and decreased expression in NPC tissues, and both of them were negatively correlated, making it a potential diagnostic marker for NPC diagnosis.

CONCLUSIONS: The hypermethylation modification of BRD7 is an important mechanism leading to the inactivation of BRD7, and targeting demethylation of BRD7 inhibits the malignant progression of NPC, which might be a promising targeted therapeutic approach for treating NPC.},
}

Humans
*Nasopharyngeal Carcinoma/genetics/pathology
Promoter Regions, Genetic/genetics
Mice
Animals
*Nasopharyngeal Neoplasms/genetics/pathology
*Chromosomal Proteins, Non-Histone/genetics/metabolism
*CRISPR-Cas Systems/genetics
Disease Progression
Cell Line, Tumor
DNA Methylation/genetics
Cell Proliferation/genetics
Female
Bromodomain Containing Proteins

@article {pmid41510755,
year = {2026},
author = {Wang, L and Su, L and Gu, W and Nie, S and Luo, J and Li, Y and Pan, A and Li, Y},
title = {A photonic crystal sensing array based on a tandem CRISPR/Cas13a system for ultra-sensitive and high-throughput detection of the CVA6 virus.},
journal = {The Analyst},
volume = {151},
number = {3},
pages = {903-912},
doi = {10.1039/d5an01178g},
pmid = {41510755},
issn = {1364-5528},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Limit of Detection ; Photons ; *Biosensing Techniques/methods ; *RNA, Viral/genetics/analysis ; Fluorescent Dyes/chemistry ; High-Throughput Screening Assays/methods ; },
abstract = {Coxsackievirus A6 (CVA6) has emerged as a major cause of hand-foot-mouth disease (HFMD), yet no standardized detection method for it is currently available. Developing a simple, sensitive, and specific CVA6 test is crucial for HFMD control and safeguarding the health of at-risk children. Herein, a photonic crystal (PC) sensing array based on a tandem CRISPR/Cas13a system has been proposed for highly specific and ultra-sensitive analysis of CVA6 RNA, without the need for reverse transcription and amplification procedures. In this strategy, two crRNAs targeting CVA 6 RNA were designed and screened, and the fluorescence signal of the tandem CRISPR/Cas13a system was found to be up to 4.2 times higher than that of the non-tandem CRISPR system. The PC array with periodic nanostructures was prepared through self-deposition and further enhanced the fluorescent signal output from the tandem CRISPR system, owing to the match of the emission wavelength of the fluorescent dyes and the photonic band gap (PBG) of the PC. Benefitting from the synergistic effect of the tandem CRISPR system and PC array, as well as the high trans-cleavage activity of Cas13a protein, this engineered sensing array enables ultra-sensitive detection with a limit of detection (LOD) as low as 24.9 fM for CVA6. Meanwhile, this sensing strategy also achieved high-throughput and rapid analysis with a detection frequency of about 96 samples every 3.4 minutes. Therefore, the proposed strategy offers a simple workflow without reverse transcription or amplification, along with high sensitivity and high throughput, demonstrating strong potential for applications in biometrics and clinical diagnostics.},
}

*CRISPR-Cas Systems/genetics
Humans
Limit of Detection
Photons
*Biosensing Techniques/methods
*RNA, Viral/genetics/analysis
Fluorescent Dyes/chemistry
High-Throughput Screening Assays/methods

@article {pmid41510784,
year = {2026},
author = {Hejlesen, R and Bakkeren, C and Damsgaard, C and Laursen, LS and Kjær-Sørensen, K and Corti, P and Malte, H and Oxvig, C and Fago, A},
title = {Myoglobin Affects Tissue-Specific Transcriptome, Heart Regeneration and Whole Animal Metabolic Rates.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {1},
pages = {e71424},
doi = {10.1096/fj.202503482RR},
pmid = {41510784},
issn = {1530-6860},
support = {NNF19OC0057938//Novo Nordisk Fonden (NNF)/ ; 3103-0011B//Danmarks Frie Forskningsfond (DFF)/ ; DMMTF24081545//Company of Biologists (Co_Biologists)/ ; //Graduate School Natural Science Aarhus University/ ; },
mesh = {Animals ; Zebrafish/genetics/metabolism ; *Myoglobin/genetics/metabolism ; *Regeneration/physiology ; *Transcriptome ; *Heart/physiology ; Muscle, Skeletal/metabolism ; Myocytes, Cardiac/metabolism ; *Myocardium/metabolism ; *Zebrafish Proteins/genetics/metabolism ; },
abstract = {Myoglobin (Mb) is a small haem-containing protein traditionally associated with oxygen carrier functions in cardiac and skeletal muscle. However, studies using Mb knockout mice have yielded conflicting results regarding its functional roles in vivo. Here, we used a CRISPR-Cas-generated zebrafish Mb knockout model to investigate the consequences of Mb loss across skeletal muscle types, transcriptomics profiles, and whole-animal metabolic rates under both resting and maximal exercise conditions. Mb deficiency did not alter skeletal muscle fiber composition or overall mitochondrial respiratory capacity but induced multiple tissue-specific transcriptomic changes, including downregulation of gene sets involved in respiration and differentiation pathways in the heart, while upregulating those associated with respiration and glycogen metabolism in the skeletal muscle. During cardiac regeneration following ventricle amputation in wild-type zebrafish, Mb expression was transiently suppressed, consistent with a role in maintaining the cardiomyocytes in a differentiated state. Physiologically, Mb knockout zebrafish displayed a reduced standard metabolic rate at rest, enhanced hypoxia tolerance (i.e., a lower critical oxygen tension), and increased maximal swimming speed, while maintaining unchanged maximal metabolic rate and aerobic scope relative to wild-type counterparts. Collectively, these findings show that loss of Mb in zebrafish elicits coordinated tissue-specific transcriptional changes, potentially facilitates cardiac regeneration, lowers standard metabolic rate, and enhances maximal swimming speed and hypoxia tolerance, thereby providing new insights into the multifaceted in vivo functions of Mb.},
}

Animals
Zebrafish/genetics/metabolism
*Myoglobin/genetics/metabolism
*Regeneration/physiology
*Transcriptome
*Heart/physiology
Muscle, Skeletal/metabolism
Myocytes, Cardiac/metabolism
*Myocardium/metabolism
*Zebrafish Proteins/genetics/metabolism

