@article {pmid40904932,
year = {2025},
author = {Wang, HM and Xu, SJ and Cai, BY and Qiu, WY and Lu, H and Tang, YD},
title = {Highly efficient gene editing of Feline herpesvirus 1 using CRISPR/Cas9 combined with FACS.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1660446},
pmid = {40904932},
issn = {2235-2988},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; *Varicellovirus/genetics ; Cats ; *Flow Cytometry/methods ; Thymidine Kinase/genetics ; Genes, Reporter ; Herpesviridae Infections/virology/veterinary ; Green Fluorescent Proteins/genetics ; },
abstract = {Feline herpesvirus 1 (FHV-1) is a major causative agent of feline viral rhinotracheitis and ocular lesions. Due to its large DNA genome, the construction of recombinant FHV-1 viruses presents considerable challenges for conventional methodologies. In this study, we implemented an integrated strategy combining CRISPR/Cas9-mediated gene editing with fluorescence-activated cell sorting (FACS) to enable the rapid and efficient generation of recombinant FHV-1 viruses. Specifically, the thymidine kinase (tk) gene was disrupted by inserting a monomeric Cherry (mCherry) reporter gene, and the glycoprotein E (gE) gene was similarly interrupted through the incorporation of a green fluorescent protein (GFP) reporter. The CRISPR/Cas9 system enables precise, site-specific genomic modifications, while FACS allows for effective enrichment and isolation of the desired recombinant viral populations. This combined approach significantly reduces the time required for recombinant virus generation from weeks to days, thereby offering substantial potential to expedite vaccine development and advance functional genomics research.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
Animals
*Varicellovirus/genetics
Cats
*Flow Cytometry/methods
Thymidine Kinase/genetics
Genes, Reporter
Herpesviridae Infections/virology/veterinary
Green Fluorescent Proteins/genetics

@article {pmid40904117,
year = {2025},
author = {Koonce, KC and Mauritzen, JJ and Hitz, IF and Vangsgaard, EF and Putz, EHM and Wajn, AS and Leth, FH and Høyland-Kroghsbo, NM},
title = {The H-NS homologues MvaT and MvaU repress CRISPR-Cas in Pseudomonas aeruginosa.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240073},
pmid = {40904117},
issn = {1471-2970},
support = {//Danmarks Frie Forskningsfond/ ; //Lundbeck Foundation/ ; },
mesh = {*Pseudomonas aeruginosa/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Bacterial Proteins/genetics/metabolism ; *DNA-Binding Proteins/genetics/metabolism ; Trans-Activators ; },
abstract = {CRISPR-Cas is an adaptive immune system of bacteria and archaea that protects against foreign genetic elements. In Escherichia coli and Salmonella, CRISPR-Cas is inhibited by the conserved global repressor the histone-like nucleoid structuring protein (H-NS), which blocks the expression of AT-rich horizontally acquired genes. While the opportunistic pathogen Pseudomonas aeruginosa harbours two partially redundant H-NS homologues, MvaT and MvaU, their role in CRISPR-Cas regulation in this bacterium remains unexplored. Here, we demonstrate that in the absence of both MvaT and MvaU, CRISPR-Cas activity increases more than tenfold, as measured by a reduction in the transformation efficiency of a CRISPR-targeted plasmid. Importantly, we find that in the absence of MvaT and MvaU, Cas proteins are already produced at low cell density prior to the onset of quorum sensing-mediated activation of CRISPR-Cas, which occurs at high cell density. Moreover, the ∆mvaT ∆mvaU mutant has a significantly reduced growth rate, known to independently increase CRISPR-Cas activity. In addition to regulating CRISPR-Cas, the absence of MvaT and MvaU affects phage-host interactions, including enhancing the adsorption of the LPS-binding phage JBD44, highlighting their broader role in coordinating bacterial defence mechanisms.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}

*Pseudomonas aeruginosa/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Bacterial Proteins/genetics/metabolism
*DNA-Binding Proteins/genetics/metabolism
Trans-Activators

@article {pmid40904116,
year = {2025},
author = {Hoikkala, V and Chi, H and Grüschow, S and Graham, S and White, MF},
title = {Diversity and abundance of ring nucleases in type III CRISPR-Cas loci.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240084},
pmid = {40904116},
issn = {1471-2970},
support = {/ERC_/European Research Council/International ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Bacteria/genetics/enzymology ; *Archaea/genetics/enzymology ; *Endonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {Most type III CRISPR-Cas systems facilitate immune responses against invading mobile genetic elements such as phages by generating cyclic oligoadenylates (cOAs). Downstream effectors activated by cOAs are typically non-specific proteins that induce damage to essential cellular components, thereby preventing phage epidemics. Owing to these toxic effects, it is crucial that the production and concentration of cOAs remain under tight regulatory control during infection-free periods or when deactivating the immune response after clearing an infection. Type III CRISPR loci often encode enzymes known as ring nucleases (RNs) that bind and degrade specific cOAs, while some effectors are auto-deactivating. Despite the discovery of several classes of RNs, a comprehensive bioinformatic analysis of type III CRISPR-Cas loci in this context is lacking. Here, we examined 38 742 prokaryotic genomes to provide a global overview of type III CRISPR loci, focusing on the known and predicted RNs. The candidate RNs Csx16 and Csx20 are confirmed as active enzymes, joining Crn1-3. Distributions and patterns of co-occurrence of RNs and associated effectors are explored, allowing the conclusion that a sizeable majority of type III CRISPR systems regulate cOA levels by degrading the signalling molecules, which has implications for cell fate following viral infection.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}

*CRISPR-Cas Systems/genetics
*Bacteria/genetics/enzymology
*Archaea/genetics/enzymology
*Endonucleases/genetics/metabolism
*Bacterial Proteins/genetics/metabolism

@article {pmid40904105,
year = {2025},
author = {Elliott, JFK and Cozens, K and Cai, Y and Waugh, G and Watson, BN and Westra, E and Taylor, TB},
title = {Phage susceptibility to a minimal, modular synthetic CRISPR-Cas system in Pseudomonas aeruginosa is nutrient dependent.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240473},
pmid = {40904105},
issn = {1471-2970},
support = {//UK Government's Horizon Europe funding guarantee/ ; //Royal Society/ ; //Philip Leverhulme Prize/ ; /BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Pseudomonas aeruginosa/virology/genetics ; *CRISPR-Cas Systems ; *Pseudomonas Phages/physiology/genetics ; *Nutrients/metabolism ; *Bacteriophages/physiology ; },
abstract = {CRISPR-Cas systems can provide adaptive, heritable immunity to their prokaryotic hosts against invading genetic material such as phages. It is clear that the importance of acquiring CRISPR-Cas immunity to anti-phage defence varies across environments, but it is less clear if and how this varies across different phages. To explore this, we created a synthetic, modular version of the type I-F CRISPR-Cas system of Pseudomonas aeruginosa. We used this synthetic system to test CRISPR-Cas interference against a panel of 13 diverse phages using engineered phage-targeting spacers. We observed complete protection against eight of these phages, both lytic and lysogenic and with a range of infectivity profiles. However, for two phages, CRISPR-Cas interference was only partially protective in high-nutrient conditions, yet completely protective in low-nutrient conditions. This work demonstrates that nutrient conditions modulate the strength of CRISPR-Cas immunity and highlights the importance of environmental conditions when screening defence systems for their efficacy against various phages.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}

*Pseudomonas aeruginosa/virology/genetics
*CRISPR-Cas Systems
*Pseudomonas Phages/physiology/genetics
*Nutrients/metabolism
*Bacteriophages/physiology

@article {pmid40904104,
year = {2025},
author = {David, E and Plantady, C and Poissonnier, S and Elliott, JFK and Kenck, E and Le Boulch, J and Gutierrez, A and Chevallereau, A},
title = {Systematic functional assessment of anti-phage systems in their native host.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240067},
pmid = {40904104},
issn = {1471-2970},
support = {//ATIP-Avenir/ ; //Agence Nationale de la Recherche/ ; //Idex Paris Cité/ ; //Emergence ville de Paris/ ; },
mesh = {*Escherichia coli/virology/genetics ; *Coliphages/physiology ; *Bacteriophages/physiology ; DNA Restriction-Modification Enzymes/genetics ; },
abstract = {Bacterial resistance to bacteriophages (phages) relies on two primary strategies: preventing phage attachment and blocking post-attachment steps. These post-attachment mechanisms are mediated by diverse defence systems, including DNA-degrading systems such as restriction-modification and CRISPR-Cas, along with abortive infection systems that induce cell death or dormancy. Computational analyses suggest that bacterial genomes encode multiple defence systems, which may act synergistically to enhance phage resistance. However, the regulation, interactions and ecological roles of these systems in native hosts remain poorly understood. This study explored the role of eight predicted defence systems in the clinical isolate NILS69 of Escherichia coli by testing its susceptibility to 93 phages. Infectivity and adsorption assays using mutants defective in these systems revealed that only PD-T4-3 and restriction-modification systems restricted phages that were able to adsorb. The restriction-modification system acted via a predicted type IV endonuclease and was also able to limit plasmid conjugation if the plasmid was transferred from a donor strain lacking a methylase, which is the hallmark of type I, II or III restriction-modification systems. Other defence systems showed no detectable activity, likely owing to phage specificity, environmental regulation or cofactor requirements. These findings underscore the need for further studies to investigate the regulation and ecological roles of bacterial defence systems in their native host contexts.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}

*Escherichia coli/virology/genetics
*Coliphages/physiology
*Bacteriophages/physiology
DNA Restriction-Modification Enzymes/genetics

@article {pmid40904102,
year = {2025},
author = {Pons, BJ and Łapińska, U and Lopes-Domingues, I and Chisnall, MAW and Westra, ER and Pagliara, S and van Houte, S},
title = {Phage provoke growth delays and SOS response induction despite CRISPR-Cas protection.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240474},
pmid = {40904102},
issn = {1471-2970},
support = {/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; /MRC_/Medical Research Council/United Kingdom ; //UK Government's Horizon Europe funding guarantee/ ; //Leverhulme Trust/ ; },
mesh = {*Pseudomonas aeruginosa/virology/growth & development/genetics ; *CRISPR-Cas Systems ; *Pseudomonas Phages/physiology ; *SOS Response, Genetics ; *Bacteriophages/physiology ; },
abstract = {Bacteria evolve resistance against their phage foes with a wide range of resistance strategies whose costs and benefits depend on the level of protection they confer and on the costs for maintainance. Pseudomonas aeruginosa can evolve resistance against its phage DMS3vir either by surface mutations that prevent phage binding or through CRISPR-Cas immunity. CRISPR immunity carries an inducible cost whose exact origin is still unknown, and previous work suggested it stems from the inability of the CRISPR-Cas system to completely prevent phage DNA injection and subsequent gene expression before clearing the phage infection. However, the bacterial processes involved are still unknown, and we hypothesize that CRISPR-immunity-associated costs could come from increased mortality rate or reduced growth ability compared with surface-resistant bacteria. To tease apart these two mechanisms with divergent ecological consequences, we use a novel microfluidics-based single-cell approach combined with flow cytometry methods to monitor the effects of phage exposure on the survival and growth of its host. We observed that while CRISPR immunity protects from phage-induced lysis, it cannot prevent phage-induced division lag, filamentation and SOS response activation in a subpopulation of the host bacteria. These results suggest that the costs associated with CRISPR immunity at the population level are caused by heterogeneity in phage-induced growth defects.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}

*Pseudomonas aeruginosa/virology/growth & development/genetics
*CRISPR-Cas Systems
*Pseudomonas Phages/physiology
*SOS Response, Genetics
*Bacteriophages/physiology

@article {pmid40903118,
year = {2025},
author = {Zhou, J and Shi, X and He, C and Zheng, X and Yuan, R and Yang, X},
title = {SERS biosensor based on the Cas13a assisted entropy-driven system and lychee-like Fe-TiO2 with excellent exciton capture and separation.},
journal = {Analytica chimica acta},
volume = {1372},
number = {},
pages = {344442},
doi = {10.1016/j.aca.2025.344442},
pmid = {40903118},
issn = {1873-4324},
mesh = {*Titanium/chemistry ; *Spectrum Analysis, Raman/methods ; *Entropy ; *Biosensing Techniques/methods ; *Iron/chemistry ; CRISPR-Cas Systems ; *MicroRNAs/analysis ; Limit of Detection ; },
abstract = {BACKGROUND: Entropy-Driven Circuits (EDC), distinguished by their spontaneous operation and absence of enzymatic reactions, represent a superior strategy for integration with CRISPR/Cas systems, as they obviate the potential for interference among various enzymes during the process of DNA amplification and CRISPR/Cas system integration. Due to the wide band gap of TiO2, its response to visible light is limited, and owing to its high crystallinity and exceptionally stable crystal lattice, the charge transfer (CT) process in TiO2 is suboptimal.

RESULTS: In this study, lychee-like Fe-TiO2 was firstly prepared to serve as Raman enhanced substrate, facilitating exciton capture and separation to exhibit an excellent Surface-enhanced Raman spectroscopy (SERS) performance. It is proven that the incorporation of Fe results in a significantly narrower band gap for TiO2, facilitating exciton resonance. The amount of Fe in TiO2 was optimized to fabricate a SERS biosensor for detection of miRNA-21 based on the Cas13a assisted entropy-driven system. The detection limit of miRNA was 43.88 fmol/L.

SIGNIFICANCE: This work proposes a sensing strategy that integrates the CRISPR/Cas system with EDC, leveraging a semiconductor substrate exhibiting superior SERS performance to provide a stable Raman signal, thereby enabling highly sensitive detection of miRNA-21, which has a potential application in disease early warning and treatment.},
}

*Titanium/chemistry
*Spectrum Analysis, Raman/methods
*Entropy
*Biosensing Techniques/methods
*Iron/chemistry
CRISPR-Cas Systems
*MicroRNAs/analysis
Limit of Detection

@article {pmid40902823,
year = {2025},
author = {Zhou, X and Diao, R and Li, X and Ziegler, CA and Gramelspacher, MJ and Freddolino, L and Hou, Z and Zhang, Y},
title = {Cas9 senses CRISPR RNA abundance to regulate CRISPR spacer acquisition.},
journal = {Nature},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41586-025-09577-9},
pmid = {40902823},
issn = {1476-4687},
abstract = {Prokaryotes create adaptive immune memories by acquiring foreign DNA snippets, known as spacers, into the CRISPR array[1]. In type II CRISPR-Cas systems, the RNA-guided effector Cas9 also assists the acquisition machinery by selecting spacers from protospacer adjacent motif (PAM)-flanked DNA[2,3]. Here, we uncover the first biological role for Cas9 that is independent of its dual RNA partners. Following depletion of crRNA and/or tracrRNA, Neisseria apoCas9 stimulates spacer acquisition efficiency. Physiologically, Cas9 senses low levels of crRNA in cells with short CRISPR arrays - such as those undergoing array neogenesis or natural array contractions - and dynamically upregulates acquisition to quickly expand the small immune memory banks. As the CRISPR array expands, rising crRNA abundance in turn reduces apoCas9 availability, thereby dampening acquisition to mitigate autoimmunity risks associate with elevated acquisition. While apoCas9's nuclease lobe alone suffices for stimulating acquisition, only full-length Cas9 responses to crRNA levels to boost acquisition in cells with low immunity depth. Finally, we show that this activity is evolutionarily conserved across multiple type II-C Cas9 orthologs. Altogether, we establish an auto-replenishing feedback mechanism in which apoCas9 safeguards CRISPR immunity depth by acting as both a crRNA sensor and a regulator of spacer acquisition.},
}

@article {pmid40902040,
year = {2025},
author = {Li, L and Dai, H and Sun, R and Zhang, Z and Zhang, B},
title = {MicroRNAs as Biotechnological Targets for Future Food Security and Agricultural Sustainability.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c07100},
pmid = {40902040},
issn = {1520-5118},
abstract = {MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate gene expression, playing key roles in plant growth, development, and stress responses. Their regulatory functions make miRNAs ideal targets for enhancing crop yield, quality, and stress tolerance using biotechnologies, such as transgenic overexpression and CRISPR/Cas genome editing. By targeting multiple genes, miRNAs address complex agricultural challenges effectively. This review focuses on the diverse roles of miRNAs in enhancing crop productivity and resilience; miRNAs are an important biotechnological target for ensuring food security and agricultural sustainability. We also highlight transgenic and CRISPR/Cas genome editing approaches to demonstrate miRNA-driven trait improvements, such as drought/salinity tolerance, pest resistance, and nutrient use efficiency. Due to the quick development of advanced biotechnology tools, both upregulated and downregulated miRNAs can be manipulated for optimizing agronomic traits. Challenges including off-target effects, regulatory barriers, and environmental concerns are analyzed with strategies proposed to overcome them. By leveraging miRNA technologies, this perspective emphasizes their transformative potentials in achieving sustainable agriculture and global food security.},
}

@article {pmid40901825,
year = {2025},
author = {Thiam, R and Ceballos, MS and Beneke, T and Kuk, N and Pasquier, G and Crobu, L and Jeffares, DC and Vergnes, B and Barckmann, B and Sterkers, Y},
title = {A novel Leishmania infantum reference strain for gene editing and the study of visceral leishmaniasis.},
journal = {PloS one},
volume = {20},
number = {9},
pages = {e0327390},
pmid = {40901825},
issn = {1932-6203},
mesh = {*Leishmania infantum/genetics/growth & development/pathogenicity ; *Leishmaniasis, Visceral/parasitology ; *Gene Editing/methods ; Animals ; Humans ; CRISPR-Cas Systems ; Mice ; Macrophages/parasitology ; Genome, Protozoan ; },
abstract = {Parasites of the Leishmania donovani complex are responsible for visceral leishmaniasis, a vector-borne disease transmitted through the bite of female phlebotomine sand flies. As well as the human hosts, these parasites infect many mammals which can serve as reservoirs. Dogs are particularly important reservoirs. Transmission is widespread across Asia, Africa, the Americas, and the Mediterranean basin, including South of France. Visceral leishmaniasis poses a fatal threat if left untreated. Research into the pathophysiology of this neglected disease is of prime importance, as is the development of new drugs. In this study, we evaluated the growth, differentiation, and macrophage infectivity of four L. donovani complex strains and identified L. infantum S9F1 (MHOM/MA/67/ITMAP263, clone S9F1) as a well-adapted strain for genetic engineering studies. We present here the genome sequence and annotation of L infantum S9F1 T7 Cas9, providing the scientific community with easy access to its genomic information. The data has been integrated into the LeishGEdit online resource to support primer design for CRISPR-Cas9 experiments. We now aim to make this strain widely available to foster studies of visceral leishmaniasis.},
}

