@article {pmid40958004,
year = {2026},
author = {Pham, DL and Cappabianca, D and Forsberg, MH and Weaver, C and Mueller, KP and Tommasi, A and Vidugiriene, J and Lauer, A and Sylvester, K and Lika, J and Bugel, M and Fan, J and Capitini, CM and Saha, K and Skala, MC},
title = {Label-free metabolic imaging monitors the fitness of chimeric antigen receptor T cells.},
journal = {Nature biomedical engineering},
volume = {10},
number = {4},
pages = {785-802},
pmid = {40958004},
issn = {2157-846X},
support = {P30 CA014520/CA/NCI NIH HHS/United States ; R35 GM119644/GM/NIGMS NIH HHS/United States ; T32 GM140935/GM/NIGMS NIH HHS/United States ; P41 EB031772/EB/NIBIB NIH HHS/United States ; NSF-EEC 1648035//National Science Foundation (NSF)/ ; R01 CA278051/CA/NCI NIH HHS/United States ; S10 OD025225/OD/NIH HHS/United States ; R01 CA278051/CA/NCI NIH HHS/United States ; R01 CA278051/CA/NCI NIH HHS/United States ; R01 CA278051/CA/NCI NIH HHS/United States ; },
mesh = {*Receptors, Chimeric Antigen/metabolism/genetics ; Humans ; Animals ; *T-Lymphocytes/metabolism/cytology/immunology ; Mice ; Immunotherapy, Adoptive/methods ; CRISPR-Cas Systems ; Cell Line, Tumor ; *Optical Imaging/methods ; Tumor Microenvironment ; Gene Editing ; },
abstract = {Chimeric antigen receptor (CAR) T cell therapy for solid tumours is challenging because of the immunosuppressive tumour microenvironment and a complex manufacturing process. Cellular manufacturing protocols directly impact CAR T cell yield, phenotype and metabolism, which correlates with in vivo potency and persistence. Although metabolic fitness is a critical quality attribute, how T cell metabolic requirements vary throughout the manufacturing process remains unexplored. Here we use optical metabolic imaging (OMI), a non-invasive, label-free method to evaluate single-cell metabolism. Using OMI, we identified the impacts of media composition on CAR T cell metabolism, activation strength and kinetics, and phenotype. We demonstrate that OMI parameters can indicate cell cycle stage and optimal gene transfer conditions for both viral transduction and electroporation-based CRISPR/Cas9. In a CRISPR-edited anti-GD2 CAR T cell model, OMI measurements allow accurate prediction of an oxidative metabolic phenotype that yields higher in vivo potency against neuroblastoma. Our data support OMI as a robust, sensitive analytical tool to optimize manufacturing conditions and monitor cell metabolism for increased CAR T cell yield and metabolic fitness.},
}

*Receptors, Chimeric Antigen/metabolism/genetics
Humans
Animals
*T-Lymphocytes/metabolism/cytology/immunology
Mice
Immunotherapy, Adoptive/methods
CRISPR-Cas Systems
Cell Line, Tumor
*Optical Imaging/methods
Tumor Microenvironment
Gene Editing

@article {pmid41638392,
year = {2026},
author = {Zhang, P and Shen, M and Ding, L and He, L and Wu, Y and Yu, S},
title = {Controlled CRISPR/Cas12a activation via DNAzyme-mediated splitting of chimeric substrate for lead detection.},
journal = {Biochimie},
volume = {244},
number = {},
pages = {1-6},
doi = {10.1016/j.biochi.2026.02.001},
pmid = {41638392},
issn = {1638-6183},
mesh = {*DNA, Catalytic/metabolism/chemistry ; *Lead/analysis ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/metabolism ; *CRISPR-Associated Proteins/metabolism ; Humans ; *Bacterial Proteins/metabolism ; },
abstract = {Chronic lead exposure poses severe threats to human health, which demands a rapid detection strategy beyond conventional instrumentation-dependent approaches. While CRISPR/Cas12a systems offer promising alternatives through trans-cleavage activity, conventional Pb[2+] biosensors relying on DNAzyme-generated intact activators suffer from high background signals due to interference from uncleaved substrates. To address this limitation, we developed a steric-hindrance-controlled activation strategy by employing a chimeric DNAzyme substrate (Sub) that prevents Cas12a binding until Pb[2+]-dependent cleavage occurs. This DNAzyme-mediated splitting releases two fragments (A1/A2) that rearrange into split activators, triggering the CRISPR/Cas12a trans-cleavage of a quenched reporter (6-FAM/BHQ1). Under the optimal condition, the sensor achieved a linear detection range of 2.5-25 μM (R[2] = 0.998) with 2.18 μM LOD and high selectivity against interferents. Validation in tap water matrices demonstrated 98.6%-102.6% recovery (RSD 3.0%-7.5%), which showed robustness in real samples. This split-activator design paradigm eliminates background from uncleaved substrates without additional pretreatment steps to provide a versatile template for converting metal ions into CRISPR-detectable signals.},
}

*DNA, Catalytic/metabolism/chemistry
*Lead/analysis
*CRISPR-Cas Systems
*Biosensing Techniques/methods
*Endodeoxyribonucleases/metabolism
*CRISPR-Associated Proteins/metabolism
Humans
*Bacterial Proteins/metabolism

@article {pmid41673138,
year = {2026},
author = {Dörr, J and Gregor, L and Lacher, SB and Oner, A and Sun, Y and Piseddu, I and Fertig, L and Spajic, S and Lesch, S and Michaelides, S and Seifert, M and Gottschlich, A and Samson, N and Majed, L and Briukhovetska, D and Simnica, D and Hartmann, V and Gabriel, K and Cohen, S and Boland, GM and Andreu-Sanz, D and Carlini, E and Stock, S and Holtermann, A and Müller, PJ and Strzalkowski, T and Trefny, MP and Endres, S and Jenkins, RW and Böttcher, JP and Kobold, S},
title = {Ablation of prostaglandin E2 signalling through dual receptor knockout in CAR T cells enhances therapeutic efficacy in solid tumours.},
journal = {Nature biomedical engineering},
volume = {10},
number = {4},
pages = {697-710},
pmid = {41673138},
issn = {2157-846X},
support = {101124203//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; },
mesh = {*Dinoprostone/metabolism ; Humans ; Animals ; *Receptors, Prostaglandin E, EP4 Subtype/genetics/metabolism/deficiency ; Mice ; *Signal Transduction ; *Receptors, Prostaglandin E, EP2 Subtype/genetics/metabolism/deficiency ; *Receptors, Chimeric Antigen/metabolism/genetics ; Cell Line, Tumor ; *Immunotherapy, Adoptive/methods ; *T-Lymphocytes/metabolism/immunology ; *Neoplasms/therapy/immunology/metabolism ; Gene Knockout Techniques ; Tumor Microenvironment ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; Female ; },
abstract = {The efficacy of chimeric antigen receptor (CAR) T cell therapy in solid cancers is limited by immunosuppression in the tumour microenvironment (TME). Prostaglandin E2 (PGE2) is a key factor locally inhibiting T cell function. We hypothesized that targeted ablation of PGE2 signalling in CAR T cells may enhance their activity in PGE2-rich solid tumours. Here we generate knockout CAR T cells double deficient for the PGE2 receptors EP2 and EP4 (EP2[-/-]EP4[-/-]) by CRISPR-Cas9 engineering. EP2[-/-]EP4[-/-] CAR T cells expanded unabatedly in the presence of PGE2. Further, they effectively controlled syngeneic and human xenograft tumour models in vivo, which was accompanied by intratumoural accumulation and persistence of modified T cells. Improved anti-tumour activity was also observed against patient-derived tumour samples from patients with pancreatic ductal adenocarcinoma (PDAC), colorectal (CRC) and neuroendocrine (NET) cancer. Our data uncovers the detrimental impact of PGE2-mediated suppression on CAR T cell efficacy and highlights EP2 and EP4 targeting as a potential strategy.},
}