@article {pmid41511319,
year = {2025},
author = {Xu, J and Zhang, J and Yang, D},
title = {Editorial: CRISPR-Based Genome Editing in Translational Research-2nd Edition.},
journal = {Cells},
volume = {15},
number = {1},
pages = {},
pmid = {41511319},
issn = {2073-4409},
support = {R42 GM122181/GM/NIGMS NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Translational Research, Biomedical ; Humans ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Genome editing technologies represented by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) have transformed biomedical research and therapeutic development [...].},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Translational Research, Biomedical
Humans
Animals
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41511442,
year = {2026},
author = {Li, Y and Li, X and Chen, Y and Wang, Y and Zuo, Z},
title = {Differential Allosteric Modulation of Cas9 Specificity.},
journal = {Journal of chemical theory and computation},
volume = {22},
number = {2},
pages = {806-817},
doi = {10.1021/acs.jctc.5c01919},
pmid = {41511442},
issn = {1549-9626},
mesh = {Allosteric Regulation ; Molecular Dynamics Simulation ; RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics ; *CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; Mutation ; },
abstract = {Both RNA- and protein-based strategies have been developed to mitigate off-target cleavage by CRISPR-Cas9, yielding noncanonical guide RNAs (gRNAs) and Cas9 variants with enhanced gene-editing precision. However, the molecular mechanisms by which such PAM-distal alterations─remote from the nuclease centers─modulate Cas9 activity and specificity remain incompletely understood. Here, we performed near-millisecond all-atom molecular dynamics simulations to elucidate how diverse PAM-distal perturbations─including gRNA truncation, base mismatching, and evolved mutations─reshape the conformational dynamics and allosteric regulation of Cas9. Despite their distinct origins, all perturbations ultimately modulate Cas9 function by altering HNH dynamics that impede the transition from the checkpoint to the catalytically active state, yet they do so through distinct allosteric routes. The 16-nt gRNA induces a pronounced REC3 reorientation toward the L2 linker and HNH domain, while PAM-distal mismatches with the 18-nt gRNA promote engagement of the unwound target DNA strand with L2─both effectively restraining HNH rotation. In contrast, evolved mutations remodel the global motional modes so that REC2 swivels inward, constraining the HNH flexibility. These perturbations delineate multiple structural paths converging on a shared allosteric outcome─HNH immobilization and catalytic suppression─thereby unifying RNA-, DNA-, and protein-level effects within a single dynamic framework linking distal structural perturbations to activity control. This work provides mechanistic insight into the regulation of Cas9 fidelity and offers principles for the design of next-generation genome editors.},
}

Allosteric Regulation
Molecular Dynamics Simulation
RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics
*CRISPR-Associated Protein 9/chemistry/metabolism/genetics
CRISPR-Cas Systems
Mutation

@article {pmid41512012,
year = {2026},
author = {Chaudhari, VR and Lin, MT and Hines, KM and Hanson, MR},
title = {Rewinding evolution in planta: A Rubisco-null platform validates high-performance ancestral enzymes.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {3},
pages = {e2523360123},
pmid = {41512012},
issn = {1091-6490},
support = {DE-SC0020142//DOE Office of Basic Energy Sciences, Chemical Sciences, Geosciences, and Biosciences Division/ ; MCB-2131582//NSF (NSF)/ ; },
mesh = {*Ribulose-Bisphosphate Carboxylase/genetics/metabolism ; *Nicotiana/genetics/enzymology ; Plants, Genetically Modified/genetics ; Photosynthesis/genetics ; CRISPR-Cas Systems ; Chloroplasts/genetics ; Evolution, Molecular ; Kinetics ; Gene Knockout Techniques ; },
abstract = {Improving the photosynthetic enzyme Rubisco is a key target for enhancing C3 crop productivity, but progress has been hampered by the difficulty of evaluating engineered variants in planta without interference from the native enzyme. Here, we report the creation of a Rubisco-null Nicotiana tabacum platform by using CRISPR-Cas9 to knock out all 11 nuclear-encoded small subunit (rbcS) genes. Knockout was achieved in a line expressing cyanobacterial Rubisco from the plastid genome, allowing the recovery of viable plants. We then developed a chloroplast expression system for coexpressing both large and small subunits from the plastid genome. We expressed two resurrected ancestral Rubiscos from the Solanaceae family. The resulting transgenic plants were phenotypically normal and accumulated Rubisco to wild-type levels. Importantly, kinetic analyses of the purified ancestral enzymes revealed they possessed a 16 to 20% higher catalytic efficiency (kcat,air/Kc,air) under ambient conditions, driven by a significantly faster turnover rate (kcat,air). We have demonstrated that our system allows robust in vivo assessment of novel Rubiscos and that ancestral reconstruction is a powerful strategy for identifying superior enzymes to improve photosynthesis in C3 crops.},
}

*Ribulose-Bisphosphate Carboxylase/genetics/metabolism
*Nicotiana/genetics/enzymology
Plants, Genetically Modified/genetics
Photosynthesis/genetics
CRISPR-Cas Systems
Chloroplasts/genetics
Evolution, Molecular
Kinetics
Gene Knockout Techniques

@article {pmid41512333,
year = {2026},
author = {Zhao, J and Sui, Z and Chen, B and Wu, R and Xu, J and Dong, H},
title = {Customizable NAND Logic-Gate Biosensing System Enabled by an Engineered Methylation-CRISPR/Cas12a Consensus Sequence for Ultrasensitive DNA Methyltransferase Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {3},
pages = {2368-2378},
doi = {10.1021/acs.analchem.5c06772},
pmid = {41512333},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism/analysis ; Limit of Detection ; Humans ; Logic ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {DNA methyltransferases (MTases) play crucial roles in epigenetic regulation, and their abnormal activity is closely associated with various human diseases. Here, we report a customizable NAND logic-gate biosensing platform for highly sensitive and intelligent detection of DNA adenine methyltransferase (Dam MTase). An engineered methylation-CRISPR/Cas12a consensus sequence (MCCS, 5'-TTTGATC-3') was rationally designed to integrate the Cas12a PAM site, Dam methylation site, and DpnI recognition sequence into a unified functional motif. Coupled with a primer-triggered hybridization chain reaction (HCR), multiple tandem MCCS units were generated to amplify the fluorescence signal output. In this logic circuit, Dam, SAM, and DpnI serve as three biochemical inputs, and their combined presence ("1,1,1") yields a low-fluorescence "OFF" output according to the NAND logic rule. The system exhibited a broad linear detection range with an ultralow detection limit of 0.00032 U mL[-1], outstanding selectivity toward nontarget MTases, and satisfactory recoveries (98.16-100.03%) in human serum samples. Furthermore, it enabled quantitative evaluation of Dam inhibitors, revealing IC50 values of 1.75 μM for 5-fluorouracil and 11.9 μM for penicillin G. This strategy provides a universal molecular computation-driven biosensing framework for enzyme activity analysis and inhibitor screening in complex biological systems.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems
*Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism/analysis
Limit of Detection
Humans
Logic
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41512493,
year = {2026},
author = {Ahmad, R and Gastoldi, G and Wong, ST and Baeza Garcia, A and Caljon, G},
title = {The EamA metabolite transporter does not affect antileishmanial drug susceptibility.},
journal = {International journal for parasitology. Drugs and drug resistance},
volume = {30},
number = {},
pages = {100632},
pmid = {41512493},
issn = {2211-3207},
mesh = {*Leishmania infantum/drug effects/genetics/metabolism ; *Antiprotozoal Agents/pharmacology ; *Protozoan Proteins/genetics/metabolism ; Drug Resistance/genetics ; Animals ; CRISPR-Cas Systems ; *Membrane Transport Proteins/genetics/metabolism ; Macrophages/parasitology ; Gene Editing ; Mice ; Humans ; Parasitic Sensitivity Tests ; Leishmaniasis, Visceral/parasitology/drug therapy ; },
abstract = {Leishmaniasis is a major neglected tropical disease, exists in 98 countries and constitutes a global public health threat. As chemotherapy is confronted with drug resistance and treatment failure, understanding the underlying mechanisms and continued drug discovery efforts are needed in the fight against leishmaniasis. A previous cosmid-based overexpression study suggested a role for EamA (LINF_020008400), annotated as a putative drug-metabolite transporter, in resistance to novel antileishmanial oxaboroles. To assess fitness cost and drug susceptibility, gene deficient Leishmania infantum lines were generated using CRISPR-Cas9 gene editing and overexpression from the ssu locus was achieved using the pLEXSY system. While in vitro parasite growth and survival were unchanged compared to control lines, the intracellular burden of the null mutant was lower. In vitro exposure to current antileishmanial drugs and several novel leads revealed an unchanged drug sensitivity profile in extracellular and intramacrophage assays. Similarly, the overexpression lines showed a significantly lower infection rate, but their drug susceptibility profiles showed no significant differences from the control. Collectively, these data suggest that -under the tested conditions- LINF_020008400 is not essential for parasite fitness, host cell infectivity and survival following exposure to antiparasitic drugs.},
}