*Leishmania infantum/genetics/growth & development/pathogenicity
*Leishmaniasis, Visceral/parasitology
*Gene Editing/methods
Animals
Humans
CRISPR-Cas Systems
Mice
Macrophages/parasitology
Genome, Protozoan

@article {pmid40788276,
year = {2025},
author = {Gao, H and Zhang, Y and Wang, Y and Wu, Y and Zheng, Z and Quan, F and Han, Q and Li, Y and Zhang, K},
title = {Integration of CRISPR/Cas12a and Cas13a in one pot for ratiometric calibration of single-nucleotide variations.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {72},
pages = {13675-13678},
doi = {10.1039/d5cc03931b},
pmid = {40788276},
issn = {1364-548X},
mesh = {*Polymorphism, Single Nucleotide ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; Calibration ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Humans ; },
abstract = {Accurate detection of single-nucleotide variations (SNVs) plays a pivotal role in medical diagnosis. Herein, by integrating Cas12a and Cas13a to simultaneously detect wild-type and mutated sites on a single RNA strand, we developed an innovative one-pot SNV analysis method, namely iCasdrop, which is capable of reducing non-specific signals induced by the wild type sequence and achieving ratiometric calibration of SNVs.},
}

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

@article {pmid40599075,
year = {2025},
author = {Zhang, T and Cai, L and Chu, Z and She, A and Yang, J and Su, X},
title = {Rapid and Sensitive Detection of miRNA by Single-Molecule Fluorescence Dequenching Assay with Target Recycled CRISPR/Cas12a Amplification System.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {35},
pages = {e2412228},
doi = {10.1002/smll.202412228},
pmid = {40599075},
issn = {1613-6829},
support = {32271521//National Natural Science Foundation of China/ ; 31971361//National Natural Science Foundation of China/ ; 2022YFC2603902//State Key Research Development Program of China/ ; PT2406//Fundamental Research Funds for the Central Universities/ ; 2023-NHLHCRF-YXHZ-ZRMS-05//National High Level Hospital Clinical Research Funding/ ; },
mesh = {*MicroRNAs/genetics/metabolism/analysis ; Humans ; *CRISPR-Cas Systems/genetics ; Fluorescence ; Carcinoma, Non-Small-Cell Lung/genetics ; Lung Neoplasms/genetics ; Female ; Cell Line, Tumor ; },
abstract = {Dysregulated miRNAs play a critical role in the development of cancers. A rapid and sensitive single-molecule fluorescence dequenching assay combined with a CRISPR/Cas12a-based target recycling amplification system for miRNA detection is developed. This single-molecule assay detects miRNAs down to ≈10 fM within 10 min. An automated single-molecule fluorescent puncta analysis procedure is also created, improving the signal-to-noise ratio by 3.76-fold compared to traditional hidden Markov model (HMM)based methods. The clinical applicability of this technique is demonstrated. Two key miRNA targets associated with non-small cell lung cancer (NSCLC) and ovarian cancer (OC) from 2867 datasets of the TCGA database are screened. Validation is initially conducted at the cell line level, followed by testing with tissue and blood samples from 10 patients with NSCLC and OC. The assay demonstrated high diagnostic accuracy, with receiver operating characteristic curves (area under the curve (AUC) > 0.93) and significant statistical differentiation (p < 0.001) between cancer and healthy samples. This method's exceptional sensitivity and speed highlight its potential for early cancer diagnostics and personalized medicine.},
}

*MicroRNAs/genetics/metabolism/analysis
Humans
*CRISPR-Cas Systems/genetics
Fluorescence
Carcinoma, Non-Small-Cell Lung/genetics
Lung Neoplasms/genetics
Female
Cell Line, Tumor

@article {pmid40536333,
year = {2025},
author = {Zhao, G and Li, Z and Zhao, MJ and Li, SY and Xia, Q and Xu, S and Zhang, Y and Wang, Y and Li, F and Liu, YL and Guo, YH and Xu, RX and Zhou, H and Zhou, H and Ding, WW and Wang, YC and Miao, Y and Wang, Z},
title = {A High-Fidelity RNA-Targeting Cas13X Downregulates Connexin43 in Macroglia: A Novel Neuroprotective Strategy for Glaucoma.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {33},
pages = {e15856},
doi = {10.1002/advs.202415856},
pmid = {40536333},
issn = {2198-3844},
support = {82301215//National Natural Science Foundation of China/ ; 32271043//National Natural Science Foundation of China/ ; 32471057//National Natural Science Foundation of China/ ; 82171047//National Natural Science Foundation of China/ ; 2018SHZDZX01//Shanghai Municipal Science and Technology Major Project/ ; },
mesh = {*Connexin 43/genetics/metabolism ; *Glaucoma/genetics/metabolism/therapy ; Animals ; Mice ; Retinal Ganglion Cells/metabolism ; Disease Models, Animal ; CRISPR-Cas Systems/genetics ; Down-Regulation ; Intraocular Pressure ; Mice, Inbred C57BL ; Humans ; Optic Nerve/metabolism ; },
abstract = {Glaucoma is a neurodegenerative disease characterized by the progressive degeneration of retinal ganglion cells (RGCs) and their axons, ultimately leading to irreversible vision loss. Elevated intraocular pressure (IOP) is one of the significant risk factors in glaucoma; however, neurodegeneration continues even after effective IOP management, underscoring the need for neuroprotective therapies. This study investigates the role of connexin43 (Cx43), which is extensively expressed in retinal macroglia, in regulating microglial activation and optic nerve degeneration in glaucoma. A high-fidelity CRISPR-Cas13 (hfCas13X) system is employed to selectively target and knock down Cx43 expression in macroglia. The findings reveal that Cx43-mediated ATP release through hemichannels exacerbates microglial activation and neuroinflammation, thereby contributing to RGC loss. Notably, in a mouse model of chronic ocular hypertension (COH) glaucoma, knocking down Cx43 in macroglia using the hfCas13X system significantly promoted the survival of RGCs and the integrity of the optic nerve, and improved visual function. The hfCas13X system, which offers high-fidelity RNA editing with minimal off-target effects, represents a novel and promising therapeutic strategy for glaucoma, highlighting the potential of gene editing technologies in the management of neurodegenerative diseases.},
}

*Connexin 43/genetics/metabolism
*Glaucoma/genetics/metabolism/therapy
Animals
Mice
Retinal Ganglion Cells/metabolism
Disease Models, Animal
CRISPR-Cas Systems/genetics
Down-Regulation
Intraocular Pressure
Mice, Inbred C57BL
Humans
Optic Nerve/metabolism

@article {pmid40534129,
year = {2025},
author = {Nicosia, L and Harrison, PT},
title = {CRISPR for cystic fibrosis: Advances and insights from a systematic review.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {9},
pages = {4091-4112},
doi = {10.1016/j.ymthe.2025.06.021},
pmid = {40534129},
issn = {1525-0024},
mesh = {*Cystic Fibrosis/genetics/therapy ; Humans ; *Gene Editing/methods ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; Mutation ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Cystic fibrosis (CF) is a severe genetic disorder caused by loss-of-function mutations in the CFTR gene. Gene-editing approaches have the potential to correct such mutations. This systematic review outlines the mechanisms of the main CRISPR-based technologies, and, through cross-study comparisons, analyzes 27 research articles that applied them to target CF-causing variants. We report and discuss the strategy design, target cell selection, editing efficiency, prevalence of editing byproducts, and levels of CFTR functional restoration achieved in each work, with the aim of providing technical insights for further exploration of CRISPR-based gene-editing approaches. Our findings show that the F508del and W1282X mutations were the most extensively studied CF-causing variants, though over fifteen mutations were targeted overall. The majority of works under review explored the use of homology-directed repair or base editing, with a growing number of studies reporting efficient prime editing. Some studies tackled multiple individual mutations, compared different editors, or tested strategies across various models, while others focused on approaches that rescue CFTR function without directly correcting a mutation. Several works also proposed strategies that could address multiple variants with a single approach, while others highlighted technical difficulties in editing certain regions of the CFTR gene. This cross-study comparison also emphasizes the need for standardized reporting of editing efficiency and functional recovery, and stresses the importance of further single-cell RNA sequencing and in vivo studies to reach clinically relevant conclusions. As gene-editing techniques continue to evolve, and with over 60 ongoing CRISPR-based clinical trials, there is growing optimism for meaningful advancements in CF gene-editing therapeutics.},
}

*Cystic Fibrosis/genetics/therapy
Humans
*Gene Editing/methods
*Cystic Fibrosis Transmembrane Conductance Regulator/genetics
*CRISPR-Cas Systems
*Genetic Therapy/methods
Mutation
Animals
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40518666,
year = {2025},
author = {Yang, Y and Fu, Z and Deng, S and Wu, G and Wang, C and Luo, X and Kang, R and Chen, Y and Peng, C and Zhang, P and Cui, K and Wan, F and Wang, J and Zhou, Q and Chen, W and Xiong, Y and Ma, W and Songyang, Z and Liang, P},
title = {RNA-DNA hybrid binding domain broadens the editing window of base editors.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {9},
pages = {4431-4446},
doi = {10.1016/j.ymthe.2025.06.024},
pmid = {40518666},
issn = {1525-0024},
mesh = {*Gene Editing/methods ; Humans ; Animals ; Mice ; *DNA/genetics/metabolism/chemistry ; CRISPR-Cas Systems ; Ribonuclease H/genetics/metabolism ; *RNA/genetics/metabolism/chemistry ; },
abstract = {Adenine base editors (ABEs) and cytosine base editors (CBEs) are prominent tools for precise genome editing but are hindered by limited editing activity at positions proximal to the protospacer adjacent motif (PAM). This study investigates the potential of enhancing base editors editing activity by fusing them with RNA-DNA hybrid binding domains (RHBDs). Specifically, fusing ABE8e with the RHBD of Homo sapiens RNaseH1 (RHBD1) significantly increased A-to-G editing efficiency in the PAM-proximal region (A9-A15) by up to 3.5-fold, while reducing off-target cytosine editing. Additionally, RHBD1 is compatible with ABEmax, BE4max, and dual base editor (eA&C-BEmax), enhancing their editing activity at the PAM-proximal bases. Notably, RHBD1-fused BE4max led to a 3.1-fold improvement in C-to-T editing efficiency at PAM-proximal region (C9-C12). Furthermore, we demonstrated that RHBD1-fused ABE8e could effectively edit disease-related single nucleotide variations (SNVs) in human cells and validated its efficacy in adult mouse liver. These findings highlight the significance of the RHBD in expanding editing window and the applicability of base editors for gene therapy and disease modeling.},
}

*Gene Editing/methods
Humans
Animals
Mice
*DNA/genetics/metabolism/chemistry
CRISPR-Cas Systems
Ribonuclease H/genetics/metabolism
*RNA/genetics/metabolism/chemistry

@article {pmid40493884,
year = {2025},
author = {Loke, J and Kim, PG and Nguyen, TTP and Boileau, M and McConkey, M and Miller, A and Shin, W and Hergott, CB and Ericsson, M and Nordstrom, A and Llopis, PM and Armstrong, SA and Mancias, JD and Ebert, BL},
title = {An in vivo barcoded CRISPR-Cas9 screen identifies Ncoa4-mediated ferritinophagy as a dependence in Tet2-deficient hematopoiesis.},
journal = {Blood},
volume = {146},
number = {10},
pages = {1174-1186},
doi = {10.1182/blood.2024028033},
pmid = {40493884},
issn = {1528-0020},
mesh = {*CRISPR-Cas Systems ; *Hematopoiesis/genetics ; *Ferritins/metabolism/genetics ; *DNA-Binding Proteins/genetics/deficiency/metabolism ; Animals ; *Nuclear Receptor Coactivators/metabolism/genetics ; Dioxygenases ; Mice ; *Proto-Oncogene Proteins/genetics/deficiency ; Hematopoietic Stem Cells/metabolism ; Mice, Knockout ; *Autophagy ; Iron/metabolism ; Humans ; },
abstract = {TET2 is among the most commonly mutated genes in both clonal hematopoiesis and myeloid malignancies; thus, the ability to identify selective dependencies in TET2-deficient cells has broad translational significance. Here, we identify regulators of Tet2 knockout (KO) hematopoietic stem and progenitor cell (HSPC) expansion using an in vivo CRISPR-Cas9 KO screen, in which nucleotide barcoding enabled large-scale clonal tracing of Tet2-deficient HSPCs in a physiologic setting. Our screen identified candidate genes, including Ncoa4, that are selectively required for Tet2 KO clonal outgrowth compared with wild type. Ncoa4 targets ferritin for lysosomal degradation (ferritinophagy), maintaining intracellular iron homeostasis by releasing labile iron in response to cellular demands. In Tet2-deficient HSPCs, increased mitochondrial adenosine triphosphate production correlates with increased cellular iron requirements and, in turn, promotes Ncoa4-dependent ferritinophagy. Restricting iron availability reduces Tet2 KO stem cell numbers, revealing a dependency in TET2-mutated myeloid neoplasms.},
}

*CRISPR-Cas Systems
*Hematopoiesis/genetics
*Ferritins/metabolism/genetics
*DNA-Binding Proteins/genetics/deficiency/metabolism
Animals
*Nuclear Receptor Coactivators/metabolism/genetics
Dioxygenases
Mice
*Proto-Oncogene Proteins/genetics/deficiency
Hematopoietic Stem Cells/metabolism
Mice, Knockout
*Autophagy
Iron/metabolism
Humans

@article {pmid40492429,
year = {2025},
author = {Meng, R and Li, J and Wang, W and Liang, D and Li, Z and Mao, C and Li, Q and Zhang, Y and Chen, H and Tang, J and Hu, P and Niu, Q and Huang, X and Shen, B and Zhang, J},
title = {Engineered Cas12j-8 is a Versatile Platform for Multiplexed Genome Modulation in Mammalian Cells.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {33},
pages = {e02593},
doi = {10.1002/advs.202502593},
pmid = {40492429},
issn = {2198-3844},
support = {2022YFC2702705//National Key Research and Development Project of China/ ; 82221005//Creative Research Groups of China/ ; 32371546//National Natural Science Foundation of China/ ; 82071434//National Natural Science Foundation of China/ ; 81971398//National Natural Science Foundation of China/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; HEK293 Cells ; *CRISPR-Associated Proteins/genetics ; },
abstract = {Cas12j-8 is a compact Cas nuclease discovered from the metagenome of giant bacteriophages, consisting of only 717 amino acids and recognizing the '5-TTN-3' protospacer adjacent motif (PAM) sequence. However, its low gene editing efficiency in mammalian cells limits its application in therapeutic gene editing. To address this limitation, structure-guided mutagenesis is employed to replace key negatively charged residues with arginine, strengthening DNA binding. The resulting quintuple mutant, engineered Cas12j-8 (enCas12j-8), demonstrates robust on-target editing efficiency comparable to LbCas12a while maintaining low off-target effects. Cytosine base editors (CBEs) and adenine base editors (ABEs) are developed using enCas12j-8, achieving up to 29.54-fold C-to-T and 36.57-fold A-to-G conversion efficiency compared with the wild-type at the dominated sites, respectively. Notably, enCas12j-8 enables multiplexed editing of three genomic loci simultaneously via a single crRNA array, achieving efficiencies comparable to single-guide approaches. Additionally, enCas12j-8-ABE facilitates the disruption of splice acceptor sites, effectively inducing exon skipping in the SOD1 gene. This strategy holds potential significance for therapeutic genome modulation. These findings establish enCas12j-8 as a versatile, high-precision tool for genome engineering, combining efficient delivery, multiplexing capability, and compatibility with diverse editing modalities.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems/genetics
Animals
HEK293 Cells
*CRISPR-Associated Proteins/genetics

@article {pmid40901634,
year = {2025},
author = {Jiang, X and Wang, X and Shen, S and Hou, S and Yu, C},
title = {3D Genome Engineering: Current Advances and Therapeutic Opportunities in Human Diseases.},
journal = {Research (Washington, D.C.)},
volume = {8},
number = {},
pages = {0865},
doi = {10.34133/research.0865},
pmid = {40901634},
issn = {2639-5274},
abstract = {Dynamic chromatin 3-dimensional (3D) conformation is a key mechanism regulating gene expression and cellular function during development and disease. Elucidating the structure, functional dynamics, and spatiotemporal organization of the 3D genome requires integrating multiple experimental approaches, including chromatin conformation capture techniques, precise genome manipulation tools, and advanced imaging technologies. Notably, CRISPR/Cas systems have emerged as a revolutionary genome-editing platform, offering unprecedented opportunities for manipulating 3D genome organization and investigating disease mechanisms. This review systematically examines recent advances in CRISPR-based mammalian 3D genome engineering and explores the therapeutic potential of 3D genome engineering strategies in disease intervention.},
}

@article {pmid40900077,
year = {2025},
author = {Yu, Y and Zhang, Z and Zhai, Z and Sun, B and Yang, D and Wang, Z and Lin, Q and Zhou, X and Zhao, J},
title = {A Novel Mouse Model of Granular Corneal Dystrophy Type II Reveals Impaired Autophagy and Recapitulates Human Pathogenesis.},
journal = {Investigative ophthalmology & visual science},
volume = {66},
number = {12},
pages = {7},
doi = {10.1167/iovs.66.12.7},
pmid = {40900077},
issn = {1552-5783},
mesh = {Animals ; *Corneal Dystrophies, Hereditary/genetics/pathology/metabolism ; *Autophagy/physiology ; *Disease Models, Animal ; Mice ; Microscopy, Electron, Transmission ; Tomography, Optical Coherence ; Transforming Growth Factor beta/genetics/metabolism ; Blotting, Western ; Extracellular Matrix Proteins/genetics/metabolism ; Humans ; *Mutation ; Mice, Inbred C57BL ; Slit Lamp Microscopy ; CRISPR-Cas Systems ; Phenotype ; *Cornea/pathology ; },
abstract = {PURPOSE: To develop and characterize a novel mouse model of granular corneal dystrophy type II (GCD2) using CRISPR/Cas9 technology and explore the underlying pathogenesis of transforming growth factor-beta-induced protein (TGFBIp) aggregation.