*Dinoprostone/metabolism
Humans
Animals
*Receptors, Prostaglandin E, EP4 Subtype/genetics/metabolism/deficiency
Mice
*Signal Transduction
*Receptors, Prostaglandin E, EP2 Subtype/genetics/metabolism/deficiency
*Receptors, Chimeric Antigen/metabolism/genetics
Cell Line, Tumor
*Immunotherapy, Adoptive/methods
*T-Lymphocytes/metabolism/immunology
*Neoplasms/therapy/immunology/metabolism
Gene Knockout Techniques
Tumor Microenvironment
Xenograft Model Antitumor Assays
CRISPR-Cas Systems
Female

@article {pmid41794998,
year = {2026},
author = {Higuchi, R and Tatara, M and Horino, S and Katori, S and Sanbo, M and Hirabayashi, M and Watanabe, M and Konno, A and Hirai, H and Yoshioka, Y and Hirabayashi, T and Kobayashi, T and Kaneko, R and Yagi, T},
title = {Protocadherin γC4 regulates neuronal survival and dendritic self-avoidance.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41794998},
issn = {2399-3642},
support = {22H05498//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 24H01237//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 25H02501//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; },
mesh = {Animals ; *Cadherins/genetics/metabolism ; *Dendrites/metabolism/physiology ; Mice ; *Neurons/metabolism ; Cell Survival ; Cadherin Related Proteins ; Protocadherins ; CRISPR-Cas Systems ; Disease Models, Animal ; Purkinje Cells/metabolism ; },
abstract = {Animal models are indispensable for linking human genetic findings to disease mechanisms. Mutations in protocadherin gamma C4 (γC4), one of the 22 isoforms encoded by the protocadherin-γ (Pcdh-γ) gene cluster, cause a human neurodevelopmental syndrome with progressive microcephaly, seizures, and intellectual disability. Here, we established a γC4 mutant mouse model that exhibits motor dysfunction, seizures, reduced brain size, and increased embryonic neuronal apoptosis. Using DOMINO (Double Mutation-Induced Open Reading Frame Switch), a two-step CRISPR/Cas9-based genome-editing strategy, we also generated γC4fl-only mice that retain full-length γC4 while truncating the other 21 Pcdh-γ isoforms. Unlike Pcdh-γ cluster-deficient mice, γC4fl-only mice were viable and fertile. Furthermore, we show that the γC4 constant region (γCR) contributes to the regulation of Purkinje cell dendritic architecture and self-avoidance. Together, these findings indicate that γCR-containing γC4 is required for neuronal survival and dendritic patterning, supporting γC4 as a principal functional isoform within the Pcdh-γ gene cluster.},
}

Animals
*Cadherins/genetics/metabolism
*Dendrites/metabolism/physiology
Mice
*Neurons/metabolism
Cell Survival
Cadherin Related Proteins
Protocadherins
CRISPR-Cas Systems
Disease Models, Animal
Purkinje Cells/metabolism

@article {pmid41812962,
year = {2026},
author = {Yogi, D and Shashikala, T and Subramanian, G and Kumar, A and Kaninika, V and Manamohan, M and Jithesh, MN and Jha, GK and Asokan, R and Ashok, K},
title = {First report of CRISPR prime editing in a globally significant non-model organism, the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae).},
journal = {Methods (San Diego, Calif.)},
volume = {250},
number = {},
pages = {36-45},
doi = {10.1016/j.ymeth.2026.03.001},
pmid = {41812962},
issn = {1095-9130},
mesh = {Animals ; *Spodoptera/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; },
abstract = {The power of CRISPR/Cas9 mediated genome editing has been harnessed in different facets of entomological research, particularly useful in developing genetic pest management strategies. The edits thus obtained are robust and results in a loss-of-function of the target gene. Recently the development of newer editing approach called Prime editing is yet another addition in the insect editing tool-box. In this regard, the prime editing offers a transformative approach to precise genome manipulation by enabling targeted insertions, deletions, and nucleotide substitutions without double-strand break or donor template. While its application has been explored in mammalian system and plants, its deployment through the delivery of ribonucleoprotein complex (RNP) has been demonstrated for the first-time in the globally significant pest, Spodoptera frugiperda. Using a Cas9 (H840A)-reverse transcriptase fusion protein (PE2) and a customized prime editing guide RNA (pegRNA), we targeted exon 3 of the Tryptophan 2,3-dioxygenase (SfTO) gene to introduce a premature stop codon. Recombinant PE2 protein was expressed in E. coli, purified, and validated functionally through RT-PCR. The Ribonucleoprotein complex was microinjected into G0 eggs and subsequent genotyping revealed successful edits, including perfect and imperfect prime edits, as well as unintended mutations. Phenotyping revealed the mutants with altered eye pigmentation, and chromatographic analysis confirmed disruption in ommochrome biosynthesis, validating functional consequences of Prime editing. This study provides a foundational proof-of-concept for Prime editing in insect pests, opening new avenues for functional genomics and designing next-generation pest management strategies.},
}

Animals
*Spodoptera/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/genetics

@article {pmid41825300,
year = {2026},
author = {Low, YC and McKnight, CL and Elliott, DA and Thorburn, DR and Frazier, AE},
title = {Generation of a pluripotent embryonic stem cell TAFAZZIN hESC model (WAe009-A-3H) of Barth syndrome.},
journal = {Stem cell research},
volume = {93},
number = {},
pages = {103948},
doi = {10.1016/j.scr.2026.103948},
pmid = {41825300},
issn = {1876-7753},
mesh = {Humans ; *Barth Syndrome/pathology/genetics/metabolism ; *Human Embryonic Stem Cells/metabolism ; Female ; *Transcription Factors/genetics/metabolism ; Cell Line ; Acyltransferases/genetics ; CRISPR-Cas Systems ; *Pluripotent Stem Cells/metabolism ; Cell Differentiation ; },
abstract = {Barth syndrome is among the most common mitochondrial diseases presenting with cardiomyopathy. We have generated a human embryonic stem cell (hESC) model of Barth syndrome (TAFAZZIN[Δ3] C15) in a female background (H9 hESC) using CRISPR/Cas9 gene editing, with compound heterozygous variants in TAFAZZIN that result in exon 3 skipping in all stable transcripts. This cell line displayed characteristics consistent with pluripotent stem cells, including typical colony morphology, expression of pluripotency markers, trilineage potential, and a normal karyotype. This TAFAZZIN[Δ3] C15 line could be used for investigation of disease mechanisms in mitochondrial cardiomyopathy and preclinical therapeutic screening.},
}

Humans
*Barth Syndrome/pathology/genetics/metabolism
*Human Embryonic Stem Cells/metabolism
Female
*Transcription Factors/genetics/metabolism
Cell Line
Acyltransferases/genetics
CRISPR-Cas Systems
*Pluripotent Stem Cells/metabolism
Cell Differentiation

@article {pmid41825380,
year = {2026},
author = {Wang, X and Zhang, X and Guo, Y and Zheng, Q and Hu, B and Liu, R and Cao, J},
title = {CRISPR/Cas12-driven portable paper-based electrochemical aptasensor based on 0D/2D Au@Ti3C2 MXene for AFB1 detection.},
journal = {Food chemistry},
volume = {511},
number = {},
pages = {148810},
doi = {10.1016/j.foodchem.2026.148810},
pmid = {41825380},
issn = {1873-7072},
mesh = {*Aflatoxin B1/analysis ; Food Contamination/analysis ; *Electrochemical Techniques/instrumentation/methods ; Gold/chemistry ; Paper ; *Aptamers, Nucleotide/chemistry ; *Biosensing Techniques/instrumentation/methods ; CRISPR-Cas Systems ; Limit of Detection ; Nitrites ; Transition Elements ; },
abstract = {Aflatoxin B1 (AFB1) is a widely distributed and potent carcinogenic small-molecule toxic contaminant, posing significant risks to food safety. Therefore, a portable, cost-effective, and field-deployable method is needed for sensitive detection of AFB1. This study developed a paper-based electrochemical aptasensor integrating CRISPR/Cas12a technology and 0D/2D Au@Ti3C2 MXene heterostructure for highly sensitive and rapid detection of AFB1. The aptasensor leveraged the Au@Ti3C2 MXene with excellent electron transfer property to amplify the electrochemical signals, while the CRISPR/Cas12a trans-cleavage activity, triggered by AFB1 specific aptamers, enabled high specificity. Utilizing a portable electrochemical workstation, the paper-based electrochemical sensing platform exhibited a linear detection range of 0.5 pg/mL-100 ng/mL with a limit of detection of 0.16 pg/mL and achieved recoveries of 93.0%-106.0% in spiked real samples, demonstrating promise as a rapid and portable tool for on-site analysis of mycotoxins in real samples.},
}