*Leishmania infantum/drug effects/genetics/metabolism
*Antiprotozoal Agents/pharmacology
*Protozoan Proteins/genetics/metabolism
Drug Resistance/genetics
Animals
CRISPR-Cas Systems
*Membrane Transport Proteins/genetics/metabolism
Macrophages/parasitology
Gene Editing
Mice
Humans
Parasitic Sensitivity Tests
Leishmaniasis, Visceral/parasitology/drug therapy

@article {pmid41512567,
year = {2026},
author = {Sushmita, and Srivastava, A and Jain, G and Singh, M and Verma, PC},
title = {Targeted disruption of a cell wall-modifying gene α-Mannosidase using CRISPR-Cas9 enhances post-harvest shelf life in tomato through ABA accumulation.},
journal = {Plant physiology and biochemistry : PPB},
volume = {231},
number = {},
pages = {111017},
doi = {10.1016/j.plaphy.2026.111017},
pmid = {41512567},
issn = {1873-2690},
mesh = {*Solanum lycopersicum/genetics/metabolism/enzymology ; *Abscisic Acid/metabolism ; *Cell Wall/metabolism/genetics ; *CRISPR-Cas Systems ; *alpha-Mannosidase/genetics/metabolism ; Plant Proteins/genetics/metabolism ; Fruit/genetics/metabolism ; Gene Expression Regulation, Plant ; Reactive Oxygen Species/metabolism ; },
abstract = {Tomato ripening is a complex process regulated by transcription factors, hormones, and physiological changes. While this intricate regulation ensures desirable traits such as color, texture, and flavor, it is often accompanied by rapid post-harvest deterioration and poor shelf life, leading to significant economic losses and limiting market potential. Various cell wall-modifying enzymes, including N-glycan-processing enzyme α-Mannosidase, play crucial roles in the softening and senescence of tomato fruits. Our study shows that α-Mannosidase knockout through CRISPR/Cas9 results in fruits with enhanced firmness, longer shelf life, and improved moisture retention. Additionally, the expression of SlRIN (Ripening Inhibitor), a key regulator of ripening and several downstream genes, including those involved in cell wall degradation, ethylene biosynthesis, and signaling, was downregulated. Interestingly, in the later stages of storage, they also exhibited higher accumulation of abscisic acid (ABA) and lower accumulation of reactive oxygen species, along with better antioxidant capacity, compared to the control fruits, which may confer delayed softening and increased shelf life. Our findings highlight α-Mannosidase as a ripening-specific regulator and a promising genetic target for extending tomato shelf life, offering a sustainable strategy to minimize post-harvest losses without compromising plant development.},
}

*Solanum lycopersicum/genetics/metabolism/enzymology
*Abscisic Acid/metabolism
*Cell Wall/metabolism/genetics
*CRISPR-Cas Systems
*alpha-Mannosidase/genetics/metabolism
Plant Proteins/genetics/metabolism
Fruit/genetics/metabolism
Gene Expression Regulation, Plant
Reactive Oxygen Species/metabolism

@article {pmid41513195,
year = {2026},
author = {Luo, Q and Huang, Y and Zheng, H and Ke, W and Zheng, H and Sun, Q and Wang, M and Weng, Z},
title = {CRISPR-engineered zebrafish expression system for human type III collagen: Therapeutic efficacy in wound healing.},
journal = {International journal of biological macromolecules},
volume = {340},
number = {Pt 1},
pages = {150161},
doi = {10.1016/j.ijbiomac.2026.150161},
pmid = {41513195},
issn = {1879-0003},
mesh = {Animals ; *Zebrafish/genetics ; Humans ; *Wound Healing/drug effects/genetics ; Animals, Genetically Modified ; *CRISPR-Cas Systems/genetics ; *Collagen Type III/genetics/pharmacology ; Mice ; Recombinant Proteins/pharmacology/genetics ; Cell Proliferation ; },
abstract = {Human type III collagen (Col III) is a critical component for skin tissue repair and anti-aging, yet its heterologous expression often faces challenges such as incomplete structure and poor thermostability. Here, we established a transgenic zebrafish expression system via CRISPR/Cas9 technology, integrating the human Col3a1 gene into a non-functional region of zebrafish chromosome 4. The extraction yield of total zebrafish collagen (Col III-TC), a composite material comprising both recombinant human Col III and endogenous zebrafish collagens, was 45.76%. Structural analysis revealed intact fibrous architecture and a thermal shrinkage temperature of 71.3 °C, significantly superior to conventional systems. Functionally, Col III-TC exhibited remarkable free radical-scavenging capacity and suppressed LPS-induced inflammation in 3T3-L1 cells (downregulating Tnfα, Il1b, and Il6, while upregulating Il10), alongside promoting fibroblast proliferation. In a murine acute wound model, Col III-TC-based dressings achieved outstanding healing efficacy (>95% closure within 15 days), with histological analysis showing improved neoskin thickness and collagen deposition. The Col3a1 transgenic zebrafish system developed in this study not only provides a novel strategy for heterologous expression of fully functional human proteins, but also highlights the broad application potential of its high-yield collagen in biomedical fields, particularly in wound healing and anti-aging therapies.},
}