METHODS: CRISPR/Cas9 technology was employed to introduce the R124H mutation in the TGFBI gene of mice. Genomic sequencing and polymerase chain reaction confirmed the mutation. Phenotypic characteristics were evaluated through slit-lamp examination, optical coherence tomography, histological analysis, electron microscopy, and immunofluorescence, comparing wild-type (WT), heterozygous (HE), and homozygous (HO) mice. Transcriptome sequencing was conducted to identify the pathogenesis of GCD2. The findings were further validated through western blotting and transmission electron microscopy.

RESULTS: The R124H mutation in TGFBI was successfully introduced, with breadcrumb-like deposits observed in the corneas of mutant mice, with HO mice displaying more severe phenotypes than HE mice. TGFBIp levels were elevated in HE and HO mice (both P < 0.001). Histological and electron microscopy analyses revealed abnormal collagen arrangement and TGFBIp deposits in the corneal stroma of the HE and HO mice. Transcriptome analysis indicated that the TGFBI-R124H mutation was associated with impaired autophagy, endocytosis, and extracellular matrix signaling. Additional experiments confirmed autophagy-related markers LC3 and SQSTM1 were upregulated in the corneas of mutant mice, accompanied by increased autophagosome formation in corneal keratocytes, indicating impaired autophagy flux in HE and HO mice.

CONCLUSIONS: We established a GCD2 mouse model caused by the R124H mutation using CRISPR/Cas9, providing a reliable platform for understanding pathogenesis for GCD2.},
}

Animals
*Corneal Dystrophies, Hereditary/genetics/pathology/metabolism
*Autophagy/physiology
*Disease Models, Animal
Mice
Microscopy, Electron, Transmission
Tomography, Optical Coherence
Transforming Growth Factor beta/genetics/metabolism
Blotting, Western
Extracellular Matrix Proteins/genetics/metabolism
Humans
*Mutation
Mice, Inbred C57BL
Slit Lamp Microscopy
CRISPR-Cas Systems
Phenotype
*Cornea/pathology

@article {pmid40897812,
year = {2025},
author = {Wolter, JM and James, LM and Boeshore, SL and Mao, H and McCoy, ES and Ryan, DF and Fragola, G and Taylor-Blake, B and Stein, JL and Zylka, MJ},
title = {AAV-dCas9 vector unsilences paternal Ube3a in neurons by impeding Ube3a-ATS transcription.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1332},
pmid = {40897812},
issn = {2399-3642},
support = {631904//Simons Foundation/ ; 1R01NS109304//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; T32HD040127//U.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; },
mesh = {Animals ; *Dependovirus/genetics ; Mice ; *Neurons/metabolism ; Humans ; *Ubiquitin-Protein Ligases/genetics/metabolism ; Genetic Vectors/genetics ; *Angelman Syndrome/genetics/therapy ; *Transcription, Genetic ; *CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Male ; *RNA, Long Noncoding/genetics ; },
abstract = {Angelman syndrome (AS) is a debilitating neurodevelopmental disorder caused by loss of maternally-inherited UBE3A. In neurons, paternally-inherited UBE3A is silenced in cis by a long non-coding RNA called Ube3a-ATS. Here, we found that Neisseria meningitidis Cas9 with two mutations (D15A and H587A) in the nuclease domains (dNmCas9) can unsilence the dormant paternal Ube3a allele in mouse and human neurons when targeted to Snord115 snoRNA genes located in introns of Ube3a-ATS. Importantly, dNmCas9 disrupted Ube3a-ATS with a non-template bias and in the absence of a chromatin modifying domain, supporting a transcriptional interference mechanism. When packaged into an adeno-associated virus (AAV) vector, dNmCas9 exhibited dose-dependent Ube3a-ATS knock-down and paternal Ube3a unsilencing in vitro and in vivo. This vector also partially rescued the hind limb clasp phenotype when delivered to neonatal AS model mice. Collectively, our study underscores the potential of dCas9-based therapeutics without chromatin repression domains to mediate transcriptional downregulation.},
}

Animals
*Dependovirus/genetics
Mice
*Neurons/metabolism
Humans
*Ubiquitin-Protein Ligases/genetics/metabolism
Genetic Vectors/genetics
*Angelman Syndrome/genetics/therapy
*Transcription, Genetic
*CRISPR-Associated Protein 9/genetics/metabolism
CRISPR-Cas Systems
Male
*RNA, Long Noncoding/genetics

@article {pmid40897312,
year = {2025},
author = {Bircheneder, M and Parniske, M},
title = {Engineering and comparison of cas12a-based genome editing systems in plants.},
journal = {The Plant journal : for cell and molecular biology},
volume = {123},
number = {5},
pages = {e70410},
pmid = {40897312},
issn = {1365-313X},
support = {401867691//Deutsche Forschungsgemeinschaft/ ; //Bayerisches Staatsministerium für Unterricht und Kultus/ ; },
mesh = {*Gene Editing/methods ; *Arabidopsis/genetics ; Nicotiana/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; *Lotus/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Plants, Genetically Modified ; },
abstract = {While Cas9 and Cas12a are both RNA-guided endonucleases used for genome editing, only Cas12a is able to process pre-crRNA via its additional ribonuclease activity. This feature reduces the complexity of Cas12a versus Cas9-based genome editing systems thus providing an attractive alternative for generating site-specific mutations in plants. Here we aimed to improve the efficiency of the cas12a-based generation of two double-strand breaks flanking the open reading frame of a target gene, leading to its full deletion. To this end, we compared the relative impact of different components on cas12a-based gene deletion efficiency in three different eudicotyledons, Arabidopsis thaliana, Lotus japonicus, and Nicotiana benthamiana. We detected the highest cas12a-based editing efficiency with a combination of suitable promoters for crRNA and cas12a expression, a tandem terminator to control cas12a expression, a re-coded cas12a, adapted to the codon usage of Arabidopsis and engineered to carry introns, and encoding a Cas12a flanked by a nuclear localization signal at both ends. Our work revealed the high potential for improving cas12a-based genome editing systems for plant genetic research.},
}

*Gene Editing/methods
*Arabidopsis/genetics
Nicotiana/genetics
CRISPR-Cas Systems/genetics
Genome, Plant/genetics
*Lotus/genetics
*CRISPR-Associated Proteins/genetics/metabolism
*Endodeoxyribonucleases/genetics/metabolism
*Bacterial Proteins/genetics/metabolism
Plants, Genetically Modified

@article {pmid40896718,
year = {2025},
author = {Madsen, CK and Hanak, T and Aronsson, H and Brinch-Pedersen, H},
title = {Rapid one-step CRISPR-cas vector assembly by isothermal spacer removal linearization and sequence-ligation independent cloning (ISRL-SLIC).},
journal = {MethodsX},
volume = {15},
number = {},
pages = {103567},
pmid = {40896718},
issn = {2215-0161},
abstract = {CRISPR-Cas genome editing is a powerful tool in various fields, but current cloning methods can be time-consuming due to the frequent use of intermediate entry vectors and multiple steps involving restriction enzymes and ligases. These multiple steps can create a bottleneck in CRISPR-Cas experiments. In response to this challenge, we propose a highly efficient streamlined approach, which enables simultaneous linearization of the acceptor plasmid and protospacer cloning in a single isothermal reaction. This eliminates the need for entry vectors, pre-linearization of vectors, and in vitro ligation, thus significantly simplifying the cloning process. The method can be applied to clone short synthetic oligos for single protospacer constructs or multiple amplicons for multiplex genome editing designs. Either way, researchers can proceed directly to Escherichia coli transformation after a one-hour isothermal reaction and recover the final construct within two days. By combining the advantages of sequence-ligation independent cloning (SLIC) cloning with a streamlined workflow, our approach facilitates rapid and efficient construction of CRISPR-Cas vectors and holds the promise of accelerating research and development in genome editing and related fields. To expedite the cloning of constructs, we propose a rapid one-step CRISPR-Cas vector assembly method that combines isothermal spacer removal with a sequence-ligation-independent cloning reaction. We could show that Isothermal Spacer Removal Linearization and Sequence-Ligation Independent Cloning (ISRL-SLIC) can create single, double and triple protospacer constructs in one reaction with scalability. The ISRL-SLIC reaction delivers clones under a broad range of oligo concentration making it a robust and time saving alternative to other methods for constructing CRISPR-Cas vectors.},
}

@article {pmid40895203,
year = {2025},
author = {Kesarwani, P and Sundar, D},
title = {Conformational changes induced by K949A mutation in the CRISPR-Cas12a complex drives an effective target-binding mechanism.},
journal = {Current research in structural biology},
volume = {10},
number = {},
pages = {100173},
pmid = {40895203},
issn = {2665-928X},
abstract = {The CRISPR/Cas system is a potential tool for genome editing, yet it faces challenges due to off-target activity caused by mismatches at specific positions. However, Off-target activity can be minimized by optimal design of guide RNA (gRNA) but there remains a possibility of unintended cleavage, highlighting the role of the Cas nuclease in off-target recognition and binding the target site. This study focuses on comparing the conformational dynamics and stability of Wildtype, RR, RVR, RRm and RVRm variants of AsCas12a with gRNA-DNA bound complexes. It was found that the cross-correlation coefficient between His1167 of the NUC domain and Thr384 of the REC II domain significantly increased after the K949A mutation compared to other variants. The extensive spread of principal components also revealed flexibility in both Cas nuclease and gRNA-DNA hybrid of RVR variant and wildtype AsCas12a whereas the confined clusters in PCA plot suggests increased stability in both the variants after mutation. This study shows the role of K949A mutation in improving stability of PAM variants and predicted critical residues such as His1167, Thr384 and Ser959, in inducing stability in mutants of PAM variants.},
}

@article {pmid40893972,
year = {2025},
author = {},
title = {Correction to "High-efficiency genome editing of an extreme thermophile Thermus thermophilus using endogenous type I and type III CRISPR-Cas systems".},
journal = {mLife},
volume = {4},
number = {4},
pages = {470},
doi = {10.1002/mlf2.70010},
pmid = {40893972},
issn = {2770-100X},
abstract = {[This corrects the article DOI: 10.1002/mlf2.12045.].},
}

@article {pmid40891977,
year = {2025},
author = {Li, X and Zhong, Y and Jin, C and Chen, X and Cui, X and Xu, Y and Fan, Y and Song, F and Cen, P and Dong, L and Yu, K and He, Q and Wang, J and Hu, S and Zhang, XY and Li, C and Tian, M and Zhang, H},
title = {CRISPR/Cas9-Engineered Triple-Fusion Reporter Gene Imaging System for Monitoring Transplanted Neural Progenitor Cells in Ischemic Stroke.},
journal = {Radiology},
volume = {316},
number = {3},
pages = {e250305},
doi = {10.1148/radiol.250305},
pmid = {40891977},
issn = {1527-1315},
mesh = {Animals ; *Neural Stem Cells/transplantation ; Rats ; Male ; *Ischemic Stroke/diagnostic imaging/therapy ; *CRISPR-Cas Systems/genetics ; *Genes, Reporter/genetics ; Magnetic Resonance Imaging/methods ; Humans ; Disease Models, Animal ; Rats, Sprague-Dawley ; Positron Emission Tomography Computed Tomography/methods ; },
abstract = {Background Neural progenitor cell therapy holds great potential for repairing brain damage induced by ischemic stroke, and molecular imaging plays a crucial role in evaluating the therapeutic efficacy of neural progenitor cell transplantation. However, the presence of the blood-brain barrier significantly limits the effectiveness of such imaging methods. Purpose To enable long-term monitoring of transplanted human neural progenitor cells (hNPCs) in a rat model of ischemic stroke by combining a clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9)-engineered triple-fusion (TF) reporter gene system with a noninvasive adenosine agonistic micelle (AM)-based probe delivery strategy. Materials and Methods Between January 2021 and May 2025, 60 male rats that were 2 months old were included. Thirty-seven rats with stroke were administered either TF human NPCs (hNPCs) or vehicle (culture media) and underwent MRI, bioluminescence imaging, PET/CT, and neurologic assessments at weeks 1, 2, 4, and 8 after transplantation. Comparisons between groups were determined by t tests, one-way analysis of variance, linear regression, and linear mixed-effects model. Results TF-hNPCs proliferated within the ischemic rat brain (week 8 vs week 1, bioluminescence imaging and PET: P < .001 and P = .02, respectively) and exhibited progressive migration and maturation by 8 weeks after transplantation (proportion of microtubule-associated protein 2-positive TF-hNPCs at week 8 vs week 4: 94.08% ± 3.02 vs 85.47% ± 6.54, respectively [P = .04]; proportion of doublecortin-positive TF-hNPCs at week 4 vs week 2: 83.90% ± 2.84 vs 59.74% ± 0.55, respectively [P = .02]). Moreover, TF-hNPC transplantation increased glucose (fluorine 18 fluorodeoxyglucose) uptake in the ischemic brain (TF-hNPCs vs vehicle at week 4, 0.58 ± 0.04 vs 0.37 ± 0.05, respectively [P = .008]; TF-hNPCs vs vehicle at week 8, 0.52 ± 0.06 vs 0.29 ± 0.02, respectively [P = .01]) and attenuated neurologic deficits compared with the vehicle group (neurologic score, TF-hNPCs vs vehicle at week 8: 9.6 ± 0.25 vs 7.6 ± 0.3, respectively; P = .003). Conclusion A CRISPR/Cas9-engineered TF reporter gene imaging system combined with a noninvasive AM-based approach enabled in vivo monitoring of transplanted human NPCs in a rat model of ischemic stroke. © RSNA, 2025 Supplemental material is available for this article. See also the editorial by Chapelin in this issue.},
}

Animals
*Neural Stem Cells/transplantation
Rats
Male
*Ischemic Stroke/diagnostic imaging/therapy
*CRISPR-Cas Systems/genetics
*Genes, Reporter/genetics
Magnetic Resonance Imaging/methods
Humans
Disease Models, Animal
Rats, Sprague-Dawley
Positron Emission Tomography Computed Tomography/methods

@article {pmid40857331,
year = {2025},
author = {Farr, GH and Reid, W and Hasegawa, EH and Azzam, A and Young, I and Li, ML and Olson, AK and Beier, DR and Maves, L},
title = {A systems genetics approach identifies roles for proteasome factors in heart development and congenital heart defects.},
journal = {PLoS genetics},
volume = {21},
number = {8},
pages = {e1011579},
pmid = {40857331},
issn = {1553-7404},
mesh = {Animals ; Zebrafish/genetics/embryology ; *Proteasome Endopeptidase Complex/genetics/metabolism ; *Heart Defects, Congenital/genetics/pathology ; *Heart/embryology/growth & development ; Humans ; *Zebrafish Proteins/genetics/metabolism ; Protein Interaction Maps/genetics ; Gene Expression Regulation, Developmental ; Transcriptome/genetics ; Mutation ; CRISPR-Cas Systems ; },
abstract = {Congenital heart defects (CHDs) occur in about 1% of live births and are the leading cause of infant death due to birth defects. While there have been remarkable efforts to pursue large-scale whole-exome and genome sequencing studies on CHD patient cohorts, it is estimated that these approaches have thus far accounted for only about 50% of the genetic contribution to CHDs. We sought to take a new approach to identify genetic causes of CHDs. By combining analyses of genes that are under strong selective constraint along with published embryonic heart transcriptomes, we identified over 200 new candidate genes for CHDs. We utilized protein-protein interaction (PPI) network analysis to identify a functionally-related subnetwork consisting of known CHD genes as well as genes encoding proteasome factors, in particular POMP, PSMA6, PSMA7, PSMD3, and PSMD6. We used CRISPR targeting in zebrafish embryos to preliminarily identify roles for the PPI subnetwork genes in heart development. We then used CRISPR to create new mutant zebrafish strains for two of the proteasome genes in the subnetwork: pomp and psmd6. We show that loss of proteasome gene functions leads to defects in zebrafish heart development, including dysmorphic hearts, myocardial cell blebbing, and reduced outflow tracts. We also identified deficits in cardiac function in pomp and psmd6 mutants. These heart defects resemble those seen in zebrafish mutants for known CHD genes and other critical heart development genes. Our study provides a novel systems genetics approach to further our understanding of the genetic causes of human CHDs.},
}

Animals
Zebrafish/genetics/embryology
*Proteasome Endopeptidase Complex/genetics/metabolism
*Heart Defects, Congenital/genetics/pathology
*Heart/embryology/growth & development
Humans
*Zebrafish Proteins/genetics/metabolism
Protein Interaction Maps/genetics
Gene Expression Regulation, Developmental
Transcriptome/genetics
Mutation
CRISPR-Cas Systems