*Aflatoxin B1/analysis
Food Contamination/analysis
*Electrochemical Techniques/instrumentation/methods
Gold/chemistry
Paper
*Aptamers, Nucleotide/chemistry
*Biosensing Techniques/instrumentation/methods
CRISPR-Cas Systems
Limit of Detection
Nitrites
Transition Elements

@article {pmid41826696,
year = {2026},
author = {Aird, EJ and Rabl, J and Knuesel, T and Groen, K and Awwad, SW and Korablev, B and Scherpe, L and Al-Herz, W and Hupfer, R and Recher, M and Jackson, SP and Hale, BG and Corn, JE},
title = {An SP110-SP100 axis is a critical regulator of promyelocytic leukaemia body dynamics and mitotic fidelity.},
journal = {Nature cell biology},
volume = {28},
number = {4},
pages = {684-695},
pmid = {41826696},
issn = {1476-4679},
support = {855741-DDREAMM-ERC-2019-SyG//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; 310030_188858//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 320030_232029//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; ALTF 144-2021//European Molecular Biology Organization (EMBO)/ ; Women's Postdoctoral Career Development Award in Science//Weizmann Institute of Science/ ; Outstanding Postdoctoral Women Fellowship//Council for Higher Education of Israel | Israeli Centers for Research Excellence (I-CORE)/ ; DRCPGM\100005//Cancer Research UK (CRUK)/ ; SEBINT-2024/100003//Cancer Research UK (CRUK)/ ; },
mesh = {Humans ; *Mitosis ; *Autoantigens/metabolism/genetics ; *Promyelocytic Leukemia Protein/metabolism/genetics ; *Antigens, Nuclear/metabolism/genetics ; *Leukemia, Promyelocytic, Acute/genetics/metabolism/pathology ; CRISPR-Cas Systems ; DNA Damage ; },
abstract = {Stimulation of the innate immune system by foreign RNA elicits a potent interferon response and can trigger cell death. The mechanisms by which cells balance a robust response with cell-intrinsic lethality are still being uncovered. Here, using genome-wide CRISPR-Cas9 genetic screens with triphosphorylated RNA stimulation, we discover that promyelocytic leukaemia (PML) nuclear body-localized speckled protein 110 (SP110) is a potent inhibitor of type 1 interferon-driven cell death. Death suppression by SP110 counteracts a toxic activity of SP100, a major constituent of PML bodies. Loss of SP110 leads to mitotic retention of SP100 and PML bodies, which associate with and perturb segregating chromosomes, leading to micronucleus formation, DNA damage and genotoxic cell death. A combination of cryo-electron microscopy, AlphaFold modelling and cellular biochemistry reveals that SP110 dissolves toxic SP100 oligomers via necessary and sufficient direct interactions between their caspase activation and recruitment domains. These data reveal the critical roles of SP100 and SP110 in governing the disassembly of PML bodies during mitosis, as well as the repercussions if this process is misregulated.},
}

Humans
*Mitosis
*Autoantigens/metabolism/genetics
*Promyelocytic Leukemia Protein/metabolism/genetics
*Antigens, Nuclear/metabolism/genetics
*Leukemia, Promyelocytic, Acute/genetics/metabolism/pathology
CRISPR-Cas Systems
DNA Damage

@article {pmid41826830,
year = {2026},
author = {Zhang, H and Zhang, P and Bindels, E and Mulugeta, E},
title = {Insights from pooled CRISPRi single-cell screens in K562 cells reveal gene functions, regulatory networks, and highlight opportunities and limitations.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {},
pmid = {41826830},
issn = {1471-2164},
mesh = {Humans ; K562 Cells ; *Gene Regulatory Networks ; *Single-Cell Analysis/methods ; *CRISPR-Cas Systems ; Transcription Factors/genetics ; Gene Knockdown Techniques ; *Clustered Regularly Interspaced Short Palindromic Repeats ; LIM Domain Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Pooled CRISPR screening combined with single-cell RNA sequencing (scRNA-seq) has emerged as a powerful strategy for dissecting gene function and reconstructing gene regulatory networks (GRNs) in complex biological systems. This approach enables high-throughput, parallel perturbation of multiple genes while providing transcriptome-wide readouts at single-cell resolution, overcoming many limitations of traditional arrayed screens. However, its broader application remains limited by technical challenges, including variable perturbation efficiency and difficulties in accurately identifying perturbed cells.In this study, we adapted and applied a modified CRISPR droplet sequencing (CROP-seq) protocol using CRISPR interference (CRISPRi) in K562 cells to knockdown six transcription factors (TFs): LMO2, TCF3, LDB1, MYB, GATA2, and RUNX1. Our modified approach, which allows direct capture of sgRNAs from the cDNA library without a separate enrichment step, significantly improved sgRNA assignment per cell. We successfully achieved reproducible knockdown of three TFs (MYB, GATA2, and LMO2), captured the impact of these perturbations on the TF target genes, and enabled us to reconstruct their GRNs and identify key regulons and transcriptional targets. These networks revealed both previously established (such as LMO2 GATA2 interaction) and novel regulatory interactions, which we independently validated, providing new insights into hematopoietic transcriptional control. To assess the efficiency of CRISPRi based pooled perturbation, we additionally analyzed publicly available Perturb-seq CRISPRi datasets and found that only ~40-50% of targeted genes led to effective knockdown, underscoring the variability in perturbation efficiency across experiments.Together, our results demonstrate both the potential and the current technical limitations of pooled CRISPRi-based single-cell screens. While this integrated approach holds great promise for high-resolution functional genomics, further optimization and standardized benchmarking are essential to improve its reliability, scalability, and reproducibility.},
}

Humans
K562 Cells
*Gene Regulatory Networks
*Single-Cell Analysis/methods
*CRISPR-Cas Systems
Transcription Factors/genetics
Gene Knockdown Techniques
*Clustered Regularly Interspaced Short Palindromic Repeats
LIM Domain Proteins/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41834297,
year = {2026},
author = {Zhu, L and Xiong, W and Yang, S and Qi, Q and Liu, X and Zhou, X and Tian, T},
title = {Dynamic Control of RNA Structure and Function through Bioorthogonal Staudinger Chemistry.},
journal = {ACS chemical biology},
volume = {21},
number = {4},
pages = {835-843},
doi = {10.1021/acschembio.6c00067},
pmid = {41834297},
issn = {1554-8937},
mesh = {*RNA/chemistry/metabolism ; Azides/chemistry ; Humans ; CRISPR-Cas Systems ; Nucleic Acid Conformation ; *Imidazoles/chemistry ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/chemistry ; },
abstract = {Here, we report a reversible chemical strategy for regulating RNA function through a Staudinger reaction-mediated postsynthetic modification. We designed a bifunctional azide reagent, 1,3-diazidopropan-2-yl 1H-imidazol-1-carboxylate (DAPIC), which specifically modifies the 2'-hydroxyl of RNA, thereby disrupting RNA structure and function. Treatment with 2-diphenylphosphinoethylamine (DPPEA) reactivates the modified RNA through an efficient Staudinger reduction. This approach enables reversible modulation of RNA folding, hybridization, and protein-binding interactions, and can be applied to guide RNAs in the CRISPR-Cas9 system. DAPIC modification completely abrogates Cas9-mediated DNA cleavage, which is restored in a DPPEA concentration-dependent manner both in vitro and in living cells. Compared with monoazide derivatives, DAPIC exhibits enhanced reactivity and reduced reagent requirements. This Staudinger-based RNA regulation platform establishes a robust and generalizable chemical tool for conditional gene editing and studies of RNA function in complex biological environments.},
}

*RNA/chemistry/metabolism
Azides/chemistry
Humans
CRISPR-Cas Systems
Nucleic Acid Conformation
*Imidazoles/chemistry
Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/chemistry