Animals
*Zebrafish/genetics
Humans
*Wound Healing/drug effects/genetics
Animals, Genetically Modified
*CRISPR-Cas Systems/genetics
*Collagen Type III/genetics/pharmacology
Mice
Recombinant Proteins/pharmacology/genetics
Cell Proliferation

@article {pmid41513250,
year = {2026},
author = {Zhu, Y and Luo, S and Cui, X and Wu, J and Cheng, W and Wang, X and Zhang, Q and Tan, G and Yang, H and Zheng, Y and Peng, W},
title = {Converting an Untransformable Vibrio parahaemolyticus Isolate into a Fast Genetic Engineering Platform.},
journal = {ACS synthetic biology},
volume = {15},
number = {2},
pages = {740-747},
doi = {10.1021/acssynbio.5c00771},
pmid = {41513250},
issn = {2161-5063},
mesh = {*Vibrio parahaemolyticus/genetics ; Plasmids/genetics ; CRISPR-Cas Systems/genetics ; *Genetic Engineering/methods ; Bacterial Proteins/genetics/metabolism ; Deoxyribonucleases/genetics/metabolism ; Gene Editing/methods ; },
abstract = {The pathogenic bacterium Vibrio parahaemolyticus represents a substantial economic and public health concern; however, elucidating its virulence mechanisms has been significantly impeded by its inherent resistant to genetic manipulation, primarily attributed to sophisticated immune defense systems including restriction-modification (R-M) modules, CRISPR-Cas systems, standalone DNases, and DdmDE systems. Paradoxically, while genetic modification is essential for overcoming these barriers, the very barriers themselves obstruct DNA introduction. Our investigation focused on the V. parahaemolyticus X1 strain, where initial plasmid transformation attempts proved unsuccessful. However, low-efficiency conjugation allowed knockout of defense genes, thereby silencing the host's defense mechanisms. Our findings revealed a standalone DNase, Vpn, as the predominant obstacle to foreign DNA entry in the X1 strain, while a DdmDE system executes elimination of invaded plasmids. Leveraging these insights, we created the V. parahaemolyticus X2 strain via sequential depletion of the Vpn nuclease and the DdmDE system. Capitalizing on the bacterium's exceptional growth rate, characterized by a generation time of approximately 10.5 min, we established a highly efficient molecular cloning platform capable of creating a new plasmid construct within a single day. This work not only presents a strategic framework for genetic manipulation of previously recalcitrant bacterial species but also underscores the potential of fast-growing marine bacteria as promising candidates for next-generation biotechnological applications.},
}

*Vibrio parahaemolyticus/genetics
Plasmids/genetics
CRISPR-Cas Systems/genetics
*Genetic Engineering/methods
Bacterial Proteins/genetics/metabolism
Deoxyribonucleases/genetics/metabolism
Gene Editing/methods

@article {pmid41516041,
year = {2025},
author = {Sahu, D and Ganguly, T and Mann, A and Gupta, Y and Nynatten, LRV and Fraser, DD},
title = {Emerging Technologies for Exploring the Cellular Mechanisms in Vascular Diseases.},
journal = {International journal of molecular sciences},
volume = {27},
number = {1},
pages = {},
pmid = {41516041},
issn = {1422-0067},
mesh = {Humans ; *Vascular Diseases/genetics/metabolism/pathology/diagnosis ; Animals ; Gene Editing/methods ; Artificial Intelligence ; CRISPR-Cas Systems ; Single-Cell Analysis/methods ; },
abstract = {Vascular diseases (VDs) and cardiovascular diseases (CVDs) are the leading causes of morbidity and mortality worldwide. Current diagnostic and therapeutic approaches are limited by insufficient resolution and a lack of mechanistic understanding at the cellular level. Traditional imaging and clinical assays do not fully capture the dynamic molecular and structural complexities underlying vascular pathology. Recent technological innovations, including single-cell and spatial transcriptomics, super-resolution and photoacoustic imaging, microfluidic organ-on-chip platforms, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based gene editing, and artificial intelligence (AI), have created new opportunities for investigating the cellular and molecular basis of VDs. These techniques enable high-resolution mapping of cellular heterogeneity and functional alterations, facilitating the integration of large-scale data for biomarker discovery, disease modeling, and therapeutic development. This review focuses on evaluating the translational readiness, limitations, and potential clinical applications of these emerging technologies. Understanding the cellular and molecular mechanisms of VDs is essential for developing targeted therapies and precise diagnostics. Integrating single-cell and multiomics approaches highlights disease-driving cell types and gene programs. Optogenetics and organ-on-chip platforms allow for controlled manipulation and physiologically relevant modeling, while AI enhances data integration, risk prediction, and clinical interpretability. Future efforts should prioritize multi-center, large-scale validation studies, harmonization of assay protocols, and integration with clinical datasets and human samples. Multi-omics approaches and computational modeling hold promise for unraveling disease complexity, while advances in regulatory science and digital simulation (such as digital twins) may further accelerate personalized medicine in vascular disease research and treatment.},
}

Humans
*Vascular Diseases/genetics/metabolism/pathology/diagnosis
Animals
Gene Editing/methods
Artificial Intelligence
CRISPR-Cas Systems
Single-Cell Analysis/methods

@article {pmid41516323,
year = {2025},
author = {Evseev, PV and Podoprigora, IV and Chaplin, AV and Khabadze, ZS and Malkov, AA and Kafarskaia, LI and Shagin, DA and Urban, YN and Borisova, OY and Efimov, BA},
title = {Bulleidia extructa PP_925: Genome Reduction, Minimalist Metabolism, and Evolutionary Insights into Firmicutes Diversification.},
journal = {International journal of molecular sciences},
volume = {27},
number = {1},
pages = {},
pmid = {41516323},
issn = {1422-0067},
mesh = {*Genome, Bacterial ; Phylogeny ; *Evolution, Molecular ; Humans ; *Bacillota/genetics/metabolism/classification ; },
abstract = {Bulleidia extructa strain PP_925, isolated from the periodontal pocket of a patient with periodontitis, is a Gram-positive Bacillota with an unusually compact genome of 1.38 Mb. Phylogenomic analyses place PP_925 within Erysipelotrichales and show close relatedness of Bulleidia to Solobacterium and Lactimicrobium, as well as the existence of previously undescribed related clades. The metabolic repertoire of PP_925 is strongly reduced: it retains glycolysis, the phosphotransacetylase-acetate kinase pathway, and arginine catabolism but lacks the tricarboxylic acid cycle and most de novo biosynthetic pathways for amino acids, nucleotides, fatty acids, cofactors, and vitamins, implying reliance on salvage and cross-feeding. Phylogenetic inference indicates independent peptidoglycan losses in multiple mycoplasma Erysipelotrichia-related lineages, while PP_925 has retained an ancestral Gram-positive cell wall despite extensive genomic reduction. The genome preserves systems crucial for host interaction and adaptability, including a horizontally acquired tad locus encoding type IV pili, a comG competence system, and several adherence-associated virulence factors. Defense mechanisms are diverse and include a CRISPR-Cas II-A system, a type II restriction-modification module adjacent to Gao_Qat-like genes, and the Wadjet system in a genome without prophages; CRISPR spacers indicate repeated encounters with Bacillota phages. Comparative genomics of PP_925 and related strains reveals a small core genome with lineage-specific adhesion and defense modules, indicating recent shared ancestry combined with adaptive flexibility under substantial genome reduction.},
}