@article {pmid40779935,
year = {2025},
author = {Wu, J and Li, X and Yin, X and Hu, J and Zhou, P and Zhong, X and Wu, M},
title = {Rapid detection of plasma exosomal LncRNA CASC9 for HCC using RT-RPA-CRISPR/Cas12a assay.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {266},
number = {},
pages = {117085},
doi = {10.1016/j.jpba.2025.117085},
pmid = {40779935},
issn = {1873-264X},
mesh = {Humans ; *Carcinoma, Hepatocellular/blood/diagnosis/genetics ; *Liver Neoplasms/blood/diagnosis/genetics ; *Exosomes/genetics/metabolism ; *RNA, Long Noncoding/blood/genetics ; Biomarkers, Tumor/blood/genetics ; Male ; Female ; Middle Aged ; ROC Curve ; CRISPR-Cas Systems/genetics ; alpha-Fetoproteins ; Real-Time Polymerase Chain Reaction/methods ; Aged ; },
abstract = {Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide. Early detection is essential for improving patient outcomes. Long non-coding RNAs (lncRNAs) in plasma exosomes have emerged as promising non-invasive biomarkers. However, sensitive detection methods remain limited. Plasma exosomes were isolated and validated using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot (WB). RNA sequencing identified CASC9 as the most significantly upregulated exosomal lncRNA in HCC patients. Its diagnostic value was evaluated using real-time quantitative PCR (RT-qPCR) and a novel RT-RPA-CRISPR/Cas12a fluorescence assay. Diagnostic performance was assessed through receiver operating characteristic (ROC) curve analysis and compared with alpha-fetoprotein (AFP). Exosomal CASC9 levels were significantly elevated in HCC patients and correlated with tumor size, stage, and number (P < 0.001). ROC analysis demonstrated that CASC9 had superior diagnostic accuracy (area under the curve [AUC] = 0.822) compared to AFP (AUC = 0.795), with further improvement when combined (AUC = 0.875). The RT-RPA-CRISPR/Cas12a assay achieved a detection limit of 0.1 copies/μL, outperforming RT-qPCR. When combined with RT-qPCR and AFP, the method achieved an AUC of 0.987 against normal controls and 0.975 against benign cases. Plasma exosomal CASC9 is a promising diagnostic biomarker for HCC. The RT-RPA-CRISPR/Cas12a assay offers a rapid, ultra-sensitive, and clinically feasible detection strategy.},
}

Humans
*Carcinoma, Hepatocellular/blood/diagnosis/genetics
*Liver Neoplasms/blood/diagnosis/genetics
*Exosomes/genetics/metabolism
*RNA, Long Noncoding/blood/genetics
Biomarkers, Tumor/blood/genetics
Male
Female
Middle Aged
ROC Curve
CRISPR-Cas Systems/genetics
alpha-Fetoproteins
Real-Time Polymerase Chain Reaction/methods
Aged

@article {pmid40764055,
year = {2025},
author = {Stewart-Ornstein, J and Irby, MJ and Lilieholm, MK and Laprise, D and Collier, MD and Aunins, T and Harjanto, D and Chang, AN and Reyon, D and Duffield, JS},
title = {3'-end ligation sequencing is a sensitive method to detect DNA nicks at potential sites of off-target activity induced by prime editors.},
journal = {Genome research},
volume = {35},
number = {9},
pages = {2064-2075},
pmid = {40764055},
issn = {1549-5469},
mesh = {Humans ; *Gene Editing/methods ; *Sequence Analysis, DNA/methods ; Genome, Human ; CRISPR-Cas Systems ; *DNA/genetics ; DNA Breaks, Double-Stranded ; },
abstract = {Gene editing makes precise changes in DNA to restore normal function or expression of genes; however, the advancement of gene editing to the clinic is limited by the potential genotoxicity of off-target editing. To comprehensively identify potential sites in the genome that may be recognized by gene editing agents, in vitro approaches, in which the editor is combined with human genomic DNA and sites where editing may occur are identified biochemically, are important tools. Existing biochemical approaches for off-target discovery recognize double-stranded breaks generated by nuclease-based gene editors such as SpCas9, but novel approaches are needed for new editing modalities, such as prime editing, that nick one strand of DNA. To fill this gap, we have developed 3'-end ligation sequencing (PEG-seq), which can identify prime editor-induced nicks throughout the genome on in vitro digested human genomic DNA to identify potential off-target sites. Here we show that PEG-seq is an important addition to the off-target detection toolkit, enabling off-target discovery for DNA nicking gene editors such as prime editors.},
}

Humans
*Gene Editing/methods
*Sequence Analysis, DNA/methods
Genome, Human
CRISPR-Cas Systems
*DNA/genetics
DNA Breaks, Double-Stranded

@article {pmid40757665,
year = {2025},
author = {Carlsson, PO and Hu, X and Scholz, H and Ingvast, S and Lundgren, T and Scholz, T and Eriksson, O and Liss, P and Yu, D and Deuse, T and Korsgren, O and Schrepfer, S},
title = {Survival of Transplanted Allogeneic Beta Cells with No Immunosuppression.},
journal = {The New England journal of medicine},
volume = {393},
number = {9},
pages = {887-894},
doi = {10.1056/NEJMoa2503822},
pmid = {40757665},
issn = {1533-4406},
mesh = {Humans ; Male ; *Diabetes Mellitus, Type 1/therapy/surgery ; *Islets of Langerhans Transplantation/methods ; Transplantation, Homologous ; Gene Editing ; Adult ; *Graft Survival ; CRISPR-Cas Systems ; Insulin/metabolism ; *Insulin-Secreting Cells/transplantation ; },
abstract = {The need to suppress a patient's immune system after the transplantation of allogeneic cells is associated with wide-ranging side effects. We report the outcomes of transplantation of genetically modified allogeneic donor islet cells into a man with long-standing type 1 diabetes. We used clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 12b (Cas12b) editing and lentiviral transduction to genetically edit the cells to avoid rejection; the cells were then transplanted into the participant's forearm muscle. He did not receive any immunosuppressive drugs and, at 12 weeks after transplantation, showed no immune response against the gene-edited cells. C-peptide measurements showed stable and glucose-responsive insulin secretion. A total of four adverse events occurred, none of which were serious or related to the study drug. (Funded by the Leona M. and Harry B. Helmsley Charitable Trust; EudraCT number, 2023-507988-19-00; ClinicalTrials.gov number, NCT06239636.).},
}

Humans
Male
*Diabetes Mellitus, Type 1/therapy/surgery
*Islets of Langerhans Transplantation/methods
Transplantation, Homologous
Gene Editing
Adult
*Graft Survival
CRISPR-Cas Systems
Insulin/metabolism
*Insulin-Secreting Cells/transplantation

@article {pmid40744866,
year = {2025},
author = {Schmal, M and Kramer, LTS and Mach, RL and Mach-Aigner, AR and Zimmermann, C},
title = {Providing a toolbox for genomic engineering of Trichoderma aggressivum.},
journal = {Microbiology spectrum},
volume = {13},
number = {9},
pages = {e0096625},
pmid = {40744866},
issn = {2165-0497},
support = {P 34036//Austrian Science Fund/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; Genome, Fungal ; Transformation, Genetic ; *Trichoderma/genetics ; *Genetic Engineering/methods ; Hypocreales/genetics ; Agaricus ; Plasmids/genetics ; Fungal Proteins/genetics ; Plant Diseases/microbiology ; Genomics/methods ; },
abstract = {Fungi belonging to the genus of Trichoderma have a long history of infecting crops of edible fungi and reducing the yield. Trichoderma aggressivum is the main causal agent of green mold disease in Agaricus bisporus. Despite its infamous role as a persistent and aggressive contamination in A. bisporus crops, T. aggressivum can also be used as a biocontrol agent or as a promoter of plant growth. In order to work efficiently with T. aggressivum on a molecular biology level, a transformation protocol is required. This study provides a detailed protocol on how to perform a transformation in T. aggressivum using plasmid DNA for ectopic integration. In addition, a Cas9-RNP-based approach has been established for genome editing. We performed two transformations to confirm the usability of the hph gene as well as the pyr4 gene from Trichoderma reesei as selection markers. First, we integrated the hph gene ectopically and determined the overall transformation efficiency. In the second transformation experiment, we disrupted the ornithine-5' phosphate decarboxylase gene of T. aggressivum by using the CRISPR-Cas9 approach. In total, four candidates showed the expected uridine auxotrophy and resistance to 5-FOA. Additionally, the genomic locus around the CRISPR target sites was sequenced to determine the exact circumstances around the gene disruption. To complete the genetic toolbox for T. aggressivum, the pyr4 gene of T. reesei was tested as a suitable selection marker in one of the generated uridine auxotrophic strains.IMPORTANCEResearchers need an efficient tool for genomic manipulation to investigate the fundamental biology of mycoparasitism of T. aggressivum and its correlation to secondary metabolites. We provide a protocol for transformation of T. aggressivum and successfully demonstrated transformation of T. aggressivum using a plasmid and genome editing applying a Cas9-RNP-based strategy. Simultaneously, we established two selection markers, the hph gene and pyr4 gene from T. reesei. By applying these methods, we give researchers the tools needed to investigate T. aggressivum on a deeper level. Possible applications include activation of biosynthetic gene clusters of secondary metabolites to determine the biosynthetic pathway and biotechnological applications of these compounds.},
}

*Gene Editing/methods
CRISPR-Cas Systems
Genome, Fungal
Transformation, Genetic
*Trichoderma/genetics
*Genetic Engineering/methods
Hypocreales/genetics
Agaricus
Plasmids/genetics
Fungal Proteins/genetics
Plant Diseases/microbiology
Genomics/methods

@article {pmid40646716,
year = {2025},
author = {Coirry, C and Manessier, J and Clot, C and Mortier, M and Fauvarque, MO and Taillebourg, E},
title = {The deubiquitinase USP36 funtions through catalytic-dependent and catalytic-independent mechanisms in Drosophila.},
journal = {Genetics},
volume = {231},
number = {1},
pages = {},
pmid = {40646716},
issn = {1943-2631},
support = {ANR-17-EURE-0003//French National Research Agency/ ; //IDEX Université Grenoble Alpes/ ; //GRAL PhD Operating Costs/ ; },
mesh = {Animals ; *Drosophila Proteins/genetics/metabolism ; Male ; *Drosophila melanogaster/genetics/growth & development ; *Ubiquitin Thiolesterase/genetics/metabolism ; Spermatogenesis/genetics ; CRISPR-Cas Systems ; Female ; Proto-Oncogene Proteins c-myc/metabolism/genetics ; Deubiquitinating Enzymes/metabolism/genetics ; },
abstract = {Deubiquitinases (DUBs) form a specific class of proteases removing ubiquitin from target proteins. They are involved in the regulation of many cellular processes including cell growth and proliferation. Among them, USP36 is a key regulator of the oncogenic transcription factor c-Myc, preventing its degradation by the proteasome. These 2 proteins form an evolutionary conserved complex providing the opportunity to investigate USP36 mechanisms of action in vivo in a genetically tractable model such as Drosophila melanogaster. Null mutants of dUsp36 die early during larval development and exhibit severe growth defects. Strikingly, we report here that flies expressing a catalytically inactive version of dUSP36 produced by CRISPR/Cas9 gene editing survive to adulthood with only minor growth defects, yet males are infertile. This finding indicates that dUSP36 deubiquitinating activity is dispensable for cell growth but essential for spermatogenesis. Our results thus reveal that dUSP36 functions through both catalytic-dependent and catalytic-independent mechanisms, highlighting a dual mode of action with implications for the understanding of DUBs mechanism of action.},
}

Animals
*Drosophila Proteins/genetics/metabolism
Male
*Drosophila melanogaster/genetics/growth & development
*Ubiquitin Thiolesterase/genetics/metabolism
Spermatogenesis/genetics
CRISPR-Cas Systems
Female
Proto-Oncogene Proteins c-myc/metabolism/genetics
Deubiquitinating Enzymes/metabolism/genetics

@article {pmid40555854,
year = {2025},
author = {Basit, A and Liu, A and Zheng, W and Zhu, J},
title = {A review on the mechanism and potential diagnostic application of CRISPR/Cas13a system.},
journal = {Mammalian genome : official journal of the International Mammalian Genome Society},
volume = {36},
number = {3},
pages = {709-726},
pmid = {40555854},
issn = {1432-1777},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Bacteria/genetics ; CRISPR-Associated Proteins/genetics ; },
abstract = {Clustered regularly Interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form a natural immune defense system in prokaryotic species, with approximately 90% of archaea and 40% of bacteria possessing these systems, highlighting their widespread role in microbial immunity. Among these, the CRISPR/Cas13a system, guided by a single-stranded RNA (crRNA), selectively targets RNA sequences and has shown immense potential in developing sensitive diagnostic tools. Recent advancements have combined Cas13a with amplification methods and lateral flow detection (CRISPR/Cas13a-LFD), improving its application for rapid and accurate RNA detection. In this review, we explore the history, structure, and functional mechanism of the CRISPR/Cas13a system, focusing on its diagnostic capabilities. We compare CRISPR/Cas13a to conventional diagnostic approaches, highlighting their advantages in sensitivity, specificity, speed, and flexibility for point-of-care application. Given the rapid development of CRISPR-based diagnostics in recent years, the Cas13a system shows great potential as a next-generation platform for accurate, portable, and cost-effective detection of viral and bacterial diseases. Furthermore, we address the existing challenges, including reliance upon amplification and off-target effects, and highlight the need for ongoing research to develop amplification-free systems suitable for clinical application.},
}

*CRISPR-Cas Systems/genetics
Humans
Animals
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Bacteria/genetics
CRISPR-Associated Proteins/genetics

@article {pmid40541099,
year = {2025},
author = {Tian, J and Wang, Y and Zhou, Z and Li, Y and Chang, C and Meng, F and Shi, W and Li, H and Wang, W and Wei, Q and Liu, H and Yang, Z and Xiao, S},
title = {Genome-wide CRISPR/Cas9 screen identifies host factors for Newcastle disease virus replication.},
journal = {Poultry science},
volume = {104},
number = {9},
pages = {105421},
pmid = {40541099},
issn = {1525-3171},
mesh = {Animals ; *Newcastle disease virus/physiology ; *Virus Replication/genetics ; *CRISPR-Cas Systems ; Mice ; *Newcastle Disease/virology/genetics ; Cell Line ; },
abstract = {Viruses rely strictly on host factors to determine their susceptibility. Newcastle disease virus (NDV), an avian pathogen, can infect a wide range of avian, mammalian and human cell lines. However, NDV can only infect certain mammals and humans, and it does not cause disease in mammals or mice. A comprehensive identification of NDV-susceptible host genes that promote NDV infection is lacking. Here, we performed a mouse genome-wide CRISPR knockout (GeCKO) screening in murine fibroblast L929 cells infected with NDV. Fifty host genes were highly enriched in the screening. Using a viral minigenome assay and gene overexpression as well as single guide RNA (sgRNA) knockout, one of the genes was shown to predominantly promote viral replication. This gene is the solute carrier family 35 member A1 (SLC35A1), which is a cytidine monophosphate (CMP)-Sia transporter involved in the synthesis of sialic acid (SA). Knockout of SLC35A1 in L929 cells decreased the expression of the NDV receptors α2,3-SA and α2,6-SA on the cell surface, resulting in significant reductions in viral adsorption, internalization and replication. Furthermore, the knockout of a six-residue stretch, [82]LGSPKE[87], of SLC35A1 in cells specifically reduced the expression of the α2,6-SA receptor but not the α2,3-SA receptor, which decreased viral adsorption and replication. The reconstitution of SLC35A1 in the knockout cells completely recovered the α2,3-SA receptor, partially recovered the α2,6-SA receptor and almost completely recovered viral replication. In chicken fibroblast DF-1 cells, siRNA-mediated knockdown of chicken SLC35A1 reduced the expression levels of both the α2,6-SA and α2,3-SA receptors, decreasing viral replication. Our research indicated that SLC35A1 is a key host factor that promotes NDV replication. The CRISPR/Cas9 system can be used to identify essential host factors for the replication of intracellular pathogens. This study provides valuable insights into host susceptibility to NDV.},
}

Animals
*Newcastle disease virus/physiology
*Virus Replication/genetics
*CRISPR-Cas Systems
Mice
*Newcastle Disease/virology/genetics
Cell Line

@article {pmid40527130,
year = {2025},
author = {Jin, S and Wang, X and Liu, X and Xu, Y and Wang, W and Chen, W and Chang, H and Li, Z and Geng, Z},
title = {Effective IHH gene knockout by CRISPR/Cas9 system in chicken DF-1 cells.},
journal = {Poultry science},
volume = {104},
number = {9},
pages = {105433},
pmid = {40527130},
issn = {1525-3171},
mesh = {Animals ; *Chickens/genetics ; *CRISPR-Cas Systems ; *Hedgehog Proteins/genetics/metabolism ; *Gene Knockout Techniques/veterinary ; *Avian Proteins/genetics/metabolism ; Cell Line ; },
abstract = {Indian hedgehog (IHH) gene codes an important signal molecule mediating chondrogenesis and bone development in chickens, which are key factors that affect body weight and several other significant economic traits. The aim of this study was to construct an IHH knockout cell model using CRISPR-associated protein 9 (CRISPR/Cas9) technology to further analyze the function of IHH. TA cloning was used to screen the single-guide RNA (sgRNA1) [45 %] and sgRNA3 (30.8 %) with the highest targeting efficiency. Monoclonal cells were selected by flow cytometry for TA cloning sequencing to construct the IHH knockout cell model. Quantitative PCR (qPCR) was used to detect the changes in downstream gene expression levels after IHH knockout. TA cloning sequencing results showed that the IHH knockout cell model was successfully constructed, and two mutation types were generated with a 100 % mutation rate. In addition, qPCR results revealed that the expression of patched 1 (PTCH1), smoothened, frizzled class receptor (Smo), glioma-associated oncogene homolog 1 (Gli1), glioma-associated oncogene homolog 2 (Gli2), and osteopontin (OPN) was significantly lower in the IHH knockout group, while that of type II collagen (Col Ⅱ) was significantly higher. These results lay a theoretical foundation for the successful application of knockout technology in poultry functional genomics research and provide a stable knockout cell line model for further study of chicken IHH gene function.},
}

Animals
*Chickens/genetics
*CRISPR-Cas Systems
*Hedgehog Proteins/genetics/metabolism
*Gene Knockout Techniques/veterinary
*Avian Proteins/genetics/metabolism
Cell Line