@article {pmid41851507,
year = {2026},
author = {Zheng, R and Lu, Z and Wei, R and Shin, YC and Du, J and Zhang, Q and Li, J and Wang, X and Wei, Y and Liu, B and Chen, Y and Ding, L and Zhang, H and Chen, H and Huang, J and Ma, L},
title = {Improving the efficiency of high-fidelity Cas9 by enhancing PAM-distal interactions.},
journal = {Nature structural & molecular biology},
volume = {33},
number = {4},
pages = {590-602},
pmid = {41851507},
issn = {1545-9985},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism/chemistry ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Cryoelectron Microscopy ; Humans ; Mutation ; Models, Molecular ; },
abstract = {Engineering CRISPR enzymes for high fidelity often impairs cleavage activity. Meanwhile, a mechanistic understanding of why high-fidelity mutations reduce Cas9's cleavage activity remains unclear, presenting a challenge in balancing nuclease specificity and efficiency for clinical applications. In this study, we show that extending the spacer region to 21 or 22 nucleotides restores the impaired cleavage activity of SuperFi-Cas9, a high-fidelity Cas9 variant with 7 mutations in the RuvC domain at the protospacer adjacent motif (PAM)-distal region. Cryo-electron microscopy structures and mutational analyses reveal that the negatively charged mutations in a protruding loop of the RuvC domain create repulsive forces that destabilize the nuclease-single guide (sg)RNA-DNA complex. Spacer extension enhances interactions in the PAM-distal region, effectively restoring cleavage activity and balancing editing efficiency with specificity. In addition, we develop a deep learning model, AIdit-SuperFi, to predict optimal sgRNA length for high-fidelity genome editing. Our findings introduce a straightforward strategy to enhance CRISPR complex stability and provide mechanistic insights into the impaired cleavage activity of engineered high-fidelity Cas9, presenting a pathway toward precise and efficient genome editing and clinical translation of CRISPR technologies.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/genetics/metabolism/chemistry
RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
Cryoelectron Microscopy
Humans
Mutation
Models, Molecular

@article {pmid41882346,
year = {2026},
author = {Wang, F and Guo, R and Zhang, S and Cui, Y and Wang, J and Hu, T and Liu, K and Wang, Q and Liu, Y and Nam, KH and Zhao, ZW and Ji, Q and Xu, X and Wang, E and Zhu, Y and Yang, Y and Luo, M and Ma, P and Ma, S and Xu, C and Hu, C},
title = {Structural insight into IscB's RNA-lid-based inactivation mechanism.},
journal = {Nature structural & molecular biology},
volume = {33},
number = {4},
pages = {603-614},
pmid = {41882346},
issn = {1545-9985},
mesh = {Cryoelectron Microscopy ; *RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism/genetics ; Models, Molecular ; *Escherichia coli Proteins/chemistry/metabolism/genetics ; Nucleic Acid Conformation ; Escherichia coli/genetics/metabolism ; CRISPR-Associated Protein 9/metabolism/chemistry/genetics ; },
abstract = {IscB, a compact Cas9 ancestor from the obligate mobile element guided activity system, has attracted growing interest as a programmable genome editor because of its small size and therapeutic delivery potential. Despite its promise, structural insights into IscB's regulation remain limited, with only a target-bound R-loop structure previously reported. Here, we present the structural trajectory of an engineered IscB, capturing its transition from a resting state to activation. Using cryo-electron microscopy, we resolve four high-resolution structures: the apo resting state, two intermediate complexes with 6-nt and 10-nt guide-target pairing and a fully paired 16-nt primed cleavage state. These structures uncover a dual inactivation mechanism mediated by RNA lids; the ωRNA lid blocks HNH domain access, while the guide RNA lid occludes the RuvC active site. As guide-target pairing progresses, the guide RNA undergoes a stepwise displacement, mimicking a 'car pedal' motion that triggers activation at 11-nt pairing. The HNH domain also contributes to R-loop stabilization through a positively charged R-wedge motif and undergoes a ~90° activation-driven rotation mediated by two hinge regions. In variants IscBHig1 and IscBHig2, engineering these hinge motifs to enhance conformational flexibility notably improved genome-editing efficiency in cells. In summary, our study reveals the molecular basis underlying IscB autoinhibition and activation, identifies previously uncharacterized regulatory features and establishes hinge elements as a target region for engineering compact, efficient genome editors.},
}

Cryoelectron Microscopy
*RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism/genetics
Models, Molecular
*Escherichia coli Proteins/chemistry/metabolism/genetics
Nucleic Acid Conformation
Escherichia coli/genetics/metabolism
CRISPR-Associated Protein 9/metabolism/chemistry/genetics

@article {pmid41910274,
year = {2026},
author = {Qiao, W},
title = {From CRISPR functional genomics to synthetic interventions: engineering antiviral strategies.},
journal = {Journal of virology},
volume = {100},
number = {4},
pages = {e0005726},
doi = {10.1128/jvi.00057-26},
pmid = {41910274},
issn = {1098-5514},
support = {C1032633001//Shenzhen Bay Laboratory/ ; },
mesh = {Humans ; *Synthetic Biology/methods ; *Antiviral Agents/pharmacology ; *Genomics/methods ; *CRISPR-Cas Systems ; Animals ; Virus Replication ; *Viruses/genetics ; *Virus Diseases/virology/therapy ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Host-Pathogen Interactions/genetics ; },
abstract = {Virus-host interactions govern infection outcomes and viral evolution, but host determinants that enable or restrict viral replication have been difficult to map comprehensively and in the right cellular contexts. Pooled CRISPR perturbation screens now provide scalable, mechanistic entry points into host dependency and restriction landscapes across diverse viruses. Recent extensions, including single-cell readouts, imaging and spatial phenotyping, organoid models, and in vivo selection, are shifting the field from static hit lists toward contextual maps that explain how host pathways and cell states shape permissiveness. In parallel, synthetic biology is translating these maps into programmable intervention classes, including receptor decoys and binders that intercept entry, conditional protein depletion systems that modulate host factors with temporal control, gene circuits that couple infection sensing to tailored responses, and engineered immune cells with tunable antiviral functions. This review highlights conceptual and technical advances that connect CRISPR functional genomics to synthetic antiviral design, summarizes emerging principles that generalize across viral families, and discusses constraints that will determine whether screen-nominated mechanisms can be engineered into effective and safe antiviral strategies.},
}

Humans
*Synthetic Biology/methods
*Antiviral Agents/pharmacology
*Genomics/methods
*CRISPR-Cas Systems
Animals
Virus Replication
*Viruses/genetics
*Virus Diseases/virology/therapy
*Clustered Regularly Interspaced Short Palindromic Repeats
Host-Pathogen Interactions/genetics

@article {pmid41922875,
year = {2026},
author = {Burr, SP and Auckland, K and Glynos, A and Dhawanjewar, A and Ryall, C and Wei, W and Hynes-Allen, A and Prater, M and Sczaniecka-Clift, M and Prudent, J and Chinnery, PF and van den Ameele, J},
title = {MitoPerturb-Seq identifies gene-specific single-cell responses to mitochondrial DNA depletion and heteroplasmy.},
journal = {Nature structural & molecular biology},
volume = {33},
number = {4},
pages = {711-723},
pmid = {41922875},
issn = {1545-9985},
mesh = {*DNA, Mitochondrial/genetics ; Animals ; Mice ; *Single-Cell Analysis/methods ; CRISPR-Cas Systems ; *Heteroplasmy/genetics ; *Mitochondria/genetics/metabolism ; DNA Polymerase gamma/genetics ; Mitochondrial Proteins/genetics ; DNA-Binding Proteins/genetics ; Mutation ; Activating Transcription Factor 4/metabolism/genetics ; Transcription Factors/genetics ; GTP Phosphohydrolases/genetics ; High Mobility Group Proteins ; },
abstract = {Mitochondria contain their own genome, mitochondrial DNA (mtDNA), which is under strict control by the cell nucleus. mtDNA occurs in many copies per cell and mutations often only affect a proportion of them, giving rise to heteroplasmy. mtDNA copy number and heteroplasmy level together shape the tissue-specific impact of mtDNA mutations, eventually giving rise to both rare mitochondrial and common neurodegenerative diseases. Here, we use MitoPerturb-Seq for CRISPR-Cas9-based, high-throughput single-cell interrogation of the nuclear genes and pathways that sense and control mtDNA copy number and heteroplasmy. We screened a panel of mtDNA maintenance genes in mouse cells with a heteroplasmic mtDNA mt-Ta mutation. This revealed both common and perturbation-specific aspects of the integrated stress response to mtDNA depletion caused by Tfam, Opa1 and Polg knockout. These responses are only partially mediated by ATF4 and cause cell-cycle stage-independent slowing of cell proliferation. MitoPerturb-Seq, thus, provides experimental insight into disease-relevant mitochondrial-nuclear interactions and may inform development of therapies targeting cell-type- and tissue-specific vulnerabilities to mitochondrial dysfunction.},
}