*Genome, Bacterial
Phylogeny
*Evolution, Molecular
Humans
*Bacillota/genetics/metabolism/classification

@article {pmid41516361,
year = {2026},
author = {Deriglazova, IO and Shepelev, MV and Kruglova, NA and Georgiev, PG and Maksimenko, OG},
title = {The Chimeric Nuclease SpRYc Exhibits Highly Variable Performance Across Biological Systems.},
journal = {International journal of molecular sciences},
volume = {27},
number = {1},
pages = {},
pmid = {41516361},
issn = {1422-0067},
support = {075-15-2024-539//Ministry of Science and Education of the Russian Federation/ ; },
mesh = {Animals ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; HEK293 Cells ; Drosophila melanogaster/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {The CRISPR-Cas9 system has significantly advanced genome editing but remains constrained by its requirement for specific protospacer adjacent motifs (PAMs). To overcome this limitation, PAM-relaxed nucleases, including the novel near-PAMless chimeric SpRYc, have been developed. Here, we evaluated SpRYc editing activity across multiple experimental systems, including human HEK293 and CEM-R5 cells, as well as Drosophila melanogaster S2 cells and embryos. In HEK293 cells, SpRYc exhibited broad PAM compatibility, enabling editing at non-canonical PAMs, albeit with reduced and variable efficiency at canonical NGG sites compared to SpCas9. This context dependency was more pronounced in CEM-R5 T cells, where SpRYc activity at endogenous CXCR4 and B2M loci was largely restricted to NGG PAMs. In contrast, unlike SpCas9, SpRYc displayed negligible genome-editing activity in Drosophila embryos in vivo. Notably, the transcriptional activator dSpRYc-VPR showed robust activity in Drosophila S2 cells at both canonical and non-canonical PAMs. Reduced chromatin occupancy of dSpRYc-VPR suggests a balance between expanded PAM recognition and DNA-binding stability, providing a mechanistic explanation for context-dependent performance of SpRYc. Overall, our results highlight that expanded targeting flexibility comes at the cost of variable efficiency, underscoring the need for extensive locus- and context-specific validation of PAM-relaxed genome-editing tools.},
}

Animals
Humans
*Gene Editing/methods
*CRISPR-Cas Systems
HEK293 Cells
Drosophila melanogaster/genetics
CRISPR-Associated Protein 9/genetics/metabolism

@article {pmid41516375,
year = {2026},
author = {Firdaus, F and Yadav, V and Ramakrishnan, M and Wasi, A and Ganie, IB and Upadhyay, A and Shahzad, A and Ahmad, Z},
title = {2-Hydroxy-4-Methoxybenzaldehyde (2H4MB): Integrating Cell Culture, Metabolic Engineering, and Intelligent Genome Editing.},
journal = {International journal of molecular sciences},
volume = {27},
number = {1},
pages = {},
pmid = {41516375},
issn = {1422-0067},
support = {Y20240114//Young Foreign Talent Program/ ; },
mesh = {*Metabolic Engineering/methods ; *Gene Editing/methods ; *Benzaldehydes/metabolism/chemistry ; Cell Culture Techniques/methods ; CRISPR-Cas Systems ; Biosynthetic Pathways ; },
abstract = {2-Hydroxy-4-Methoxybenzaldehyde (2H4MB) is a valuable aromatic compound with applications in flavour, fragrance, and pharmaceuticals. Because of its endangered status and root-specific accumulation, its production in native plants is restricted. In order to increase 2H4MB yield, this study emphasises recent developments in metabolic engineering, synthetic biology, in vitro culture methods, and AI-assisted route prediction. This review discussed about how CRISPR-based genome editing can be used to modify important biosynthetic genes and regulatory components, as well as how predictive machine learning techniques can be used to improve production conditions. Inadequate genetic resources, poorly understood biosynthetic pathways, and a dearth of reliable transformation systems are among the present constraints. The work highlights the importance of using integrative plant biotechnology techniques to fully realise the industrial and medicinal potential of this underutilised chemical.},
}

*Metabolic Engineering/methods
*Gene Editing/methods
*Benzaldehydes/metabolism/chemistry
Cell Culture Techniques/methods
CRISPR-Cas Systems
Biosynthetic Pathways

@article {pmid41516423,
year = {2026},
author = {Kongsomboonchoke, P and Ariyachet, C and Kaewsapsak, P and Sirichindakul, P and Tangkijvanich, P},
title = {Development of a Sensitive and Specific RPA-CRISPR/Cas12a Assay for Intrahepatic Quantification of HBV cccDNA.},
journal = {International journal of molecular sciences},
volume = {27},
number = {1},
pages = {},
pmid = {41516423},
issn = {1422-0067},
support = {//Second Century Fund (C2F)/ ; //Center of Excellence in Hepatitis and Liver Cancer/ ; },
mesh = {Humans ; *Hepatitis B virus/genetics ; *DNA, Circular/genetics/analysis ; *CRISPR-Cas Systems/genetics ; *DNA, Viral/genetics/analysis ; *Hepatitis B/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; *Liver/virology ; Sensitivity and Specificity ; Recombinases/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Hepatitis B virus (HBV) persists in infected hepatocytes through covalently closed circular DNA (cccDNA), a stable episomal form that serves as the transcriptional template for viral replication. Accurate and sensitive quantification of intrahepatic cccDNA is crucial for evaluating antiviral therapies, particularly those targeting a functional cure. Here, we report the development of a novel, cccDNA-specific detection system combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a-based fluorescence detection. We designed and validated CRISPR RNAs (crRNAs) targeting HBV cccDNA-specific regions conserved across genotypes A-D. Reaction conditions for both RPA and Cas12a detection were optimized to enhance sensitivity, specificity, and accuracy. The system reliably detected as few as 10 copies of cccDNA-containing plasmid per reaction and showed no cross-reactivity with non-cccDNA forms in serum or plasma, indicating assay specificity. When applied to liver tissue samples from 10 HBV-infected and 6 non-HBV patients, the RPA-CRISPR/Cas12a assay exhibited a high sensitivity (90%) and a strong correlation with qPCR results (R[2] = 0.9155), confirming its accuracy. In the conclusion, the RPA-CRISPR/Cas12a system provides a robust, cost-effective, and scalable platform for sensitive and specific quantification of intrahepatic HBV cccDNA. This method holds promises for research and high-throughput therapeutic screening applications targeting cccDNA clearance.},
}