@article {pmid40891974,
year = {2025},
author = {Chapelin, F},
title = {CRISPR-based Triple-Modality Imaging Ushers a New Era for Stem Cell Tracking in Stroke.},
journal = {Radiology},
volume = {316},
number = {3},
pages = {e252546},
doi = {10.1148/radiol.252546},
pmid = {40891974},
issn = {1527-1315},
mesh = {Humans ; *Cell Tracking/methods ; *Stroke/diagnostic imaging/therapy ; *Multimodal Imaging/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Stem Cells ; *CRISPR-Cas Systems ; },
abstract = {"Just Accepted" papers have undergone full peer review and have been accepted for publication in Radiology. This article will undergo copyediting, layout, and proof review before it is published in its final version. Please note that during production of the final copyedited article, errors may be discovered which could affect the content.},
}

Humans
*Cell Tracking/methods
*Stroke/diagnostic imaging/therapy
*Multimodal Imaging/methods
*Clustered Regularly Interspaced Short Palindromic Repeats
*Stem Cells
*CRISPR-Cas Systems

@article {pmid40891659,
year = {2025},
author = {Takizawa, A and Foeckler, J and Knapp, E and Grzybowski, M and Geurts, AM and Carroll, J and Merriman, DK},
title = {Successful Generation of Germline Tyrosinase Gene Edited Thirteen-Lined Ground Squirrels (Ictidomys tridecemlineatus Mitchill 1821).},
journal = {Molecular reproduction and development},
volume = {92},
number = {9},
pages = {e70055},
doi = {10.1002/mrd.70055},
pmid = {40891659},
issn = {1098-2795},
support = {//This study was supported by the National Eye Institute of the National Institutes of Health (NIH) under award number T32EY014537, U24EY029891 and by the National Center for Advancing Translational Sciences, National Institutes of Health, Award Number UL1TR001436./ ; },
mesh = {Animals ; *Monophenol Monooxygenase/genetics/metabolism ; *Sciuridae/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; Female ; Male ; *Germ-Line Mutation ; *Germ Cells/metabolism ; },
abstract = {Thirteen-lined ground squirrels (Ictidomys tridecemlineatus Mitchill 1821; 13-LGS) are useful diurnal rodent models of human cone-mediated vision due to their cone photoreceptor-dominant retinas. To develop the 13-LGS as a better model of inherited human visual disorders, we report a gene-editing protocol targeting the 13-LGS tyrosinase (Tyr) gene. CRISPR/Cas9 microinjection into donor embryos, followed by transfer to pseudo-pregnant recipients, yielded two Tyr-mutated founders. Mating these two to wild-type 13-LGS resulted in 22 offspring, of which five were genotyped with either a 17-bp deletion, 1-bp insertion, or 7-bp deletion Tyr mutation. These results demonstrated that this valuable mammalian model is amenable to germline gene editing by conventional methods.},
}

Animals
*Monophenol Monooxygenase/genetics/metabolism
*Sciuridae/genetics
*Gene Editing/methods
CRISPR-Cas Systems
Female
Male
*Germ-Line Mutation
*Germ Cells/metabolism

@article {pmid40891290,
year = {2025},
author = {Deng, Z and Sha, R and Qin, H and Shang, Y and Yuan, A and Xie, W and Peng, H},
title = {A CRISPR Cas protein coronated AuNP nanostructure for enhanced uptake efficiency into cells.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc03658e},
pmid = {40891290},
issn = {1364-548X},
abstract = {The effectiveness of nanotechnologies is often limited by their non-specific aggregation in biological environments. We developed a protein coronated nanostructure by functionalizing AuNPs with nucleic acid scaffolds and CRISPR Cas proteins, significantly enhancing nanoparticle stability and cellular uptake efficiency, making it a promising tool for imaging and biomedical applications.},
}

@article {pmid40890415,
year = {2025},
author = {Laidoudi, Y and Davoust, B and Lepidi, H and Levasseur, A},
title = {Emergence of the zoonotic bacterium Necropsobacter rosorum in nutria Myocastor coypus with implications for wildlife and human health.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {32252},
pmid = {40890415},
issn = {2045-2322},
mesh = {Animals ; Humans ; Phylogeny ; Genome, Bacterial ; France/epidemiology ; *Pasteurellaceae/genetics/isolation & purification/classification/pathogenicity ; Animals, Wild/microbiology ; *Zoonoses/microbiology ; Whole Genome Sequencing ; *Rodentia/microbiology ; *Bacterial Zoonoses/microbiology/epidemiology ; },
abstract = {The nutria (Myocastor coypus), a semi-aquatic rodent native to South America, poses significant ecological and agricultural threats as an invasive species in France, where it continues to proliferate despite sustained control efforts. A fatal case of pneumonia in a nutria from Marseille (France) prompted a microbiological investigation that led to the isolation, taxonomic classification, genomic characterization, and phylogenetic analysis of Necropsobacter rosorum. Whole-genome sequencing of the N. rosorum strain RG01 revealed a genome size of 2,505,657 base pairs and 2303 predicted open reading frames, showing high similarity to other publicly available N. rosorum genomes. Comparative pan-genomic analysis indicated a high level of genomic conservation among N. rosorum strains. The presence of putative virulence factors and a CRISPR-Cas system suggests both pathogenic potential and adaptive defense mechanisms against bacteriophage predation. This study also explored the genetic epidemiology of members of the Pasteurellaceae family, highlighting a considerable overlap between species infecting animals and humans. Among the 408,387 sequence records retrieved from GenBank, 62.1% were deemed suitable for genomic epidemiological analysis. Notably, N. rosorum was underrepresented, with only 13 entries spanning nine countries and three host types, revealing critical gaps in current surveillance and research. Collectively, these findings contribute to a better understanding of the microbiology and epidemiology of N. rosorum and Pasteurellaceae-associated infections, and underscore the importance of integrated, genomics-informed approaches for the monitoring, control, and prevention of zoonotic diseases.},
}

Animals
Humans
Phylogeny
Genome, Bacterial
France/epidemiology
*Pasteurellaceae/genetics/isolation & purification/classification/pathogenicity
Animals, Wild/microbiology
*Zoonoses/microbiology
Whole Genome Sequencing
*Rodentia/microbiology
*Bacterial Zoonoses/microbiology/epidemiology

@article {pmid40890128,
year = {2025},
author = {Álvarez-Pérez, JC and Sanjuán-Hidalgo, J and Arenas, AM and Hernández-Navas, I and Benitez-Cantos, MS and Andrades, A and Calabuig-Fariñas, S and Jantus-Lewintre, E and Paz-Ares, L and Ferrer, I and Medina, PP},
title = {High-fidelity Cas9-mediated targeting of KRAS driver mutations restrains lung cancer in preclinical models.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7080},
pmid = {40890128},
issn = {2041-1723},
support = {LAB-AECC-2018//Fundación Científica Asociación Española Contra el Cáncer (Scientific Foundation, Spanish Association Against Cancer)/ ; B-CTS-480-UGR20, C-EXP-051-UGR23, C-CTS-149-UGR23//Universidad de Granada (University of Granada)/ ; },
mesh = {*Lung Neoplasms/genetics/therapy/pathology ; Animals ; Humans ; *Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Carcinoma, Non-Small-Cell Lung/genetics/therapy/pathology ; Mice ; Xenograft Model Antitumor Assays ; Cell Line, Tumor ; Gene Editing/methods ; *Genetic Therapy/methods ; Mutation ; Female ; Adenoviridae/genetics ; },
abstract = {Missense mutations in the 12[th] codon of KRAS are key drivers of lung cancer, with glycine-to-cysteine (G12C) and glycine-to-aspartic acid (G12D) substitutions being among the most prevalent. These mutations are strongly associated with poor survival outcomes. Given the critical role of KRAS in lung cancer and other cancers, it remains as a major target for the development of new and complementary treatments. We have developed a CRISPR-High Fidelity (HiFi)-Cas9-based therapy strategy that can effectively and specifically target KRAS[G12C] and KRAS[G12D] mutants, avoiding KRAS[WT] off-targeting and affecting KRAS downstream pathways, thereby significantly reducing tumorgenicity. The delivery of HiFiCas9 components via ribonucleoprotein particles (RNPs) and adenovirus (AdV) effectively abrogates cell viability in KRAS-mutant Non-Small Cell Lung Cancer (NSCLC) preclinical models, including 2D and 3D cell cultures, cell-derived xenografts (CDX), and patient-derived xenograft organoids (PDXO). Our in vitro studies demonstrate that HiFiCas9-based therapy achieves superior KRAS inhibition compared to Sotorasib and effectively circumvents certain resistance mechanisms associated with Sotorasib treatment. Moreover, in vivo delivery using adenoviral particles significantly suppresses tumor growth in preclinical NSCLC models. Collectively, our findings establish HiFiCas9 as an effective therapeutic strategy with promising clinical applications, especially if in vivo delivery methods are further optimized.},
}

*Lung Neoplasms/genetics/therapy/pathology
Animals
Humans
*Proto-Oncogene Proteins p21(ras)/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Carcinoma, Non-Small-Cell Lung/genetics/therapy/pathology
Mice
Xenograft Model Antitumor Assays
Cell Line, Tumor
Gene Editing/methods
*Genetic Therapy/methods
Mutation
Female
Adenoviridae/genetics

@article {pmid40825745,
year = {2025},
author = {Zhuang, S and Huang, X and Diao, M and Dong, R and Dong, Y and Liu, J and Liu, Y},
title = {CONVERT: Dynamic crRNA Reconfiguration for Universal One-Pot CRISPR/Cas12a-Based Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {34},
pages = {18776-18785},
doi = {10.1021/acs.analchem.5c03577},
pmid = {40825745},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *RNA/genetics ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {Current one-pot CRISPR diagnostics necessitate meticulous control of nucleic acid hybridization parameters or extensive modification of CRISPR components to achieve complete enzymatic silencing, a fundamental bottleneck limiting their robustness and generalizability. Here, we resolve this challenge through dynamic crRNA reconfiguration, a paradigm-shifting strategy that exploits the intrinsic structural duality of CRISPR RNA. We present CONVERT (CRISPR One-Pot Nucleic acid detection Via Engineered crRNA Reconfiguration Technology), a universal platform where nontarget intact crRNA acts as a universal suppressor, achieving complete Cas12a inhibition during RPA (recombinase polymerase amplification) by irreversible enzyme sequestration. Target detection is initiated through programmable conversion to split crRNA activators, bypassing sequence-specific design constraints entirely. This crRNA conformational switching mechanism is implemented via photocleavable linker cleavage and subsequent assembly with presupplied truncated crRNA (tcrRNA), activating trans-cleavage signal amplification. Three transformative advances emerge: First, the endogenous crRNA engineering eliminates dependency on exogenous nucleic acid blockers or chemical modifications, reducing optimization costs and time. Second, near-total enzymatic suppression ensures zero cross-talk between amplification and detection phases. Third, the unified workflow enables contamination-resistant operation through spatiotemporal control, requiring no physical partitioning. Validated for Enterocytozoon bieneusi detection, the system demonstrates attomolar sensitivity of 1 aM, 100% diagnostic concordance with qPCR, and absolute specificity against related pathogens. By effectively decoupling CRISPR regulation from sequence-context constraints through rational crRNA structural plasticity, this work establishes a generalized framework for next-generation molecular diagnostics.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism/genetics
*Endodeoxyribonucleases/metabolism/genetics
*RNA/genetics
*Bacterial Proteins/metabolism/genetics

@article {pmid40742143,
year = {2025},
author = {Liu, G and Wu, J and Yang, B and Kong, J and Li, Y and Chen, C and Hu, J and Sun, Z and Hou, Y and Sun, R and Li, D},
title = {Ultrasensitive eccDNA Detection for Tumor Diagnostics by Using CRISPR/Cas12a-Coupled Nested PCR.},
journal = {Analytical chemistry},
volume = {97},
number = {34},
pages = {18527-18536},
doi = {10.1021/acs.analchem.5c02316},
pmid = {40742143},
issn = {1520-6882},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Polymerase Chain Reaction/methods ; *DNA, Circular/genetics/blood/analysis ; Limit of Detection ; *Biomarkers, Tumor/genetics/blood ; *Neoplasms/diagnosis/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Extrachromosomal circular DNA (eccDNA) has emerged as a novel biomarker for cancer detection due to its tumor-specific amplification and stable structure in circulation. However, its clinical application is hindered by extremely low abundance in biofluids and the lack of robust detection techniques. To address this, we screened for tumor-associated eccDNA biomarkers and developed NPCC (Nested PCR-CRISPR/Cas12a), a novel method combining nested PCR for ultrasensitive amplification with CRISPR/Cas12a for sequence-specific detection. The assay employs two rounds of junction-specific PCR to enrich eccDNA, followed by CRISPR/Cas12a-mediated cleavage guided by target-specific crRNA. Validation using synthetic circular DNA standards demonstrated a limit of detection (LoD) of 10[-6] fM, representing a >100-fold improvement over conventional PCR, with no cross-reactivity to linear or genomic DNA fragments. In plasma samples from 88 cancer patients, NPCC successfully detected multiple tumor-specific eccDNAs, including the hepatocellular carcinoma marker eccDNA-HCC-1 (AUC = 0.8977). NPCC overcomes key technical barriers in liquid biopsy, offering a cost-effective, highly sensitive, and specific platform for noninvasive cancer diagnostics.},
}

Humans
*CRISPR-Cas Systems/genetics
*Polymerase Chain Reaction/methods
*DNA, Circular/genetics/blood/analysis
Limit of Detection
*Biomarkers, Tumor/genetics/blood
*Neoplasms/diagnosis/genetics
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40614014,
year = {2025},
author = {Shan, L and He, Q and Zhang, W and Zheng, X and Zhao, J and Yang, P and Mao, Q and Xia, H},
title = {CRISPR/Cas9-mediated promoterless gene targeting reduces lysosome storage in MPS VII mice.},
journal = {Science China. Life sciences},
volume = {68},
number = {9},
pages = {2697-2706},
pmid = {40614014},
issn = {1869-1889},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; Glucuronidase/genetics/metabolism/blood ; *Mucopolysaccharidosis VII/therapy/genetics/metabolism ; *Gene Targeting/methods ; *Lysosomes/metabolism ; Disease Models, Animal ; Genetic Vectors/genetics ; Promoter Regions, Genetic ; Genetic Therapy/methods ; Actins/genetics ; Adenoviridae/genetics ; Liver/metabolism ; },
abstract = {Targeted gene integration mediated by CRISPR/Cas9 is a promising therapeutic strategy for monogenic autosomal recessive diseases. In this study, we established a novel all-in-one high-capacity adenovirus (HCAd) that can pack both CRISPR/Cas9 and donor DNA into the same vector and tested it on a mouse model of mucopolysaccharidosis type VII (MPS VII) caused by mutations in the β-glucuronidase (GUSB) gene. This system allowed targeted integration of promoterless GUSB in the mouse beta-actin gene (mActb) locus and the co-expression of GUSB with the self-cleaving peptide T2A (T2A) controlled by a strong endogenous mActb promoter. The in vivo results indicated that the serum GUSB level of MPS VII mice treated with a single intraperitoneal injection of the HCAd vector achieved 14% of that of wild-type mice, resulting in significant amelioration of lysosomal storage in the liver and spleen. Furthermore, the HCAd was injected intraventricularly in the brain of newborn MPS VII mice, leading to strongly positive GUSB enzyme staining in the choroid plexus and perivascular spaces of the periventricular regions and reduced lysosome storage. In summary, by using an all-in-one vector, the study provides a universal, one-for-all therapeutic for MPSVII, a disease caused by different mutations of the GUSB gene.},
}

Animals
*CRISPR-Cas Systems/genetics
Mice
Glucuronidase/genetics/metabolism/blood
*Mucopolysaccharidosis VII/therapy/genetics/metabolism
*Gene Targeting/methods
*Lysosomes/metabolism
Disease Models, Animal
Genetic Vectors/genetics
Promoter Regions, Genetic
Genetic Therapy/methods
Actins/genetics
Adenoviridae/genetics
Liver/metabolism

@article {pmid40377870,
year = {2025},
author = {Amen, RA and Hassan, YM and Essmat, RA and Ahmed, RH and Azab, MM and Shehata, NR and Elgazzar, MM and El-Sayed, WM},
title = {Harnessing the Microbiome: CRISPR-Based Gene Editing and Antimicrobial Peptides in Combating Antibiotic Resistance and Cancer.},
journal = {Probiotics and antimicrobial proteins},
volume = {17},
number = {4},
pages = {1938-1968},
pmid = {40377870},
issn = {1867-1314},
mesh = {Humans ; *Gene Editing/methods ; *Neoplasms/microbiology/therapy/drug therapy ; *Microbiota ; *Antimicrobial Peptides/pharmacology ; CRISPR-Cas Systems ; Animals ; *Drug Resistance, Microbial ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The growing crisis of antibiotic resistance and the increasing incidence of cancer have prompted the exploration of innovative approaches, such as gene editing and antimicrobial peptides (AMPs). The human microbiome is integral to various aspects of health, disease, and therapeutic development, influencing metabolic pathways, immune function, and pathogen resistance. Recent advances in gene editing technologies, particularly CRISPR (clustered regularly interspaced short palindromic repeats), have opened new avenues for leveraging the microbiome to address complex medical challenges, including combating multidrug-resistant pathogens and cancer. The microbiome plays a crucial role in combating antibiotic resistance by modulating microbial communities, influencing pathogen survival and susceptibility to treatments. This review explores the microbiome's dynamic role in metabolic regulation, its contribution to cancer management, and how AMPs help maintain homeostasis and exhibit emerging anticancer properties, supported by both preclinical findings and clinical evidence. Additionally, CRISPR-based microbiome engineering offers potential to enhance host-microbiome interactions, optimizing therapeutic outcomes. The integration of microbiome metagenomics and proteomics has led to the discovery of novel AMPs with targeted anticancer effects. Innovative strategies, such as engineered probiotics and CRISPR-based microbiome engineering, present exciting prospects for next-generation therapies. Despite these advances, the translation of microbiome-based therapies into clinical settings remains challenging due to ethical, regulatory, and ecological hurdles. This review underscores the transformative potential of microbiome-based interventions, emphasizing the role of personalized medicine in maximizing therapeutic efficacy. Furthermore, we also address critical research gaps, limitations, and future directions, including optimizing AMP stability, delivery, and bioavailability, as well as overcoming the regulatory and ethical challenges in clinical translation.},
}