*DNA, Mitochondrial/genetics
Animals
Mice
*Single-Cell Analysis/methods
CRISPR-Cas Systems
*Heteroplasmy/genetics
*Mitochondria/genetics/metabolism
DNA Polymerase gamma/genetics
Mitochondrial Proteins/genetics
DNA-Binding Proteins/genetics
Mutation
Activating Transcription Factor 4/metabolism/genetics
Transcription Factors/genetics
GTP Phosphohydrolases/genetics
High Mobility Group Proteins

@article {pmid41928564,
year = {2026},
author = {Topfer, SK and Rutherford, M and Zewe, F and Strive, T and Oh, KP},
title = {Viability-Based Assessment Reveals True Efficiency of CRISPR-Cas9 Transfection Methods in Rabbit Spermatozoa.},
journal = {The CRISPR journal},
volume = {9},
number = {2},
pages = {59-70},
doi = {10.1177/25731599261424870},
pmid = {41928564},
issn = {2573-1602},
mesh = {Animals ; Rabbits ; Male ; *Spermatozoa/metabolism ; *CRISPR-Cas Systems/genetics ; *Transfection/methods ; Electroporation/methods ; *Gene Editing/methods ; Cell Survival/genetics ; Plasmids/genetics ; Sperm Motility ; Lipids ; },
abstract = {Modern genome editing tools such as CRISPR-Cas9 have revolutionized mammalian genome engineering, yet translation to in vivo applications remains limited by low efficiency and frequent occurrence of mosaicism. Sperm-mediated delivery of editing reagents is one proposed alternative that may mitigate these issues. This method depends on efficient transfection of genome editing materials into viable spermatozoa, a critical yet frequently overlooked parameter. Using FACS, we compared electroporation (Neon NxT) and lipofection (CRISPRMAX) for introducing CRISPR-Cas9 ribonucleoproteins into viable rabbit spermatozoa. Electroporation, shown to enable Cas9 and plasmid transfection in spermatozoa from other species, performed poorly once dead spermatozoa were excluded. In contrast, Lipofectamine CRISPRMAX improved transfection efficiency with minimal effects on spermatozoa viability and motility. These findings emphasize the importance of distinguishing true transfection (transfection of viable spermatozoa) from total transfection and highlight lipofection as a promising alternative to electroporation for sperm-based genome editing, with potential applications in rabbit genome engineering.},
}

Animals
Rabbits
Male
*Spermatozoa/metabolism
*CRISPR-Cas Systems/genetics
*Transfection/methods
Electroporation/methods
*Gene Editing/methods
Cell Survival/genetics
Plasmids/genetics
Sperm Motility
Lipids

@article {pmid41928587,
year = {2026},
author = {Gibson, JR and Dhungana, A and Pokhrel, M and Arthur, BB and Adebayo, O and Hossack, D and Cottle, RN},
title = {Toward Next-Gen Cell Therapy for Pediatric Patients: Neonatal Hepatocytes Tolerate Electroporation-Mediated Gene Editing and Engraft in the Liver.},
journal = {The CRISPR journal},
volume = {9},
number = {2},
pages = {103-114},
doi = {10.1177/25731599261430830},
pmid = {41928587},
issn = {2573-1602},
support = {R01 HL168093/HL/NHLBI NIH HHS/United States ; },
mesh = {*Hepatocytes/transplantation/metabolism/cytology ; *Gene Editing/methods ; Animals ; *Electroporation/methods ; Mice ; CRISPR-Cas Systems ; *Tyrosinemias/therapy/genetics ; Humans ; *Liver/metabolism ; Animals, Newborn ; *Cell- and Tissue-Based Therapy/methods ; Mice, Knockout ; Disease Models, Animal ; Hydrolases/genetics ; },
abstract = {Hepatocyte transplantation (HTx) offers a safer, less invasive alternative to orthotopic liver transplantation for inherited metabolic liver diseases, especially in high-risk pediatric patients. Combining HTx with ex vivo gene editing is a promising autologous therapeutic strategy using the patient's cells. We investigated the feasibility of this approach by applying CRISPR-Cas9 gene knock-out to neonatal mouse hepatocytes and comparing their engraftment potential with that of mature adult cells in the Fah[-/-] mouse model of hereditary tyrosinemia type I (HT1). Electroporation-mediated gene editing did not significantly impair the ability of neonatal hepatocytes to engraft in vivo. Quantitative histological analysis revealed comparable liver repopulation levels between recipients of gene-edited neonatal cells and adult cells after hepatoxicity-mediated selection, providing a benchmark for electroporation-mediated gene editing in neonatal hepatocytes, and supporting the development of genetically corrected neonatal hepatocyte products as a crucial long-term or bridge-to-transplant therapeutic strategy for pediatric liver disease.},
}

*Hepatocytes/transplantation/metabolism/cytology
*Gene Editing/methods
Animals
*Electroporation/methods
Mice
CRISPR-Cas Systems
*Tyrosinemias/therapy/genetics
Humans
*Liver/metabolism
Animals, Newborn
*Cell- and Tissue-Based Therapy/methods
Mice, Knockout
Disease Models, Animal
Hydrolases/genetics

@article {pmid41928679,
year = {2026},
author = {Li, D and Feng, J and Wang, X and Zhao, E and Liang, Y and Li, K and Li, Y},
title = {An Anaerobic Fluorescent Reporter System and CRISPR-Cas12a Enable High-Throughput Metabolic Engineering of Clostridium butyricum.},
journal = {ACS synthetic biology},
volume = {15},
number = {4},
pages = {1669-1680},
doi = {10.1021/acssynbio.6c00158},
pmid = {41928679},
issn = {2161-5063},
mesh = {*Metabolic Engineering/methods ; *CRISPR-Cas Systems/genetics ; *Clostridium butyricum/genetics/metabolism ; Anaerobiosis ; Gene Editing/methods ; Plasmids/genetics/metabolism ; Promoter Regions, Genetic/genetics ; Green Fluorescent Proteins/genetics/metabolism ; Genes, Reporter ; High-Throughput Screening Assays/methods ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Clostridium butyricum is an important probiotic and industrial organism with significant potential for anaerobic bioproduction. However, the lack of efficient genetic tools, particularly for high-throughput screening under strict anaerobic conditions, has hindered its metabolic engineering. To address this, we first established a highly efficient conjugation method, significantly improving exogenous DNA transformation efficiency. Leveraging time-resolved transcriptomic data, we then mapped the dynamic activity of native promoters and developed a robust anaerobic fluorescent protein reporter system. This system overcomes the oxygen-dependent limitation of traditional reporters like GFP, enabling high-throughput and quantitative screening of promoter strength in live anaerobes. Furthermore, we constructed a CRISPR-Cas12a-based genome editing platform for scarless gene manipulation and a two-plasmid curing strategy to generate markerless and genetically stable engineered strains. The power of this integrated toolkit was demonstrated by engineering the acetaldehyde metabolic pathway. Overexpression of aldehyde dehydrogenase (ALDH) resulted in a 79.29% increase in enzyme activity, indicating an enhanced catalytic capacity for acetaldehyde oxidation. This proof-of-concept module, together with the anaerobic fluorescent reporter and CRISPR-Cas12a platform, supports a streamlined workflow for genetic part characterization and metabolic engineering in C. butyricum under strict anaerobic conditions.},
}

*Metabolic Engineering/methods
*CRISPR-Cas Systems/genetics
*Clostridium butyricum/genetics/metabolism
Anaerobiosis
Gene Editing/methods
Plasmids/genetics/metabolism
Promoter Regions, Genetic/genetics
Green Fluorescent Proteins/genetics/metabolism
Genes, Reporter
High-Throughput Screening Assays/methods
Bacterial Proteins/genetics/metabolism