Humans
*Hepatitis B virus/genetics
*DNA, Circular/genetics/analysis
*CRISPR-Cas Systems/genetics
*DNA, Viral/genetics/analysis
*Hepatitis B/virology/diagnosis
*Nucleic Acid Amplification Techniques/methods
*Liver/virology
Sensitivity and Specificity
Recombinases/metabolism
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41517680,
year = {2026},
author = {Rehman, SU and Abbas, GH},
title = {CRISPR/CAS9-based gene editing in cancer therapy: A systematic review and meta-analysis on current status and future directions.},
journal = {Medicine},
volume = {105},
number = {2},
pages = {e47114},
pmid = {41517680},
issn = {1536-5964},
mesh = {*Gene Editing/methods/trends ; *CRISPR-Cas Systems ; *Neoplasms/therapy/genetics ; Humans ; *Genetic Therapy/methods/trends ; },
abstract = {BACKGROUND: The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has recently been discovered for gene editing and cancer therapy and its applications are expanding. This review and meta-analysis aim to assess the present and future of CRISPR/Cas9 based gene editing in cancer treatment and the way forward.

METHODS: The search was conducted in PubMed from 2015 to 2025 and 89 relevant studies were identified. The study design, CRISPR/Cas9 targets, delivery methods, therapeutic efficacy and limitations were extracted from the studies.

RESULTS: We reviewed the efficacy, challenges, and potential for translation of CRISPR/Cas9 in oncogene and tumor suppressor gene targeting and immune modulation. Several preclinical researches showed that CRISPR/Cas9 mediated disruption of oncogenes or restoration of tumor suppressor genes led to significant tumor regression. The evaluation was also extended to off target effects and integration with immunotherapy.

CONCLUSION: From the findings of this work, it can be concluded that CRISPR/Cas9 is a promising tool, but there are several limitations including off target effects, delivery systems and ethical issues that need to be solved in order to improve the clinical significance.},
}

*Gene Editing/methods/trends
*CRISPR-Cas Systems
*Neoplasms/therapy/genetics
Humans
*Genetic Therapy/methods/trends

@article {pmid41517973,
year = {2026},
author = {Jin, G and Li, H and Yu, H and Gu, Z and Cui, H and Chen, J and Li, X},
title = {Asp-2078-Gly mutation in ACCase confers quizalofop-p-ethyl resistance in Eleusine indica and establishment of a LAMP-CRISPR/Cas12a visual genotyping assay for the target mutation.},
journal = {Pest management science},
volume = {82},
number = {4},
pages = {3374-3383},
doi = {10.1002/ps.70468},
pmid = {41517973},
issn = {1526-4998},
support = {//the Key Project in Soybean Bio breeding and Commercialization/ ; //the China Agriculture Research System/ ; 2022ZD04021//the Sci-Tech Innovation 2030 Agenda/ ; },
mesh = {*Herbicide Resistance/genetics ; *Acetyl-CoA Carboxylase/genetics/metabolism ; *Eleusine/genetics/drug effects/enzymology ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; *Herbicides/pharmacology ; Mutation ; *Genotyping Techniques/methods ; *Plant Proteins/genetics/metabolism ; Molecular Diagnostic Techniques ; },
abstract = {BACKGROUND: Eleusine indica is a widespread, competitive weed causing yield losses in major crops. Repeated use of acetyl-CoA carboxylase (ACCase)-inhibiting herbicides in cotton fields has led to the evolution of resistant populations, posing a growing threat to cotton production in China. This study aimed to elucidate the target-site resistance mechanism of E. indica to quizalofop-p-ethyl and establish a rapid visual detection method based on the identified mutation.

RESULTS: The NJC-R population showed resistance to quizalofop-p-ethyl (resistance index = 5.5). Gene sequencing revealed that an Asp-2078-Gly mutation in ACCase was one of the mechanisms underlying resistance. Loop-mediated isothermal amplification (LAMP) combined with the CRISPR/Cas12a system was developed to detect this mutation in E. indica. This method not only enabled genotype discrimination (wild-type, heterozygous, homozygous mutant), but also provided visual results within 70 min, exhibiting superior performance compared with the derived cleaved amplified polymorphic sequences assay. In addition, this method eliminated false positives from nonspecific LAMP amplification, was ~100-fold more sensitive than a polymerase chain reaction, and the assay results were 100% concordant with Sanger sequencing for the 50 samples tested.

CONCLUSION: This study confirmed that the Asp-2078-Gly mutation confers quizalofop-p-ethyl resistance in E. indica from the cotton field in China, and LAMP-CRISPR/Cas12a was first applied for detecting ACCase target-site mutations in E. indica. Given its rapidity and high accuracy, this technique has the potential to be applied for resistance monitoring and to guide rational herbicide application. © 2026 Society of Chemical Industry.},
}

*Herbicide Resistance/genetics
*Acetyl-CoA Carboxylase/genetics/metabolism
*Eleusine/genetics/drug effects/enzymology
*Nucleic Acid Amplification Techniques/methods
CRISPR-Cas Systems
*Herbicides/pharmacology
Mutation
*Genotyping Techniques/methods
*Plant Proteins/genetics/metabolism
Molecular Diagnostic Techniques

@article {pmid41518077,
year = {2026},
author = {Liu, Y and Wang, H and Chen, L and Wu, X and Xu, Z and Huang, Q and Zhang, H and Cao, X and Liang, X and Zhong, X and Luo, C},
title = {CRISPR-Cas9-Generated TXNDC15 c.560delA Homozygous Mouse Model Exhibits Meckel-Gruber Syndrome Phenotype.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {64},
number = {1},
pages = {e70040},
pmid = {41518077},
issn = {1526-968X},
support = {SZSM202311005//Sanming Project of Medicine in Shenzen Municipality/ ; 2021A1515012495//Natural Science Foundation of Guangdong Province/ ; JCYJ20210324135009024//Shenzhen Science and Technology Innovation Commission/ ; },
mesh = {Animals ; Mice ; *Polycystic Kidney Diseases/genetics/pathology ; CRISPR-Cas Systems ; *Ciliary Motility Disorders/genetics/pathology ; *Encephalocele/genetics/pathology ; Disease Models, Animal ; Phenotype ; Homozygote ; *Retinitis Pigmentosa/genetics/pathology ; Frameshift Mutation ; },
abstract = {To determine whether TXNDC15 variation causes Meckel-Gruber syndrome (MKS), we assessed the pathogenicity of the frameshift variant c.560delA. A CRISPR-Cas9 generated mouse model carrying the equivalent Txndc15 c.512delA mutation was analyzed at embryonic day 15.5. Homozygous Txndc15[mt/mt] embryos displayed the complete MKS phenotype-fetal lethality, exencephaly, omphalocele, post-axial polydactyly, and polycystic kidneys-together with markedly reduced TXNDC15 protein in brain, liver, and kidney. These findings confirm TXNDC15 as a bona fide MKS disease gene.},
}