Humans
*Gene Editing/methods
*Neoplasms/microbiology/therapy/drug therapy
*Microbiota
*Antimicrobial Peptides/pharmacology
CRISPR-Cas Systems
Animals
*Drug Resistance, Microbial
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40326293,
year = {2025},
author = {Lazarides, K and Engel, JL and Meseonznik, M and Feng, T and Choi, AH and Yu, Y and Liu, S and Meier, SR and Zhang, H and Shen, B and Tjin Tham Sjin, R and Whittington, DA and McMillan, BJ and Doyon, B and Pan, X and Wilker, E and Huang, A and Andersen, JN and Mallender, WD and Bandi, M},
title = {CRISPR Screens Identify POLB as a Synthetic Lethal Enhancer of PARP Inhibition Exclusively in BRCA-Mutated Tumors.},
journal = {Molecular cancer therapeutics},
volume = {24},
number = {9},
pages = {1466-1479},
doi = {10.1158/1535-7163.MCT-24-0822},
pmid = {40326293},
issn = {1538-8514},
mesh = {Humans ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; Animals ; Mice ; *BRCA2 Protein/genetics ; *Synthetic Lethal Mutations ; Female ; Cell Line, Tumor ; *BRCA1 Protein/genetics ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; Mutation ; Apoptosis/drug effects ; },
abstract = {PARP inhibitors (PARPi) are an approved class of anticancer therapeutics that inhibit the activities of PARP1/2 and produce synthetic lethality in BRCA1/2-mutated cancers because of the absence of a functional homologous recombination-dependent DNA repair pathway. Although PARPis have led to successful clinical outcomes, two thirds of patients develop acquired resistance, limiting long-term utility as maintenance therapy. Motivated by this clinical need, we utilized a CRISPR target discovery screening platform to identify DNA polymerase beta (POLB) as a gene that acts selectively and synergistically with PARPis in BRCA1/2-mutated cancers and found that POLB knockout (KO) along with PARPi treatment enhanced loss of viability in BRCA1/2-mutant and BRCA2-null cells but not in isogenic BRCA1/2 wild-type cells. Overexpression of either POLB wild-type or catalytically inactive mutants confirmed that perturbation of both the polymerase and lyase catalytic activities of POLB are required for synergistic PARP-BRCA synthetic lethality. Mechanistically, POLB KO was associated with an increase in single- and double-strand DNA breaks, cell-cycle arrest, and apoptosis when in combination with PARP inhibition. The translational nature of this interaction was further examined using murine xenograft models of BRCA1-mutant and BRCA2-null cell lines, wherein the combination of POLB KO and niraparib led to profound tumor regression and prevented tumor regrowth even after cessation of treatment. Together, these results suggest that POLB is a synergistic enhancer of the synthetic lethal interaction between PARP and BRCA and support POLB as a promising therapeutic target for improving antitumor responses to PARPis in homologous recombination-deficient cancers.},
}

Humans
*Poly(ADP-ribose) Polymerase Inhibitors/pharmacology
Animals
Mice
*BRCA2 Protein/genetics
*Synthetic Lethal Mutations
Female
Cell Line, Tumor
*BRCA1 Protein/genetics
Xenograft Model Antitumor Assays
CRISPR-Cas Systems
Mutation
Apoptosis/drug effects

@article {pmid40889353,
year = {2025},
author = {Yu, Y and Yuan, Q and Liu, Z and Tong, B and Shi, S},
title = {Enhancement of Free Fatty Acids Production in Rhodotorula toruloides Using the CRISPR/Cas9-Based Base Editor.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00359},
pmid = {40889353},
issn = {2161-5063},
abstract = {Rhodotorula toruloides is a promising cell factory to produce various value-added chemicals, including fatty acid derivatives. However, their metabolic engineering development has been hindered by the limited availability of genetic tools. In this study, an accurate and specific gene-editing tool, CRISPR/Cas-based cytidine base editor (CBE) system, was developed for the first time in R. toruloides to broaden its genetic toolbox. The target gene was disrupted by introducing a premature stop codon via C to T mutation. This system achieved single-gene disruption efficiencies of up to 90% and successfully disrupted four genes in parallel with 5% efficiency, marking a breakthrough in multiplexed editing for this yeast. To enable iterative engineering, an inducible Cre-loxP system was integrated, achieving an over 70% selection marker recycling efficiency. Application of this system enabled the construction of uracil-auxotrophic strains. Furthermore, the CBE system was employed to disrupt four genes involved in lipid metabolism, resulting in an engineered strain capable of producing 512.3 mg/L of free fatty acids, thereby demonstrating the utility of the CBE system as an efficient genome editing tool in R. toruloides. The study provides valuable tools to expand the genetic toolbox of R. toruloides and paves the way for fully exploiting its metabolic engineering potential.},
}

@article {pmid40888633,
year = {2025},
author = {Kelleher, AM and Kim, HI and Bayammagari, GS and Davis, DJ and Spencer, TE},
title = {A Cxcl15 Cre Recombinase Mouse Model Useful to Study Gland Development in the Uterus.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {63},
number = {5},
pages = {e70026},
doi = {10.1002/dvg.70026},
pmid = {40888633},
issn = {1526-968X},
support = {HD042311/NH/NIH HHS/United States ; HD112315/NH/NIH HHS/United States ; },
mesh = {Animals ; Female ; *Integrases/genetics/metabolism ; Mice ; Hepatocyte Nuclear Factor 3-beta/genetics/metabolism ; *Uterus/metabolism/growth & development ; *Endometrium/metabolism/growth & development ; Pregnancy ; CRISPR-Cas Systems ; },
abstract = {The mammalian uterus contains glands in the endometrium that develop only or primarily after birth. In the mouse, endometrial glands govern post implantation pregnancy establishment via regulation of blastocyst implantation, stromal cell decidualization, and placental development. Here, we describe a new uterine glandular epithelium (GE) specific Cre recombinase mouse line that is useful to study endometrial gland development and function. Utilizing CRISPR-Cas9 genome editing, improved Cre recombinase (iCre) was inserted into the endogenous C-X-C motif chemokine ligand 15 (Cxcl15) gene. Cxcl15 mRNA, Cxcl15 protein, and Cxcl15-iCre recombinase activity were specific to the developing GE of the uterus. Cxcl15-iCre mice were crossed with floxed Foxa2 mice to conditionally delete Foxa2 specifically in the glands of the neonatal mouse uterus. This conditional deletion of Foxa2 in the developing neonatal uterus resulted in adult mice that lacked Foxa2 in the GE of the uterus, and the adult mice were infertile. The studies described here establish that Cxcl15-iCre mice are a valuable resource to elucidate and explore mechanisms regulating the development and function of glands in the uterus.},
}

Animals
Female
*Integrases/genetics/metabolism
Mice
Hepatocyte Nuclear Factor 3-beta/genetics/metabolism
*Uterus/metabolism/growth & development
*Endometrium/metabolism/growth & development
Pregnancy
CRISPR-Cas Systems

@article {pmid40887498,
year = {2025},
author = {Gwon, LW and Badon, IW and Lee, Y and Kim, HJ and Lee, SH},
title = {Advances in large-scale DNA engineering with the CRISPR system.},
journal = {Experimental & molecular medicine},
volume = {},
number = {},
pages = {},
pmid = {40887498},
issn = {2092-6413},
support = {the Chung-Ang University Research Grants in 2024//Chung-Ang University (CAU)/ ; },
abstract = {In recent years, DNA engineering technology has undergone significant advancements, with clustered regularly interspaced short palindromic repeats (CRISPR)-based target-specific DNA insertion emerging as one of the most rapidly expanding and widely studied approaches. Traditional DNA insertion technologies employing recombinases typically involve introducing foreign DNA into genes in vivo by either pre-engineering recognition sequences specific to the recombinase or through genetic crossing to incorporate the requisite recognition sequence into the target gene. However, CRISPR-based gene insertion technologies have advanced to streamline this engineering process by combining the CRISPR-Cas module with recombinase enzymes. This process enables accurate and efficient one-step insertion of foreign DNA into the target gene in vivo. Here we provide an overview of the latest developments in CRISPR-based gene insertion technologies and discusses their potential future applications.},
}

@article {pmid40885801,
year = {2025},
author = {Zirman, A and Abed El-Nabi, M and Samuel, E and Anavy, L and Yakhini, Z and Nachman, I and Rak, R},
title = {Pooled CRISPR screens identifies key regulators of bovine stem cell expansion for cultured meat.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1313},
pmid = {40885801},
issn = {2399-3642},
support = {20-04-0001//Ministry of Agriculture and Rural Development (Israeli Ministry of Agriculture and Rural Development)/ ; },
mesh = {Animals ; Cattle ; Cell Proliferation/genetics ; *Mesenchymal Stem Cells/cytology/metabolism/physiology ; *CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cells, Cultured ; *Meat ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knockout Techniques ; PTEN Phosphohydrolase/genetics ; Tumor Suppressor Protein p53/genetics ; In Vitro Meat ; },
abstract = {Cultured meat presents a sustainable alternative to traditional meat production but faces significant challenges in scalability and cost efficiency. A key limitation is the restricted proliferation capacity of bovine mesenchymal stem cells (bMSCs), a widely used cell source in the field. Using a pooled, lentiviral CRISPR knockout screen, we interrogated 3000 CRISPR guides targeting 600 genes involved in stem cell regulation or proliferation. Notably, knockouts of TP53 and PTEN significantly increased proliferation rates and delayed senescence. Validation with individual gene knockouts confirms these effects, showing enhanced growth but reduced differentiation potential. We also identified chondrogenic differentiation as a promising target whose repression may further promote MSC expansion. These findings demonstrate the utility of CRISPR screening for optimizing bovine stem cell traits and offer a path toward more scalable cultured meat production in the future.},
}

Animals
Cattle
Cell Proliferation/genetics
*Mesenchymal Stem Cells/cytology/metabolism/physiology
*CRISPR-Cas Systems
Cell Differentiation/genetics
Cells, Cultured
*Meat
*Clustered Regularly Interspaced Short Palindromic Repeats
Gene Knockout Techniques
PTEN Phosphohydrolase/genetics
Tumor Suppressor Protein p53/genetics
In Vitro Meat

@article {pmid40669350,
year = {2025},
author = {Suvá, M and Bastón, JI and Wiedenmann, EA and Pose Ortiz de Rozas, MB and Jordán, R and Ghetti, A and Viale, DL and Moro, LN and Vichera, GD},
title = {Use of an exogenous DNA-free system to generate MSTN-KO calves by CRISPR/Cas9 and SCNT.},
journal = {Reproductive biology},
volume = {25},
number = {3},
pages = {101050},
doi = {10.1016/j.repbio.2025.101050},
pmid = {40669350},
issn = {2300-732X},
mesh = {Animals ; Cattle/genetics ; *Nuclear Transfer Techniques/veterinary ; *Myostatin/genetics ; *CRISPR-Cas Systems ; *Gene Editing/veterinary/methods ; *Gene Knockout Techniques/veterinary ; Animals, Genetically Modified ; Female ; Fibroblasts ; Male ; },
abstract = {This study aimed to obtain myostatin (MSTN)-knockout calves, while avoiding the risk of exogenous DNA integration during gene editing. To achieve this, we combined CRISPR/Cas9 ribonucleoprotein gene editing with somatic cell nuclear transfer (SCNT) technology. In the first experiment (E1), we compared the gene editing efficiency of four gRNAs targeting different coding regions of the MSTN gene using plasmid-based CRISPR/Cas9 in bovine fetal fibroblasts (BFF-E1 cells). The highest bioinformatically-predicted editing rate (BPE) was obtained with gRNA2 (96 %), which was subsequently used for further experiments. Next, embryos were produced by SCNT using BFF-E1-edited cells as nuclear donors. Sanger sequencing of the embryos showed biallelic MSTN editing. In the second experiment (E2), plasmid-based editing was replaced with CAS9 protein and trac:crRNA oligoribonucleotides. Editing efficiency was assessed on one edited bovine fetal fibroblast line (BFF-E2-male[ed]) and two edited bovine mesenchymal stem lines (MSC-E2-male[ed] and MSC-E2-fem[ed]) derived from price-winning animals. BPEs were 58.8 %, 31 % and 59 % in fibroblast and MSC cells, and 64 %, 73.3 %, and 66.6 % in SCNT embryos derived from BFF-E2-male[ed], MSC-E2-male[ed] and MSC-E2-fem[ed], respectively. Transfer of 35 MSC-E2-fem[ed] embryos to recipient cows, resulted in the birth of one MSTN-edited calf with a heterozygous genotype. A second-generation clone was subsequently produced, using a fibroblast sample as nuclear donor. In conclusion, we established an efficient protocol for generating high rates of edited blastocysts with a desirable genetic background, resulting in the birth of two MSTN-knockout calves. This study provides a foundation for gene editing to improve productive or biomedical traits.},
}

Animals
Cattle/genetics
*Nuclear Transfer Techniques/veterinary
*Myostatin/genetics
*CRISPR-Cas Systems
*Gene Editing/veterinary/methods
*Gene Knockout Techniques/veterinary
Animals, Genetically Modified
Female
Fibroblasts
Male

@article {pmid40639438,
year = {2025},
author = {Bhatia, P and Mohd, A and Agrawal, I and Katiyar, H and Goel, A and Aich, M and Chakraborty, D and Veerapu, NS},
title = {Rapid and efficient generation of viral genome knock-in cell lines using the CRISPR-Cas9 system to produce infectious virus.},
journal = {Journal of virological methods},
volume = {338},
number = {},
pages = {115219},
doi = {10.1016/j.jviromet.2025.115219},
pmid = {40639438},
issn = {1879-0984},
mesh = {*CRISPR-Cas Systems ; Humans ; *Genome, Viral ; Cell Line ; *Gene Knock-In Techniques/methods ; *Hepatitis B virus/genetics ; Genotype ; Virus Replication ; Hepacivirus/genetics ; Antiviral Agents/pharmacology ; Gene Editing ; },
abstract = {Several medically significant viruses are difficult to propagate with conventional laboratory host systems, limiting their availability for detailed characterization, antiviral screening, and functional studies. A range of methods can be used to generate viruses, such as creating sophisticated cell lines, organoid cultures, and the utilization of animal models. Here, we report the generation and characterization of CRISPR-Cas9 edited Huh7 stable cell lines engineered to carry and express overlength HBV genotypes A, B, C and D and full HEV genomes in the AAVS1 site. Viral polymerase inhibitors and IFN-α significantly reduced the production of viral genomes and proteins from the edited cells. The virus released by the edited cells was infectious in permissive cell lines and could be blocked by neutralizing antibodies. This approach can extend to other viruses, like HCV genotype 3, that are hard to culture or to culturable viruses, like Dengue, for vaccine production.},
}

*CRISPR-Cas Systems
Humans
*Genome, Viral
Cell Line
*Gene Knock-In Techniques/methods
*Hepatitis B virus/genetics
Genotype
Virus Replication
Hepacivirus/genetics
Antiviral Agents/pharmacology
Gene Editing

@article {pmid40629854,
year = {2025},
author = {Lin, ZC and Yang, SC and Tran, TTP and Fang, JY},
title = {Animal models of psoriasis for novel drug discovery: a literature update.},
journal = {Expert opinion on drug discovery},
volume = {20},
number = {9},
pages = {1193-1208},
doi = {10.1080/17460441.2025.2528959},
pmid = {40629854},
issn = {1746-045X},
mesh = {*Psoriasis/drug therapy/physiopathology/genetics/pathology ; Animals ; Humans ; *Disease Models, Animal ; *Drug Discovery/methods ; Imiquimod/administration & dosage ; Mice ; Gene Editing/methods ; CRISPR-Cas Systems ; Cytokines/metabolism ; Mice, Knockout ; },
abstract = {INTRODUCTION: Psoriasis is a chronic, immune-mediated inflammatory skin disorder with a multifactorial pathogenesis involving keratinocyte proliferation, dysregulated immune responses, and vascular remodeling. The development of effective therapeutics mainly relies on preclinical models that can reproduce disease-relevant mechanisms.

AREAS COVERED: This review outlines current in vivo psoriasis models, including spontaneous mutation models, transgenic and knockout mice, xenotransplantation systems, and cytokine-induced and imiquimod-induced models. Each model is evaluated for its ability to replicate key histological and immunological features of human psoriasis, such as acanthosis, immune cell infiltration, and cytokine network activation. The utility of CRISPR/Cas9 gene editing in generating targeted models is also discussed, thus highlighting its potential use for mechanistic studies. Finally, this review also emphasizes the limitations in translational applicability and the need for multimodel validation strategies regarding psoriasis. This article was based on a comprehensive literature search using PubMed, Scopus, and Google Scholar databases, covering publications from January 2015 to March 2025.