@article {pmid41930529,
year = {2026},
author = {Pan, H and Zhao, J and Yang, J and Cao, W and Liu, G},
title = {A ∆ssaV deletion attenuates Salmonella Choleraesuis to generate a self-limiting, immunogenic vaccine candidate.},
journal = {Vaccine},
volume = {80},
number = {},
pages = {128546},
doi = {10.1016/j.vaccine.2026.128546},
pmid = {41930529},
issn = {1873-2518},
mesh = {Animals ; Vaccines, Attenuated/immunology/genetics/administration & dosage ; *Salmonella Vaccines/immunology/genetics/administration & dosage ; Mice ; *Salmonella enterica/immunology/genetics/pathogenicity ; Antibodies, Bacterial/blood ; Gene Deletion ; Female ; *Type III Secretion Systems/genetics ; Immunity, Humoral ; Virulence ; *Salmonella Infections, Animal/prevention & control/immunology ; Macrophages/microbiology/immunology ; Mice, Inbred BALB C ; Bacterial Proteins/genetics/immunology ; Immunity, Cellular ; Immunogenicity, Vaccine ; CRISPR-Cas Systems ; },
abstract = {Live attenuated Salmonella enterica serovar Choleraesuis (S. Choleraesuis) strains, a host-adapted swine pathogen with well-characterized virulence and strong immunogenicity, represents a rational choice for constructing live bacterial vaccines. The ssaV gene, a key component of the SPI-2 T3SS, was knocked out to construct a vaccine candidate designed to strike an optimal balance between attenuation and immunogenicity using CRISPR-Cas9. This mutant retained the ability to adhere and invade epithelial cells but showed markedly reduced survival in macrophages, indicating impaired SPI-2-dependent persistence. The ∆ssaV mutant exhibited a > 1000-fold increase in LD50 and underwent rapid systemic clearance, collectively confirming its marked attenuation and efficient elimination in vivo. The ∆ssaV mutant vaccine elicited strong humoral and cellular immune responses in mice and achieved a 70% survival rate upon lethal challenge with the WT strain. These results demonstrate that ssaV deletion creates a rationally designed vaccine strain that is attenuated, immunogenic, and self-limiting, positioning it as a promising live vaccine candidate in the murine model.},
}

Animals
Vaccines, Attenuated/immunology/genetics/administration & dosage
*Salmonella Vaccines/immunology/genetics/administration & dosage
Mice
*Salmonella enterica/immunology/genetics/pathogenicity
Antibodies, Bacterial/blood
Gene Deletion
Female
*Type III Secretion Systems/genetics
Immunity, Humoral
Virulence
*Salmonella Infections, Animal/prevention & control/immunology
Macrophages/microbiology/immunology
Mice, Inbred BALB C
Bacterial Proteins/genetics/immunology
Immunity, Cellular
Immunogenicity, Vaccine
CRISPR-Cas Systems

@article {pmid41934379,
year = {2026},
author = {Lin, WW and Su, J and Li, JX and Chen, YW and Chen, JY and Wu, B and Cai, NQ and Gan, LJ and Liu, ZJ},
title = {Enhanced SNV Detection of HLA-B*15:02TA by Integrating Blocking RPA and Inhibiting CRISPR-Cas12a.},
journal = {Analytical chemistry},
volume = {98},
number = {15},
pages = {11234-11245},
doi = {10.1021/acs.analchem.5c08035},
pmid = {41934379},
issn = {1520-6882},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Polymorphism, Single Nucleotide ; *CRISPR-Associated Proteins/antagonists & inhibitors/genetics ; *Endodeoxyribonucleases/antagonists & inhibitors/genetics/metabolism ; Bacterial Proteins ; },
abstract = {With the introduction of the RPA-based CRISPR-Cas12a method, crRNA allostery and Cas12a protein engineering have been applied to the identification of single-nucleotide variants (SNVs). However, the complicated testing procedure often falls short of the intended simple and rapid objectives of CRISPR-based diagnostics (CRISPR-Dx), and the discrimination factor (DF) for SNV distinction remains suboptimal (DF = 2-5). In this study, we proposed a one-tube "Blocking RPA-coupled Inhibiting CRISPR-Cas12a" (BRIC) cascade strategy that features a dual recognition of SNVs. In the BRIC cascade, a reduction of RPA amplification products of wild-type target (WT) and WT-activated Cas12a cleavage lowered the WT detection signal to near-background levels, without compromising the efficiency of RPA or CRISPR-Cas12a. With HLA-B*15:02TA (rs3909184, G > C), a gene associated with drug-induced cutaneous rash, as the SNV target, the proposed one-tube BRIC strategy achieved a DF of 32.51, with the WT signal approximating the background level, thereby greatly enhancing SNV detection specificity. On the other hand, MT can be detected in a large number of WT (with a resolution of 0.1%). Within the HLA-B*15:02TA concentration (CMT) range of 5.0 × 10[-7] to 1.0 × 10[-3] nM (500 aM to 1 pM), a linear relationship was evident between the fluorescence signal (FMT) and lgCMT (FMT = 4146.55 + 578.09lgCMT; R[2] = 0.9904), with a detection limit of 67.6 aM (>3σ). Combined with a 3D-printed portable fluorimeter, the proposed method reported high consistency with next-generation sequencing in detecting HLA-B*15:02TA in 22 clinical plasma samples, validating the reliability of the one-tube BRIC cascade assay. These improvements in assay design, process, and DF highlight the promising potentials of the BRIC strategy in clinical settings.},
}

Humans
*CRISPR-Cas Systems/genetics
*Polymorphism, Single Nucleotide
*CRISPR-Associated Proteins/antagonists & inhibitors/genetics
*Endodeoxyribonucleases/antagonists & inhibitors/genetics/metabolism
Bacterial Proteins

@article {pmid41937727,
year = {2026},
author = {Kriete, A and Basika, T and Novas, R and Mathieson, OL and Belikoff, EJ and Kleiner, M and Scott, MJ},
title = {Conditional Expression of Cas9 and dCas9 in Lucilia cuprina Reveals dCas9-Associated Lethality.},
journal = {The CRISPR journal},
volume = {9},
number = {2},
pages = {71-88},
doi = {10.1177/25731599261436373},
pmid = {41937727},
issn = {2573-1602},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Female ; Male ; *Diptera/genetics ; *CRISPR-Associated Protein 9/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Sex Determination Processes/genetics ; Lucilia cuprina ; },
abstract = {Conditional sex transformation systems could improve genetic control strategies against insect pests. Here, we developed and tested CRISPR-based, tetracycline-repressible sex transformation strains in the Australian sheep blowfly, Lucilia cuprina. Using Tet-Off-regulated expression of Cas9 and dCas9, we targeted the sex-determining gene transformer with the goal of converting females into males. Conditional Cas9 expression enabled knockout of a visual marker gene, confirming inducible genome editing. However, strains expressing transformer-targeting sgRNA arrays did not undergo sex transformation. Embryonic microinjection of transformer-targeting sgRNAs into Cas9-expressing embryos produced intersex individuals, indicating that sgRNA expression from the integrated arrays was insufficient to disrupt the sex determination pathway. In contrast, high-level dCas9 expression was associated with developmental delays, reduced body weight, and lethality. These findings establish the first conditional CRISPR expression system in L. cuprina and demonstrate that Cas9 is compatible with inducible gene editing, whereas dCas9 is poorly tolerated at high expression levels.},
}

Animals
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Female
Male
*Diptera/genetics
*CRISPR-Associated Protein 9/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Sex Determination Processes/genetics
Lucilia cuprina