Animals
Mice
*Polycystic Kidney Diseases/genetics/pathology
CRISPR-Cas Systems
*Ciliary Motility Disorders/genetics/pathology
*Encephalocele/genetics/pathology
Disease Models, Animal
Phenotype
Homozygote
*Retinitis Pigmentosa/genetics/pathology
Frameshift Mutation

@article {pmid41518477,
year = {2026},
author = {Ghazaei, C},
title = {The role of bacteriophages and CRISPR-Cas in combating multidrug-resistant bacteria.},
journal = {Natural products and bioprospecting},
volume = {16},
number = {1},
pages = {14},
pmid = {41518477},
issn = {2192-2195},
abstract = {The alarming increase of multidrug-resistant (MDR) bacteria presents a serious global health crisis, reducing the effectivenessof traditional antibiotics and requiring alternative therapeutic strategies. Among the most promising innovations are bacteriophages-viruses that specifically infect bacteria-and CRISPR-Cas systems, molecular tools enabling precise genome editing. These technologies individually offer targeted antibacterial activity with minimal disturbance to the host microbiota. When combined, they forma synergistic platform capable of overcoming many limitations of conventional antibiotics, including broad-spectrum activity, resistance development, and limited adaptability. This review examinesmechanisms of bacterial resistance, the biological foundation of bacteriophages and CRISPR-Cas systems, and their application in fighting MDR pathogens. However, significant challenges remain, including delivery barriers, off-target effects, regulatory uncertainty, and public acceptance of gene-editing tools. Antimicrobial resistance now tanks among the top threats to global health, with an estimated burden exceeding one million deaths annually, surpassing many other infectious diseases. The article concludes with a discussion of the clinical prospects of phage-CRISPR therapies and highlights key areas for future research. By merging the specificity of phages with the programmable strength of CRISPR, these biotechnological advances provide a powerful and approach to address the growing threat of antibiotic resistance.},
}

@article {pmid41518500,
year = {2026},
author = {Álvarez, E and Franco-Zorrilla, JM},
title = {Targeted DNA Affinity Purification for Quantifying DNA-Binding Specificities of Transcription Factors.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2985},
number = {},
pages = {89-104},
pmid = {41518500},
issn = {1940-6029},
mesh = {*Transcription Factors/metabolism/genetics ; Binding Sites ; *DNA, Plant/metabolism/genetics ; High-Throughput Nucleotide Sequencing/methods ; Protein Binding ; *Chromatography, Affinity/methods ; Gene Expression Regulation, Plant ; *DNA/metabolism ; Arabidopsis/genetics/metabolism ; },
abstract = {Plant growth relies on flexible gene regulation to adapt to environmental changes. This process is ultimately controlled by transcription factors (TFs), which bind to specific DNA motifs, known as TF-binding sites (TFBS), located in the gene regulatory regions to regulate their expression. These interactions play crucial roles in plant development and responses to environmental cues, as well as in plant evolution and domestication, making both cis- (i.e., TFBS) and trans-regulatory factors as potential molecular targets in plant breeding for traits such as yield, quality, and stress resilience. These biotechnological approaches require precise knowledge of the target gene sets and TFBS specifically recognized by TFs. Recent advances in high-throughput sequencing techniques have enabled precise identification of TF target genes, especially thanks to methodologies that combine the main features of both in vitro and in vivo approaches. However, small scale and targeted approaches are still needed to evaluate the relative contribution of specific nucleotide positions in TF recognition. In this chapter, we describe a modified version of DNA Affinity Purification sequencing (DAP-seq) that replaces genomic DNA with a PCR-generated library of TFBS variants. This approach, termed targeted-DAP, allows a flexible and quantitative analysis of TF-binding using next-generation sequencing. Additionally, expressing TFs in Escherichia coli provides an economical source of proteins, enabling scalable and cost-effective analysis of DNA-binding specificities. We showed the benefits of this technique to demonstrate the contribution of the genomic context around the TFBS for specific recognition of a bHLH TF. Development of targeted DAP-seq would be of interest for the evaluation of nucleotide variation-either allelic or generated by CRISPR/Cas-within TFBS in TF recognition with predictable consequences on plant phenotypes.},
}

*Transcription Factors/metabolism/genetics
Binding Sites
*DNA, Plant/metabolism/genetics
High-Throughput Nucleotide Sequencing/methods
Protein Binding
*Chromatography, Affinity/methods
Gene Expression Regulation, Plant
*DNA/metabolism
Arabidopsis/genetics/metabolism

@article {pmid41519779,
year = {2026},
author = {Lim, MYT and Tan, C and Subhramanyam, CS and Teo, SJ and DeFalco, L and Pasaribu, SK and Koh, CH and Rayamajhi, D and Chi, J and Li, S and Wee, KB and Roy, S and Huber, RG and Aw, SS},
title = {A programmable ribozyme for RNA signal transduction.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1428},
pmid = {41519779},
issn = {2041-1723},
support = {H20C6a0034//Agency for Science, Technology and Research (A*STAR)/ ; UIBR//Agency for Science, Technology and Research (A*STAR)/ ; Cell and Gene Therapy Flagship Grant C253623010//Agency for Science, Technology and Research (A*STAR)/ ; IAF-PP H20C6a0034//Agency for Science, Technology and Research (A*STAR)/ ; OFYIRG16may045//MOH | National Medical Research Council (NMRC)/ ; Core funding//A*STAR | Institute of Molecular and Cell Biology (Institute of Molecular and Cell Biology - A STAR)/ ; },
mesh = {*RNA, Catalytic/metabolism/genetics/chemistry ; Humans ; Animals ; Zebrafish/genetics/embryology ; Aptamers, Nucleotide/metabolism/genetics ; CRISPR-Cas Systems/genetics ; *Signal Transduction/genetics ; Gene Editing/methods ; HEK293 Cells ; MicroRNAs/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *RNA/metabolism/genetics ; },
abstract = {RNA detection applications can be augmented if a sensed RNA can be directly functionally transduced. However, there is no generalisable approach that allows an RNA trigger itself to directly activate diverse non-coding RNA effectors. Here, we report engineering of a programmable, RNA trigger-activated, dual-site self-cleaving ribozyme with modular sensing domain and cleavage product. This platform, UNlocked by Activating RNA (UNBAR), is entirely encoded within one RNA strand. The ribozyme can be designed to be almost completely inactive in absence of trigger, and to exhibit single-nucleotide trigger specificity. UNBAR ribozymes carry out cell-free sensing and protein-free amplification of microRNA and viral RNA sequences, and trigger-dependent release of ncRNA effectors sgRNA, shRNA and aptamer. We demonstrate RNA detection and functional transduction by a cleaved aptamer, whose fluorescence can be directly read out as a function of trigger RNA. We further engineer the ribozyme for function in cells, and demonstrate trigger-dependent regulation of CRISPR-Cas9 editing by sgRNA-embedded ribozymes in zebrafish embryos and human cells. UNBAR is a first-in-class modality with potential to be developed into a versatile platform for synthetic biology, diagnostics and gene regulation.},
}