EXPERT OPINION: Despite extensive model development, no single system fully mimics human psoriatic disease. The imiquimod-induced model remains widely used due to its practicality, although it better reflects acute inflammation compared with chronic pathology. The combination of complementary models and the incorporation of human-derived tissues or immune components may improve translational relevance. Advances in genome editing and humanized systems are likely to shape the future of psoriasis research and therapeutic discovery.},
}

*Psoriasis/drug therapy/physiopathology/genetics/pathology
Animals
Humans
*Disease Models, Animal
*Drug Discovery/methods
Imiquimod/administration & dosage
Mice
Gene Editing/methods
CRISPR-Cas Systems
Cytokines/metabolism
Mice, Knockout

@article {pmid40885742,
year = {2025},
author = {Valdez, I and O'Connor, I and Patel, D and Gierer, K and Harrington, J and Ellis, E and Caponetti, SA and Sebra, RP and Valley, HC and Coote, K and Mense, M and Marro, SG and Jiang, T},
title = {A streamlined base editor engineering strategy to reduce bystander editing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8115},
pmid = {40885742},
issn = {2041-1723},
support = {R00HL153940//U.S. Department of Health & Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; 005363I223//Cystic Fibrosis Foundation (CF Foundation)/ ; },
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; HEK293 Cells ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; Adenosine Deaminase/genetics/metabolism ; },
abstract = {Base editing (BE) can permanently correct over half of known human pathogenic genetic variants without requiring a repair template, thus serving as a promising therapeutic tool to treat a broad spectrum of genetic diseases. However, the broad activity windows of current base editors pose a major challenge to their therapeutic application. Here, we show that integrating a naturally occurring oligonucleotide binding module into the deaminase active center of TadA-8e, a highly active deoxyadenosine deaminase, enhances its editing specificity. When conjugated with a Cas9 nickase or alternative PAM Cas9 variants, the engineered TadA variant-TadA-NW1-consistently achieves robust A-to-G editing efficiencies within an editing window consisting of four nucleotides, substantially narrower than the 10-bp editing window of the TadA-8e-derived ABEs. Moreover, compared to ABE8e, ABE-NW1 shows significantly decreased Cas9-dependent and -independent off-target activity while maintaining similar on-target editing efficiency. Further, TadA-NW1 can be reprogrammed to perform desired cytidine deamination and adenine transversion within a restricted editing window. Finally, in a cystic fibrosis (CF) cell model, ABE-NW1 outperforms existing ABEs in accurately and efficiently correcting the CFTR W1282X variant, one of the most common CF-causing mutations. In all, we engineered a suite of base editors with refined activity windows, enabling more precise base editing. Importantly, this study presents a streamlined genome editor re-engineering strategy to accelerate the development of therapeutic base editing.},
}

*Gene Editing/methods
Humans
CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/metabolism/genetics
HEK293 Cells
Cystic Fibrosis Transmembrane Conductance Regulator/genetics
Adenosine Deaminase/genetics/metabolism

@article {pmid40784045,
year = {2025},
author = {Abdel-Malek, K and von Eisenhart-Rothe, F and Stiegmann, S and Gottmann, I and Doppler, SA and Schneider, S and Preisler, H and Lahm, H and Dreßen, M},
title = {Generation of two B2M knockout induced pluripotent stem cell lines (DHMi005-A-8 and DHMi005-A-9) using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103785},
doi = {10.1016/j.scr.2025.103785},
pmid = {40784045},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Humans ; *beta 2-Microglobulin/genetics/metabolism/deficiency ; *Gene Knockout Techniques ; Cell Line ; },
abstract = {The transplantation of cells into a recipient organism has many hurdles to overcome, including the problem of T-cell-triggered cellular immune defense. Cellular rejection is based on antigen presentation by the MHC-I-complex, which is recognized by cytotoxic T lymphocytes. Elimination of the MHC-I-complex by knocking out the B2M (Beta-2-microglobulin) subunit may be one way of reducing or even completely preventing the initial cellular immune response during transplantation. Using CRISPR/Cas9 we established one heterozygous and one homozygous B2M knockout induced pluripotent stem cell (iPSC) line as a first step towards more effective cell transplantation.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
Humans
*beta 2-Microglobulin/genetics/metabolism/deficiency
*Gene Knockout Techniques
Cell Line

@article {pmid40752255,
year = {2025},
author = {Ramachandran, H and Thomas, AC and Binder, S and Hildebrandt, B and Spitali, P and Rossi, A},
title = {Generation of two iPSC lines with pathogenic DMD nonsense mutations c.4729C>T and c.5713G>T.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103789},
doi = {10.1016/j.scr.2025.103789},
pmid = {40752255},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Codon, Nonsense/genetics ; *Muscular Dystrophy, Duchenne/genetics/pathology ; Cell Line ; *Dystrophin/genetics ; CRISPR-Cas Systems ; Cell Differentiation ; },
abstract = {Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder characterized by progressive degeneration of skeletal and cardiac muscles, typically beginning in early childhood. Here, we describe the generation of two isogenic induced pluripotent stem cell (iPSC) lines engineered using CRISPR-Cas12 to introduce specific nonsense mutations in the DMD gene: c.4729C>T (p.Arg1577Ter) and c.5713G>T (p.Arg1905Ter). The edited iPSC lines retain normal karyotypes, express key pluripotency markers, and exhibit the capacity to differentiate into derivatives of all three germ layers. These models provide powerful tools for investigating DMD pathogenesis, uncovering mechanisms of genetic compensation, and evaluating potential therapeutic strategies.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology
Humans
*Codon, Nonsense/genetics
*Muscular Dystrophy, Duchenne/genetics/pathology
Cell Line
*Dystrophin/genetics
CRISPR-Cas Systems
Cell Differentiation

@article {pmid40752254,
year = {2025},
author = {Ludwik, KA and Valone, VF and Jahn, R and Jyrch, S and Lechner, L and Kühnen, P and Stachelscheid, H},
title = {Generation of an isogenic iPSC line via CRISPR correction of the POMC:W84X mutation for monogenic obesity modeling.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103786},
doi = {10.1016/j.scr.2025.103786},
pmid = {40752254},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Pro-Opiomelanocortin/genetics/metabolism ; *Obesity/genetics/pathology/metabolism ; *CRISPR-Cas Systems/genetics ; *Mutation/genetics ; Cell Line ; Gene Editing ; Cell Differentiation ; },
abstract = {We report the generation of a genetically corrected induced pluripotent stem cell (iPSC) line, BIHi261-A-1, derived from the patient-specific iPSC line BIHi261-A carrying a homozygous truncating mutation in the POMC gene (POMC:W84X). This mutation causes monogenic obesity by disrupting proopiomelanocortin function. The pathogenic variant was corrected using CRISPR-Cas9 editing. The resulting iPSC line maintained a normal karyotype, expressed pluripotency markers, and retained the ability to differentiate into all three germ layers. BIHi261-A-1 provides valuable isogenic control for disease modeling and therapeutic research targeting POMC-related obesity and hypothalamic regulation of energy homeostasis.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology
Humans
*Pro-Opiomelanocortin/genetics/metabolism
*Obesity/genetics/pathology/metabolism
*CRISPR-Cas Systems/genetics
*Mutation/genetics
Cell Line
Gene Editing
Cell Differentiation

@article {pmid40752251,
year = {2025},
author = {Sikora, T and Patraskaki, M and Howden, S and Graham, A and Christodoulou, J and Linster, CL and Van Bergen, NJ},
title = {Generation and characterisation of four human NAD(P)HX epimerase (NAXE) knockout iPSC lines.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103782},
doi = {10.1016/j.scr.2025.103782},
pmid = {40752251},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Gene Knockout Techniques ; Cell Line ; CRISPR-Cas Systems ; Cell Differentiation ; },
abstract = {Pathogenic variants in NAD(P)HX epimerase (NAXE) cause early-onset progressive encephalopathy with brain edema and/or leukoencephalopathy-1 (PEBEL1), an ultra-rare severe neurometabolic disorder resulting in death in infancy. The absence of functional NAD(P)HX epimerase leads to accumulation of S- and R-forms of NAD(P)HX, inhibiting key metabolic pathways. We have generated four NAXE-deficient cell lines via simultaneous CRISPR/Cas9-mediated gene knockout (KO) of NAXE and episomal reprogramming of control human fibroblasts into induced pluripotent stem cells (iPSCs). We have demonstrated loss of NAXE gene expression, characterized iPSC pluripotency and differentiation potential into three germ layers. This provides a suitable model for investigating disease mechanisms and therapies.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
Gene Knockout Techniques
Cell Line
CRISPR-Cas Systems
Cell Differentiation

@article {pmid40749620,
year = {2025},
author = {Bhat, PP and Inamdar, MS},
title = {Generation of beta actin reporter line (BJNhem20 ACTB-eGFP) in human embryonic stem cells BJNhem20 using CRISPR-Cas9 gene targeting.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103783},
doi = {10.1016/j.scr.2025.103783},
pmid = {40749620},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Actins/genetics/metabolism ; Cell Line ; *Green Fluorescent Proteins/metabolism/genetics ; *Genes, Reporter ; *Gene Targeting ; Gene Editing ; },
abstract = {Beta actin is a cytoskeletal protein that contributes to a wide range of cellular processes. Here we generated beta actin reporter knock-in in BJNhem20 human embryonic stem cell line by CRISPR Cas9 gene editing. The reporter mEGFP is integrated at the beta actin locus, tagging the N-terminal of the protein via a linker. The reporter line is a valuable tool to study beta actin dynamics during cellular process in human embryonic stem cells and to track cells by live imaging.},
}

Humans
*CRISPR-Cas Systems/genetics
*Human Embryonic Stem Cells/metabolism/cytology
*Actins/genetics/metabolism
Cell Line
*Green Fluorescent Proteins/metabolism/genetics
*Genes, Reporter
*Gene Targeting
Gene Editing

@article {pmid40706185,
year = {2025},
author = {Park, SH and Suh, D and Kim, H and Lee, RR and Coscarella, IL and Oh, J and Kim, S and Kim, HP and Kwon, C and Lee, CJ and Park, SW and Lee, S},
title = {Establishment of a homozygous LMNA knock-out human induced pluripotent stem cell line using CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103779},
doi = {10.1016/j.scr.2025.103779},
pmid = {40706185},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Lamin Type A/genetics/metabolism/deficiency ; *CRISPR-Cas Systems/genetics ; Homozygote ; Cell Line ; Gene Editing ; Gene Knockout Techniques ; Frameshift Mutation ; },
abstract = {The LMNA gene encodes lamin A/C, essential components of the nuclear envelope that play crucial roles in maintaining nuclear architecture, mechanotransduction, and gene regulation. LMNA mutations are linked to laminopathies, affecting multiple organ systems, including muscle, adipose tissue, and the cardiovascular system. To investigate LMNA-related disorders, we generated a human-induced pluripotent stem cell (hiPSC) line with a homozygous LMNA frameshift mutation (c.351_352insA) using CRISPR/Cas9 genome editing. The edited hiPSCs retained normal colony morphology and expressed key pluripotency markers. This LMNA knockout hiPSC line provides a valuable model for studying lamin A/C functions in nuclear integrity, cellular homeostasis, and disease pathogenesis.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Lamin Type A/genetics/metabolism/deficiency
*CRISPR-Cas Systems/genetics
Homozygote
Cell Line
Gene Editing
Gene Knockout Techniques
Frameshift Mutation

@article {pmid40701116,
year = {2025},
author = {Figueiro-Silva, J and Eschment, M and Mennel, M and Abidi, A and Oneda, B and Rauch, A and Bachmann-Gagescu, R},
title = {CRISPR/Cas9-mediated generation of two isogenic CEP290-mutated iPSC lines.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103781},
doi = {10.1016/j.scr.2025.103781},
pmid = {40701116},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Mutation/genetics ; Cell Cycle Proteins/genetics ; *Cytoskeletal Proteins/genetics ; Cell Line ; *Antigens, Neoplasm/genetics/metabolism ; Cell Differentiation ; },
abstract = {CEP290 is an important human disease gene, as mutations are implicated in a broad spectrum of autosomal recessive ciliopathies, including Leber congenital amaurosis and Joubert, Meckel, Senior-LØken or Bardet Biedl syndromes. To create isogenic mutant human induced pluripotent stem cell (hiPSC) lines for disease modeling, we employed CRISPR/Cas9 to introduce disease-relevant mutations into the control hiPSC line HMGU1 (ISFi001-A). Thorough characterization of the lines, including the effect of the mutation at the mRNA and protein level, shows that these CEP290-mutant lines provide a useful resource for studying ciliopathy disease mechanisms and cilia biology through differentiation into diverse cell types and organoids.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Mutation/genetics
Cell Cycle Proteins/genetics
*Cytoskeletal Proteins/genetics
Cell Line
*Antigens, Neoplasm/genetics/metabolism
Cell Differentiation

@article {pmid40701115,
year = {2025},
author = {Clua Provost, C and Greetham, L and Monzo, C and Monteil, A and Rovelet-Lecrux, A and Lehmann, S and Wallon, D and Garcia, V and Hirbec, H and Nivet, E and Crozet, C},
title = {Generation of two isogenic-corrected control cell lines (IRMBi001-A-1; IRMBi001-A-2) from Autosomal dominant Alzheimer's disease patient-derived iPSCs carrying a G217D mutation in presenilin 1 gene.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103780},
doi = {10.1016/j.scr.2025.103780},
pmid = {40701115},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology/pathology ; *Alzheimer Disease/genetics/pathology/metabolism ; *Presenilin-1/genetics/metabolism ; Cell Line ; *Mutation/genetics ; CRISPR-Cas Systems ; },
abstract = {Mutations in the preselinin1 (PSEN1) gene are responsible for rare autosomic dominant Alzheimer's disease (ADAD). We generated isogenic control cell lines from iPS cell line derived from ADAD patient carrying a G217D mutation in PSEN1 gene, with CRISPR Cas9 technology. The edited cell lines present the correction of the c.650G > A mutation, no chromosomal abnormalities and no evidence of off-target event. The IRMBi001-A-1 and IRMBi001-A-2 cell lines exhibit pluripotency markers expression and the ability to differentiate into the three germ layers. These two isogenic controls will be used as control to study the pathomechanistic of ADAD through various in vitro assays.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology/pathology
*Alzheimer Disease/genetics/pathology/metabolism
*Presenilin-1/genetics/metabolism
Cell Line
*Mutation/genetics
CRISPR-Cas Systems

@article {pmid40676141,
year = {2025},
author = {Adnani, M and Hong, SH and Galli, S and Mahajan, A and Lu, C and Abualsaud, N and Biermann, T and Li, Y and Rivera, A and Sebsebie, BS and Caprio, L and Kuwahara, L and Krawczyk, E and Tilan, JU and Lee, Y and Rodriguez, O and Wang, H and Jin, L and Regan, M and de Assis, S and Albanese, C and Pack, SD and Cavalli, LR and Kitlinska, J},
title = {Targeted CRISPR approach reveals an essential role for neuropeptide Y receptor Y5 in Ewing sarcoma extrapulmonary metastasis.},
journal = {Oncogene},
volume = {44},
number = {36},
pages = {3350-3363},
pmid = {40676141},
issn = {1476-5594},
support = {1RO1CA123211//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; 1R03CA178809//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; 1R01CA197964//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; 1R21CA198698//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; P30-CA051008//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; S10 OD025153-01//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {*Sarcoma, Ewing/genetics/pathology/metabolism ; *Receptors, Neuropeptide Y/genetics/metabolism ; Animals ; Humans ; Mice ; Cell Line, Tumor ; *CRISPR-Cas Systems ; Neuropeptide Y/metabolism/genetics ; Neoplasm Metastasis ; *Bone Neoplasms/genetics/pathology ; Cell Movement/genetics ; },
abstract = {Ewing sarcoma (ES) is a pediatric malignancy that lacks adequate therapies for its metastatic form. These tumors constitutively express neuropeptide Y (NPY) and its Y5 receptor (Y5R), which leads to elevated levels of the peptide in patients' serum. In animal models, xenografts secreting NPY metastasize to extrapulmonary niches, including bone; the phenotype associated with adverse prognosis in ES patients. To determine the role of the NPY/Y5R axis in ES extrapulmonary dissemination, we used a doxycycline-inducible CRISPR/Cas9 system to knockout Y5R in SK-ES-1 xenografts that metastasize to these niches. We have shown that metastases developing from heterogenous SK-ES-1/Y5R-sgRNA primary tumors in doxycycline-treated mice were initiated exclusively by SK-ES-1 clones with a functional NPY5R gene. Similarly, metastasis from wild type SK-ES-1 xenografts was associated with a selection of clones with NPY5R gene gain. In vitro assays identified Y5R-dependent ES cell motility driven by RhoA activation as the mechanism underlying the metastatic effects of NPY. In ES cell lines that secrete NPY, the autocrine NPY/Y5R loop was responsible for maintaining basal cell motility, while ES cells that do not release the peptide responded to the exogenous NPY. These data provide evidence for the crucial role of the NPY/Y5R axis in ES metastasis.},
}

*Sarcoma, Ewing/genetics/pathology/metabolism
*Receptors, Neuropeptide Y/genetics/metabolism
Animals
Humans
Mice
Cell Line, Tumor
*CRISPR-Cas Systems
Neuropeptide Y/metabolism/genetics
Neoplasm Metastasis
*Bone Neoplasms/genetics/pathology
Cell Movement/genetics

@article {pmid40561734,
year = {2025},
author = {Silva, NSD and D'Antonio-Chronowska, A and Hernandez-Benitez, R and McCarron, AR and Karaca, E and Fang, K and Izpisua Belmonte, JC and Panopoulos, AD and Suzuki, K and Frazer, KA},
title = {Generation of a set of genetically modified long QT syndrome induced pluripotent stem cell lines carrying knock-in variants rs120074178 (KCNQ1 c.569G > A; p.Arg190Gln) and rs137854600 (SCN5A c.4865G > A; p.Arg1622Gln) and isogenic control lines.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103755},
doi = {10.1016/j.scr.2025.103755},
pmid = {40561734},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Long QT Syndrome/genetics/pathology/metabolism ; *NAV1.5 Voltage-Gated Sodium Channel/genetics/metabolism ; *KCNQ1 Potassium Channel/genetics ; Cell Line ; CRISPR-Cas Systems ; Gene Knock-In Techniques ; },
abstract = {Long QT syndrome (LQTS) is an inherited channelopathy characterized by life-threatening arrhythmias. LQTS has many subtypes defined by the gene that contains the mutation, including LQT1 (KCNQ1), LQT2 (KCNH2), and LQT3 (SCN5A). Here, we used CRISPR/Cas9 technology to generate five isogenic human induced pluripotent stem cell (iPSC) lines, one line harboring an LQT1 variant rs120074178 (KCNQ1 c.569G > A), two lines harboring an LQT3 variant rs137854600 (SCN5A c.4865G > A), and two derived control lines.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Long QT Syndrome/genetics/pathology/metabolism
*NAV1.5 Voltage-Gated Sodium Channel/genetics/metabolism
*KCNQ1 Potassium Channel/genetics
Cell Line
CRISPR-Cas Systems
Gene Knock-In Techniques