@article {pmid41952066,
year = {2026},
author = {Chatla, K and Ayalew, L and Yim, M and Ko, P and Lippold, S and Hernandez, G and Chua, BA and Patil, DP and Camperi, J},
title = {Analytical Assessment of sgRNA Impurities and Their Impact on Functional Performance.},
journal = {Analytical chemistry},
volume = {98},
number = {15},
pages = {11438-11447},
doi = {10.1021/acs.analchem.6c00747},
pmid = {41952066},
issn = {1520-6882},
mesh = {*RNA, Guide, CRISPR-Cas Systems/analysis/genetics ; Gene Editing ; Humans ; CRISPR-Cas Systems ; Chromatography, Reverse-Phase ; Chromatography, Gel ; Mass Spectrometry ; },
abstract = {Single guide RNA (sgRNA) is a critical component of the clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing system, guiding Cas9 to specific genomic loci for precise DNA modification. With its growing clinical potential, sgRNAs have emerged as a promising modality for gene editing-based therapeutics, underscoring the need for robust analytical characterization to ensure quality, safety, efficacy, and regulatory compliance. Here, we present an integrated strategy for deep profiling of sgRNA impurities, combining high-resolution ion-pairing reversed-phase liquid chromatography (IP-RPLC) and size-exclusion chromatography (SEC) with advanced technologies, namely, native mass spectrometry and nanopore direct RNA sequencing. The isolation and characterization of isolated chromatographic peaks revealed truncated, chemically modified, and deletion-prone species. Notably, early eluting fractions in IP-RPLC exhibited elevated deletion frequencies in the target-specific region, correlating with reduced gene-editing efficiency and increased variability in T cells when tested using the Cas-CLOVER system. In contrast, late-eluting fractions in IP-RPLC revealed polyphosphorylated variants with minimal functional impact, while off-target analyses suggested that early eluting impurities may paradoxically reduce off-target editing. Lastly, even at high levels, sgRNA aggregates showed only a limited impact on activity: fully aggregated preparations displayed ∼10% lower knockout efficiency; however, an increase in cumulative off-target frequencies was observed. Overall, this study highlights the value of combining advanced analytical tools to achieve deep profiling of sgRNA impurities. By linking specific impurity profiles to functional outcomes, these findings provide actionable insights for improving sgRNA quality control and advancing the development of safe and effective gene editing-based therapeutics.},
}

*RNA, Guide, CRISPR-Cas Systems/analysis/genetics
Gene Editing
Humans
CRISPR-Cas Systems
Chromatography, Reverse-Phase
Chromatography, Gel
Mass Spectrometry

@article {pmid42007994,
year = {2026},
author = {Tiwari, P and Rathinasabapathi, P},
title = {Programmable CRISPR-Cas diagnostic platforms for rapid detection of uropathogens and antimicrobial resistance.},
journal = {Archives of microbiology},
volume = {208},
number = {7},
pages = {},
pmid = {42007994},
issn = {1432-072X},
}

@article {pmid42007998,
year = {2026},
author = {Sharma, D and Khan, M and Khan, JA},
title = {Genome editing‑based strategies to combat geminiviruses: CRISPR/Cas9 and emerging high‑fidelity tools.},
journal = {Archives of microbiology},
volume = {208},
number = {7},
pages = {},
pmid = {42007998},
issn = {1432-072X},
}

@article {pmid42008182,
year = {2026},
author = {Azhar, F and Mazhari, BBZ and Ibrahim, MN and Alanazi, A and Islam, F},
title = {CRISPR-based tools in food safety and microbiology: applications for pathogen detection and control.},
journal = {Archives of microbiology},
volume = {208},
number = {7},
pages = {},
pmid = {42008182},
issn = {1432-072X},
}

@article {pmid42009546,
year = {2026},
author = {Xu, T and Zhao, L and Dai, Y and Duan, J and Wang, Y and Shao, L and Chen, D and Zhu, L and Xu, Z},
title = {[A rapid visual detection method for porcine circovirus type 4 based on enzymatic recombinase amplification and CRISPR/EsCas13d].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {4},
pages = {1755-1768},
doi = {10.13345/j.cjb.250568},
pmid = {42009546},
issn = {1872-2075},
support = {2024YFD1800500 and 2024YFD1800102//the National Key Research and Development Program of China/ ; sccxtd-2024-08 and sccxtd-2024-18//the National Modern Agricultural Industrial Technology System/ ; },
mesh = {*Circovirus/isolation & purification/genetics ; Animals ; Swine ; *Recombinases/genetics/metabolism ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Circoviridae Infections/diagnosis/veterinary/virology ; *Swine Diseases/virology/diagnosis ; },
abstract = {The emerging outbreaks of infectious diseases in humans and animals worldwide pose serious threats to public health and cause substantial economic losses. This has raised the demand for more efficient and sensitive diagnostic methods to strengthen disease surveillance and early warning. In this study, we developed a portable visual platform based on ERA-CRISPR/EsCas13d for the rapid detection of porcine circovirus type 4 under resource-limited conditions. We optimized the platform by integrating enzymatic recombinase amplification (ERA), T7 transcription, and CRISPR/EsCas13d cleavage in a single-tube reaction, thereby simplifying the workflow and shortening the total detection time to 30 min. In addition, a rapid nucleic acid release method was employed, eliminating the need for laboratory-based extraction and complex heating steps, which further improved the simplicity and usability. In addition, lyophilized reagents were modified to enhance stability, thus reducing cold-chain and storage requirements and enabling cost-effective transport and field deployment. The method achieved a visual limit of detection of 50 cp/μL for porcine circovirus type 4 and demonstrated no cross-reactivity with six other common swine pathogens. Moreover, the platform supported two visual readout formats-UV light (470 nm) and lateral flow assay (LFA)-providing flexible options for interpretation in different application scenarios. Validation with 60 clinical samples showed 100% concordance with quantitative PCR results, highlighting the practical utility and application potential of the ERA-CRISPR/EsCas13d-based portable visual platform for rapid on-site diagnostics.},
}

*Circovirus/isolation & purification/genetics
Animals
Swine
*Recombinases/genetics/metabolism
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
*Circoviridae Infections/diagnosis/veterinary/virology
*Swine Diseases/virology/diagnosis

@article {pmid42009554,
year = {2026},
author = {Li, Y and Wen, D and Zhou, Z and Liao, C and Zhao, Q and Shuai, J and Zhang, X and Yu, X and Huang, J},
title = {[A CRISPR-Cas13a-based amplification-free electrochemical biosensor for rapid detection of bovine viral diarrhea virus].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {4},
pages = {1868-1880},
doi = {10.13345/j.cjb.250329},
pmid = {42009554},
issn = {1872-2075},
support = {2021YFF0600805 and 2021YFF0602801//the National Key Research and Development Program of China/ ; 2024SNJF044//the Zhejiang Provincial Department of Agriculture and Rural Affairs Project/ ; 20241203A23//the Key Research Program in the Field of Agriculture and Social Development of Hangzhou/ ; },
mesh = {*Biosensing Techniques/methods ; Cattle ; Animals ; *CRISPR-Cas Systems/genetics ; *Diarrhea Viruses, Bovine Viral/isolation & purification/genetics ; *Electrochemical Techniques/methods ; Nucleic Acid Amplification Techniques ; Bovine Virus Diarrhea-Mucosal Disease/diagnosis/virology ; },
abstract = {Bovine viral diarrhea virus (BVDV), a major pathogen in the global bovine industry, causes diarrhea, fever, and reproductive disorders, leading to substantial economic losses. Developing the methods for rapid and accurate detection of BVDV is crucial for epidemic control. Current detection methods have notable limitations. PCR-based nucleic acid amplification techniques rely on sophisticated instruments and complex procedures. CRISPR-Cas13a systems, despite their high specificity, still require nucleic acid pre-amplification, which results in cumbersome workflows and contamination risks. To establish a simpler and more efficient on-site detection method for BVDV, this study integrated the CRISPR-Cas13a system characterized by specific recognition with electrochemical sensing praised for efficient signal transduction to establish a novel nucleic acid amplification-free method for the detection of BVDV. Through optimization of key parameters, including CRISPR RNA (crRNA) combination, buffer components, and Cas13a/crRNA concentration ratio, the biosensor achieved a detection limit of 3 090 copies/μL-representing a 4-5 order of magnitude improvement in sensitivity compared with conventional Cas13a fluorescence-based detection-and completed the entire process from sample loading to result output within 35 min. Specificity tests demonstrated that the sensor exclusively detected BVDV without cross-reactivity to other common bovine viruses (bovine parainfluenza virus type 3, bovine respiratory syncytial virus, bluetongue virus, and foot-and-mouth disease virus). Clinical validation with 22 samples demonstrated 100% specificity and sensitivity. The developed CRISPR-Cas13a-based electrochemical biosensor offers the advantages of being nucleic acid amplification-free and operationally simple, serving as a powerful new tool for rapid on-site BVDV detection with significant potential for veterinary diagnostics and epidemic prevention and control.},
}