*RNA, Catalytic/metabolism/genetics/chemistry
Humans
Animals
Zebrafish/genetics/embryology
Aptamers, Nucleotide/metabolism/genetics
CRISPR-Cas Systems/genetics
*Signal Transduction/genetics
Gene Editing/methods
HEK293 Cells
MicroRNAs/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*RNA/metabolism/genetics

@article {pmid41519853,
year = {2026},
author = {Michael Deans, PJ and Retallick-Townsley, KG and Li, A and Seah, C and Johnson, J and Garcia Gonzalez, J and Cao, E and Schrode, N and Yu, A and Cartwright, S and Voloudakis, G and Zhang, W and Wang, M and Fullard, JF and Girdhar, K and Stahl, E and Akbarian, S and Zhang, B and Roussos, P and O'Reilly, P and Huckins, LM and Brennand, KJ},
title = {Functional implications of polygenic risk for schizophrenia in human neurons.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1355},
pmid = {41519853},
issn = {2041-1723},
support = {R01ES033630//U.S. Department of Health & Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS)/ ; R56MH101454//U.S. Department of Health & Human Services | NIH | National Institute of Mental Health (NIMH)/ ; T35 DK104689/DK/NIDDK NIH HHS/United States ; R01 ES033630/ES/NIEHS NIH HHS/United States ; R56 MH101454/MH/NIMH NIH HHS/United States ; },
mesh = {Humans ; *Schizophrenia/genetics ; *Multifactorial Inheritance/genetics ; *Neurons/metabolism ; Induced Pluripotent Stem Cells/metabolism ; *Genetic Predisposition to Disease ; Genome-Wide Association Study ; CRISPR-Cas Systems ; Transcriptome ; Polymorphism, Single Nucleotide ; },
abstract = {Genome wide association studies of schizophrenia reveal a complex polygenic risk architecture comprised of hundreds of risk variants; most are common in the population, non-coding, and act by genetically regulating the expression of one or more gene targets ("eGenes"). It remains unclear how the myriad genetic variants that are predicted to confer individually small effects combine to yield substantial clinical impacts in aggregate. Here, we demonstrate that convergence (i.e., the shared downstream transcriptomic changes with a common direction of effect), resulting from one-at-a-time perturbation of schizophrenia eGenes, influences the outcome when eGenes are manipulated in combination. In total, we apply pooled and arrayed CRISPR approaches to target 21 schizophrenia eGenes in human induced pluripotent stem cell-derived glutamatergic neurons, finding that functionally similar eGenes yield stronger and more specific convergent effects. Points of convergence constrain additive relationships between polygenic risk loci: consistent with a liability threshold model, combinatorial perturbations of these same schizophrenia eGenes reveal that pathway-level convergence predicts when observed effects will fail to sum to levels predicted by an additive model. Targeting points of convergence as novel therapeutic targets may prove more efficacious than individually reversing the effects of multiple risk loci.},
}

Humans
*Schizophrenia/genetics
*Multifactorial Inheritance/genetics
*Neurons/metabolism
Induced Pluripotent Stem Cells/metabolism
*Genetic Predisposition to Disease
Genome-Wide Association Study
CRISPR-Cas Systems
Transcriptome
Polymorphism, Single Nucleotide

@article {pmid41519897,
year = {2026},
author = {Boutin, J and Fayet, S and Marin, V and Bergès, C and Riandière, M and Toutain, J and Lamrissi-Garcia, I and Thibault, C and Cappellen, D and Dabernat, S and Poulet, A and Francillette, M and Droin, N and Debeissat, C and Brunet de la Grange, P and Moreau-Gaudry, F and Bedel, A},
title = {Single-cell multiplex approaches deeply map ON-target CRISPR-genotoxicity and reveal its mitigation by palbociclib and long-term engraftment.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1429},
pmid = {41519897},
issn = {2041-1723},
mesh = {*Piperazines/pharmacology ; Animals ; *Pyridines/pharmacology ; Humans ; *Single-Cell Analysis/methods ; Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Mice ; Polymorphism, Single Nucleotide ; Hematopoietic Stem Cells/drug effects/metabolism ; Loss of Heterozygosity ; Female ; },
abstract = {Genome editing by CRISPR-Cas9-nuclease is promising for gene therapy. However, safety concerns remain. Monitoring ON-target genotoxicity is essential, especially to assay megabasic rearrangements at the targeted locus. Here, we developed a combined single-cell resolution approach with DNA sequencing focused on single nucleotide polymorphism (scSNP-DNAseq), micronuclei and LOH cytometry-reporter assays. This sensitive multiplexed strategy enables the sensitive monitoring of CRISPR-mediated genotoxicity in primary cells. Using this approach, we detect, map and characterize various types of induced-losses of heterozygosity and assess editing-associated chromosomal instability. Importantly, palbociclib prevents the appearance of such genomic rearrangements in hematopoietic stem cells without impairing cell fate or graft capability. Conversely, short-term risk is significantly increased with DNA-PKcs inhibitor AZD7648. Fortunately, targeting HBG1/2p, scSNP-DNA-seq reveals that ON-target genotoxic events are no longer detectable after long-term xenografts. This work demonstrates that scSNP-DNA-seq should be routinely implemented to monitor chromosomal rearrangements before and after CRISPR-edited cell infusions.},
}

*Piperazines/pharmacology
Animals
*Pyridines/pharmacology
Humans
*Single-Cell Analysis/methods
Gene Editing/methods
*CRISPR-Cas Systems/genetics
Mice
Polymorphism, Single Nucleotide
Hematopoietic Stem Cells/drug effects/metabolism
Loss of Heterozygosity
Female