*Biosensing Techniques/methods
Cattle
Animals
*CRISPR-Cas Systems/genetics
*Diarrhea Viruses, Bovine Viral/isolation & purification/genetics
*Electrochemical Techniques/methods
Nucleic Acid Amplification Techniques
Bovine Virus Diarrhea-Mucosal Disease/diagnosis/virology

@article {pmid42009663,
year = {2026},
author = {Batty, P and Beneder, H and Schätz, C and Onea, G and Zaczek, M and Kutschat, AP and Abele, M and Müller, S and Superti-Furga, G and Winter, GE and Seruggia, D},
title = {Disruption of the SAGA CORE triggers collateral degradation of KAT2A.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42009663},
issn = {2041-1723},
support = {947803//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 10.55776/P36302//Austrian Science Fund (Fonds zur Förderung der Wissenschaftlichen Forschung)/ ; },
mesh = {*Histone Acetyltransferases/metabolism/genetics ; Humans ; Proteolysis ; Proteasome Endopeptidase Complex/metabolism ; Acetylation ; Histones/metabolism ; Ubiquitin-Protein Ligases/metabolism/genetics ; HEK293 Cells ; Proteomics ; *Trans-Activators/metabolism/genetics ; CRISPR-Cas Systems ; TATA-Binding Protein Associated Factors/metabolism/genetics ; p300-CBP-Associated Factor ; },
abstract = {The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex regulates gene expression through histone acetylation at promoters, mediated by its histone acetyl transferase (HAT), KAT2A. While SAGA structure and function are well characterised, mechanisms controlling the stability of individual subunits, including KAT2A, remain unclear. Here, using a fluorescence-based KAT2A stability reporter, we systematically dissect the molecular dependencies controlling KAT2A protein abundance, and identify the non-enzymatic SAGA CORE module subunits-TADA1, TAF5L, and TAF6L- as necessary for KAT2A stability. Loss of these subunits disrupts SAGA complex integrity, leading to non-chromatin-bound KAT2A that is degraded by the proteasome and consequent reduced H3K9 acetylation. Proteomic profiling reveals progressive loss of components from the CORE and HAT modules upon acute SAGA CORE disruption, indicating that an intact CORE is required for the stability of numerous SAGA components. Finally, a focused CRISPR screen of ubiquitin-proteasome system genes identifies the E3 ligase UBR5, a known regulator of orphan protein degradation, and the deubiquitinase OTUD5, as regulators of KAT2A degradation when the SAGA CORE is perturbed. Together, these findings reveal a dependency of KAT2A protein stability on SAGA CORE integrity and define an orphan quality control mechanism targeting unassembled KAT2A, revealing a potential vulnerability in SAGA-driven malignancies.},
}

*Histone Acetyltransferases/metabolism/genetics
Humans
Proteolysis
Proteasome Endopeptidase Complex/metabolism
Acetylation
Histones/metabolism
Ubiquitin-Protein Ligases/metabolism/genetics
HEK293 Cells
Proteomics
*Trans-Activators/metabolism/genetics
CRISPR-Cas Systems
TATA-Binding Protein Associated Factors/metabolism/genetics
p300-CBP-Associated Factor

@article {pmid42009664,
year = {2026},
author = {Corazzi, L and Ing, A and Benito, E and Cosenza, MR and Hasenfeld, P and Weber, T and Marx, AJM and Ionasz, VS and Trausch, N and Benedetto, S and Di Muzio, G and Ding, B and Berlanda, J and Giaisi, M and Claudino, N and Höfer, T and Korbel, JO and Wei, PC},
title = {Recurrent DNA break clusters drive replication-stress-induced copy number variants and genome diversification.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42009664},
issn = {2041-1723},
support = {949990//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 101098056//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; YIP-DKFZ//Helmholtz Association/ ; },
mesh = {*DNA Copy Number Variations/genetics ; Animals ; *DNA Replication/genetics ; Mice ; Neural Stem Cells/metabolism ; *DNA Breaks ; *Genome ; CRISPR-Cas Systems ; Whole Genome Sequencing ; Single-Cell Analysis ; DNA End-Joining Repair/genetics ; Humans ; DNA Repair ; },
abstract = {Copy number variants (CNVs) are strongly implicated in neurological and psychiatric disorders and brain cancer, yet the process by which replication stress generates CNVs-and why some recur while others remain rare-remains poorly understood. Here, we show that recurrent DNA-break clusters (RDCs) act as common initiating lesions that drive both recurrent and non-recurrent CNVs. In murine neural progenitor cells subjected to chemically induced replication stress, bulk whole-genome sequencing identifies recurrent CNVs enriched at late-replicating RDCs within actively transcribed genes. Single-cell genome sequencing further uncovers frequent, non-recurrent CNVs associated with RDCs that arise during the transition from early to late DNA replication. These CNVs represent stable, heritable structural variants with breakpoints consistently enriched at RDCs. CRISPR/Cas9-mediated transcriptional suppression abolishes both RDC formation and CNV generation, establishing RDC-associated breaks as a shared upstream source. Mechanistically, CNV formation depends on DNA repair context: CNVs are Pol θ-dependent in NHEJ-deficient cells but arise independently of Pol θ in NHEJ-proficient cells. Together, these findings define RDCs as central drivers of replication-stress-induced genome diversification.},
}

*DNA Copy Number Variations/genetics
Animals
*DNA Replication/genetics
Mice
Neural Stem Cells/metabolism
*DNA Breaks
*Genome
CRISPR-Cas Systems
Whole Genome Sequencing
Single-Cell Analysis
DNA End-Joining Repair/genetics
Humans
DNA Repair

@article {pmid42011779,
year = {2026},
author = {Biczók, Z and Krausz, SL and Simon, DA and Tóth, E and Varga, É and Annus, T and Huba, F and Varga, M and Bakos, É and Fodor, E and Welker, E},
title = {Disrupting pegRNA intramolecular complementarity via PBS and spacer sequence alterations can enhance prime editing efficiency.},
journal = {Nucleic acids research},
volume = {54},
number = {7},
pages = {},
doi = {10.1093/nar/gkag292},
pmid = {42011779},
issn = {1362-4962},
support = {K134968//Hungarian Scientific Research Fund/ ; K142322//Hungarian Scientific Research Fund/ ; RRF-2.3.1-21-2022-00015//PharmaLab/ ; ELKH-PoC-2023//National Research, Development, and Innovation Office of Hungary/ ; //Hungarian Research Network/ ; //Ministry of National Economy/ ; },
mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; CRISPR-Cas Systems ; Binding Sites ; Plasmids/genetics ; Base Sequence ; Humans ; Base Pair Mismatch ; },
abstract = {The length and sequence of the primer binding site (PBS) are critical for efficient prime editing, and its intramolecular complementarity with the prime editing guide RNA (pegRNA) spacer is a major drawback. We investigated the effects of these factors by literature analyses and by testing over 300 modified pegRNAs with weakened PBS-spacer interactions. It has been suggested that the effective PBS length for plasmid-delivered pegRNAs without end protection is considerably longer than what efficient priming requires due to exonuclease digestion of the PBS ends; however, analysing literature data of over 3000 pegRNAs revealed no significant shift in the optimal PBS length for epegRNAs compared to conventional pegRNAs. We also found improvement in editing efficiency with up to seven-fold when mismatches were introduced in the spacer or PBS sequence disrupting complementarity, although this effect is more pronounced with non-optimal PBS lengths. A combination of spacer mismatches and PBS deletions led to further editing improvements, even compared to the optimal PBS, although finding the best combination requires extensive optimization. Here, we achieved near-optimal editing efficiency in the majority of cases without the need for prior pegRNA optimization by using SPELL (Streamlined Prime Editing with fixed-Length PBS Leverage), a prime editing approach that employs a 17-20 nucleotide-long PBS with a single nucleotide deletion.},
}

*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry
CRISPR-Cas Systems
Binding Sites
Plasmids/genetics
Base Sequence
Humans
Base Pair Mismatch

@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