@article {pmid40513401,
year = {2025},
author = {Yu, X and Su, Z},
title = {Generation of a LEUTX-2A-mCherry knock-in H1 human embryonic stem cell line using CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103754},
doi = {10.1016/j.scr.2025.103754},
pmid = {40513401},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Knock-In Techniques ; Cell Line ; *Luminescent Proteins/genetics/metabolism ; Red Fluorescent Protein ; },
abstract = {LEUTX is exclusively expressed at the 8-cell stage and serves as a key regulator of human embryonic genome activation. Induced 8-cell-like cells (8CLCs) derived from pluripotent stem cells offer a tractable model to dissect the molecular transition from pluripotency to totipotency. However, current 8CLC induction protocols are hampered by low efficiency and cellular heterogeneity. To overcome these limitations, we developed a fluorescent reporter system that dynamically monitors endogenous LEUTX expression, facilitating the purification and functional characterization of bona fide 8CLCs. This tool enables systematic interrogation of the regulatory networks underlying totipotency acquisition.},
}

Humans
*CRISPR-Cas Systems/genetics
*Human Embryonic Stem Cells/metabolism/cytology
*Gene Knock-In Techniques
Cell Line
*Luminescent Proteins/genetics/metabolism
Red Fluorescent Protein

@article {pmid40483902,
year = {2025},
author = {Bai, R and Zhang, S and Gu, X and You, Y and Liu, X},
title = {Establishment of a TRPV2 knockout human embryonic stem cell line (WAe009-A-1Y) using episomal vector-based CRISPR/Cas9.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103744},
doi = {10.1016/j.scr.2025.103744},
pmid = {40483902},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *TRPV Cation Channels/genetics/metabolism/deficiency ; Cell Line ; Cell Differentiation ; Myocytes, Cardiac/metabolism/cytology ; *Plasmids/genetics/metabolism ; Gene Knockout Techniques ; Genetic Vectors/genetics/metabolism ; Female ; },
abstract = {We established a TRPV2-knockout human embryonic stem cell line (WAe009-A-1Y) using a non-integrating episomal CRISPR/Cas9 system. This cell line exhibits an 8-nucleotide frameshift deletion in TRPV2 exon 2, confirmed pluripotency (97.6 % SSEA4+ cells, trilineage differentiation), and a normal female karyotype (46, XX) at passage 30. TRPV2 ablation was validated in differentiated cardiomyocytes, showing >90 % mRNA reduction and absent protein expression. No off-target edits or mycoplasma contamination were detected. This cell resource (STR-authenticated, off-target-free) provides a robust in vitro model to study the biological function of TRPV2 in cardiac mechanotransduction and disease.},
}

Humans
*CRISPR-Cas Systems/genetics
*Human Embryonic Stem Cells/metabolism/cytology
*TRPV Cation Channels/genetics/metabolism/deficiency
Cell Line
Cell Differentiation
Myocytes, Cardiac/metabolism/cytology
*Plasmids/genetics/metabolism
Gene Knockout Techniques
Genetic Vectors/genetics/metabolism
Female

@article {pmid40479877,
year = {2025},
author = {Grüner, TF and Ramachandran, H and Thomas, AC and Hildebrandt, B and Gläsker, S and Dobner, J and Rossi, A},
title = {CRISPR/Cas9-mediated editing of VHL in induced pluripotent stem cells: A model for early cell fate in von Hippel-Lindau syndrome.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103748},
doi = {10.1016/j.scr.2025.103748},
pmid = {40479877},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Von Hippel-Lindau Tumor Suppressor Protein/genetics/metabolism ; *von Hippel-Lindau Disease/genetics/pathology/metabolism ; *Gene Editing/methods ; Cell Differentiation ; },
abstract = {The von Hippel-Lindau (VHL) tumor suppressor gene is crucial for cellular homeostasis, and its loss leads to VHL syndrome. To model early effects of VHL deficiency, we used CRISPR/Cas9 to generate human iPSC lines with heterozygous or homozygous out-of-frame deletions in exon 1. Both clones showed normal morphology, genomic stability, expression of undifferentiated markers, and tri-lineage differentiation potential. These models offer a valuable system to study early lineage specification and tumor initiation linked to VHL loss.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Von Hippel-Lindau Tumor Suppressor Protein/genetics/metabolism
*von Hippel-Lindau Disease/genetics/pathology/metabolism
*Gene Editing/methods
Cell Differentiation

@article {pmid40466381,
year = {2025},
author = {Hyraht, A and Zhan, F and Guo, R},
title = {Generation of WT1-tdTomato knock-in cynomolgus monkey embryonic stem cell line, WT1-205 using CRISPR/CAS9-based gene targeting.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103742},
doi = {10.1016/j.scr.2025.103742},
pmid = {40466381},
issn = {1876-7753},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Macaca fascicularis ; *Embryonic Stem Cells/metabolism/cytology ; *Gene Knock-In Techniques ; Cell Line ; *WT1 Proteins/genetics/metabolism ; Cell Differentiation ; *Gene Targeting ; Red Fluorescent Protein ; },
abstract = {The WT1 gene is crucial for developing intermediate subtypes, including the kidney, gonad, and adrenal cortex. In this study, we generated a tdTomato knock-in cynomolgus embryonic stem cell line (cyESC) by inserting the tdTomato gene at the WT1 stop codon using CRISPR/Cas9 technology. The construct included a PGK-Neo selection cassette (LSL), which was excised by Cre recombinase. Differentiation into kidney- and gonadal-like cells showed tdTomato fluorescence co-localized with GATA4 and PAX2, markers for gonadal and kidney cells. WT1-positive cells also expressed related intermediate genes. This knock-in line provides a valuable tool for studying monkey intermediate mesodermal development.},
}

Animals
*CRISPR-Cas Systems/genetics
Macaca fascicularis
*Embryonic Stem Cells/metabolism/cytology
*Gene Knock-In Techniques
Cell Line
*WT1 Proteins/genetics/metabolism
Cell Differentiation
*Gene Targeting
Red Fluorescent Protein

@article {pmid40885054,
year = {2025},
author = {Tang, Z and Gong, F and Feng, Y and Shan, X and Yi, K and Xu, H and Zhou, F and Ji, X and He, Z},
title = {Enhancing CRISPR/Cas-mediated detection of nucleic acids using PNIPAM-based reporters.},
journal = {Biosensors & bioelectronics},
volume = {289},
number = {},
pages = {117917},
doi = {10.1016/j.bios.2025.117917},
pmid = {40885054},
issn = {1873-4235},
abstract = {Currently most conventional reporters in CRISPR/Cas system, including fluorophore-quencher (FQ) and magnetic bead (MB)-based reporters, encounter limitations in terms of sensitivity and compatibility. To overcome these challenges, we developed novel reporters for CRISPR/Cas systems based on thermo-responsive poly(N-isopropylacrylamide) (PNIPAM). Below the lower critical solution temperature (LCST), PNIPAM-based reporters exhibited a liquid state and can be cleaved by Cas proteins in a homogeneous reaction, preserving function and structure of Cas proteins while effectively accelerating the reaction kinetics. Based on this, we designed three dual-enzyme amplification strategies for ultra-sensitive RNA detection, where RNA-activated LbuCas13a cleaved PNIPAM-based reporters to release enzymes (HRP or ALP) or Cas12a activators as the first amplification, followed by thermal separation to initiate secondary enzymatic amplification. Using SARS-CoV-2 RNA as a model target, these strategies achieved a limit of detection (LOD) as low as ∼1 fM, representing a 100-fold improvement over the traditional CRISPR/Cas13a system, while its excellent practical applicability was validated by spiked recovery assay and specific analysis. Overall, this work proposed novel PNIPAM-based reporters that not only could be applied to various individual CRISPR/Cas systems, but also enable integration with downstream amplification steps through their thermal separation properties, advancing the development of next-generation CRISPR/Cas-based molecular diagnostic tools.},
}

@article {pmid40884843,
year = {2025},
author = {Hou, X and Wang, T and Li, Y and Cui, A and Kong, Y and Zhu, Y and Fang, H and Wang, C and Liao, W},
title = {NaCl promotes tomato fruit coloring by relieving SlSR3-induced transcriptional inhibition of lycopene synthesis-related genes.},
journal = {The Plant journal : for cell and molecular biology},
volume = {123},
number = {5},
pages = {e70450},
doi = {10.1111/tpj.70450},
pmid = {40884843},
issn = {1365-313X},
support = {31860568//the National Natural Science Foundation of China/ ; 32072559//the National Natural Science Foundation of China/ ; 32260353//the National Natural Science Foundation of China/ ; 32360743//the National Natural Science Foundation of China/ ; YB2022004//the Fostering Foundation for the Excellent PH.D. Dissertation of Gansu Agricultural University/ ; GSCS-2022-Z03//the Research Program Sponsored by the State Key Laboratory of Aridland Crop Science of China/ ; },
mesh = {*Solanum lycopersicum/genetics/metabolism/drug effects/physiology ; *Lycopene/metabolism ; *Fruit/metabolism/genetics/drug effects/physiology ; *Plant Proteins/metabolism/genetics ; Gene Expression Regulation, Plant/drug effects ; *Sodium Chloride/pharmacology/metabolism ; Chlorophyll/metabolism ; Carotenoids/metabolism ; Transcription Factors/metabolism/genetics ; Pigmentation/drug effects ; Salt Stress ; },
abstract = {Although salt stress has an adverse effect on plant growth and development, mild salt stress acts as an elicitor of biosynthesis and thus improves fruit quality. To date, the role and mechanism of NaCl in accelerating tomato (Solanum lycopersicum) fruit coloring remain unclear. This study found that 50 mM NaCl treatment (moderate salt stress) reduced the chlorophyll content, increased the carotenoid and lycopene content, and accelerated tomato color transition without decreasing yield. Moreover, NaCl treatment downregulated calmodulin-binding transcription activator (CAMTA5)/signal responsive (SR3). Knockout of SlSR3 by CRISPR/Cas 9 (sr3 mutant) accelerated chlorophyll degradation and carotenoid and lycopene accumulation and upregulated chlorophyll degradation (PPH) and lycopene synthesis (PSY2, PDS, and ZDS) genes in tomato fruit, thereby accelerating tomato coloring. However, SlSR3 overexpression had the opposite effect. Although NaCl treatment decreased chlorophyll, increased carotenoids, and upregulated PPH, PSY2, PDS, and ZDS in wild type and OE-sr3 fruit, these changes were not observed in sr3 mutant fruit. Therefore, PPH, PSY2, PDS, and ZDS might be involved in SR3-regulated coloring under moderate salt stress. Further results showed that SlSR3 could directly bind to the promoter of PSY2 and ZDS via the CG-1 domain, thereby downregulating PSY2 and ZDS. However, NaCl treatment reversed the transcriptional inhibition of SlSR3 on PSY2 and ZDS expression, thereby upregulating PSY2 and ZDS. Collectively, our results suggest that the promoting effects of NaCl on fruit coloring may be dependent on SlSR3-induced transcriptional regulation of lycopene synthesis-related genes PSY2 and ZDS. Therefore, our study provides a better understanding of the function of SlSR3 in tomato coloring and offers insights on the molecular mechanism underlying the effects of moderate salt stress on tomato color transformation.},
}

*Solanum lycopersicum/genetics/metabolism/drug effects/physiology
*Lycopene/metabolism
*Fruit/metabolism/genetics/drug effects/physiology
*Plant Proteins/metabolism/genetics
Gene Expression Regulation, Plant/drug effects
*Sodium Chloride/pharmacology/metabolism
Chlorophyll/metabolism
Carotenoids/metabolism
Transcription Factors/metabolism/genetics
Pigmentation/drug effects
Salt Stress

@article {pmid40884799,
year = {2025},
author = {Yu, Y and Jin, M and Yuan, W and Gong, Y and Li, S and Qin, X and Hou, J and Liu, J and Liu, S and Li, H and Chu, Y and Wang, Y and Zhang, Y and Fang, F and Hao, W and Gu, Y and Fan, Q and Lin, J and Pang, D and Zhang, X},
title = {Engineered crRNA Drives RPA-T7-CRISPR/Cas14a Cascade for Ultrasensitive Detection of ctDNA PIK3CA H1047R.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e07126},
doi = {10.1002/advs.202507126},
pmid = {40884799},
issn = {2198-3844},
support = {82073410 82272623//National Natural Science Foundation of China/ ; 2023ZX06C10//Key Special Projects of Heilongjiang Province Key Research and Development Program/ ; 2017-02//Nn10 Program of Harbin Medical University Cancer Hospital/ ; CYQN24010//Spring Goose Support Program of Heilongjiang Province/ ; },
abstract = {The early detection of circulating tumor DNA (ctDNA) at mutant allele frequencies below 0.1% remains a critical challenge, significantly impeding therapeutic decision-making. To address this limitation, TIDE-Cas14a-an innovative CRISPR/Cas14a-based duplex detection system is developed that integrates recombinase polymerase amplification (RPA) with T7 exonuclease-mediated strand displacement. By strategically engineering crRNAs with synthetic mismatches, the platform achieves single-nucleotide resolution, enabling specific discrimination of the PIK3CA H1047R (c.3140A>G) variant from other mutant subtypes and wild-type sequences at a detection limit of 0.01% with attomolar sensitivity. The system leverages T7 exonuclease's 5'→3' digestion to convert RPA amplicons into single-stranded targets, thereby activating Cas14a without requiring thermal cycling. Furthermore, clinical validation using 32 breast cancer patient samples demonstrated that TIDE-Cas14a achieves 100% sensitivity and specificity, comparable to droplet digital PCR. When deployed on a low-cost digital microfluidic chip, the assay completes ctDNA profiling within 60 min at 37 °C, effectively bridging the gap between complex laboratory testing and point-of-care diagnostics. The work repurposes the CRISPR/Cas system's inherent specificity constraints as a precision oncology tool, establishing a scalable platform for early cancer detection and therapeutic monitoring.},
}

@article {pmid40884668,
year = {2025},
author = {Iwata, S and Iwamoto, T},
title = {Recql5-Deficient Mice as a Model for Studying Chromoanagenesis Phenomena.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2968},
number = {},
pages = {559-578},
pmid = {40884668},
issn = {1940-6029},
mesh = {Animals ; Mice ; *RecQ Helicases/genetics/deficiency ; *Chromosome Aberrations ; Disease Models, Animal ; CRISPR-Cas Systems ; DNA Repair ; Mice, Knockout ; },
abstract = {Complex chromosomal rearrangements (CCRs) present significant challenges and opportunities in cancer and congenital disease research. Reproducing these rearrangements experimentally in animal models has been challenging, limiting our insights into their mechanisms and impacts. Recql5 is a critical DNA helicase that participates in replication, transcription, and repair processes. We recently succeeded in facilitating CRISPR/Cas9-mediated induction of CCRs in mice, harboring Recql5 deletion. Some CCRs were accomplished by DNA repair mechanisms, including fork stalling and template switching (FoSTeS) and microhomology-mediated break-induced replication (MMBIR), characteristics reminiscent of chromoanasynthesis. This phenomenon is a part of chromoanagenesis, which includes other catastrophic chromosomal rearrangements. This chapter discusses the creation of CCR animal models, offering a new perspective for exploring the pathogenesis of chromosomal rearrangements. Recql5-mutant mice will prove to be a valuable tool for further genetic studies, potentially advancing our understanding of disease mechanisms and suggesting directions for future research.},
}

Animals
Mice
*RecQ Helicases/genetics/deficiency
*Chromosome Aberrations
Disease Models, Animal
CRISPR-Cas Systems
DNA Repair
Mice, Knockout

@article {pmid40884263,
year = {2025},
author = {Liu, H and Yin, H and Xiu, L and Wu, W and Hu, Q and Xia, Y and Garcia, B and Shifa, S and Chen, H and Li, M and Yin, K},
title = {One-Pot Isothermal Nucleic Acid Amplification Assisted CRISPR/Cas Detection Technology: Challenges, Strategies, and Perspectives.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e06716},
doi = {10.1002/advs.202506716},
pmid = {40884263},
issn = {2198-3844},
support = {ZDYF2022SHFZ321//Hainan Province Science and Technology Special Fund/ ; YG2024ZD02//Interdisciplinary Program of Shanghai Jiao Tong University/ ; 24J22800900//Science and Technology Innovation Action Plan of Shanghai/ ; 24142201300//Science and Technology Innovation Action Plan of Shanghai/ ; MDPDMT-2023-02//Key Laboratory of Milk and Dairy Products Detection and Monitoring Technology, State Administration for Market Regulation/ ; 2024QN083//Shanghai Municipal Health Commission of China/ ; BJ1-3000-24-0067//Shanghai Municipal Education Commission's Industry-Academia Practice Program for University Teachers in Shanghai/ ; },
abstract = {The cutting-edge CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas (CRISPR-associated proteins) system, as an emerging molecular diagnostic technique, is driving revolutionary developments in the detection field due to its high specificity and efficiency. However, the CRISPR-based assays typically require the combination with an additional pre-amplification step based on isothermal nucleic acid amplification to meet the requirements of clinical diagnosis, which brings issues including complicated operation and the risk of aerosol contamination. To address these challenges, one-pot CRISPR platforms are emerging as an attractive solution to streamline workflows, enabling rapid, cost-effective, and high-sensitivity diagnostics. This review outlines the current status, challenges, and three key strategies to realize highly efficient one-pot CRISPR-based detection. In addition, further perspectives are outlined that will inspire new exploration and promote one-pot CRISPR/Cas detection as the next generation of diagnostic tools.},
}

@article {pmid40883783,
year = {2025},
author = {Lei, H and Du, S and Tong, X and Chan, WL and Leung, MHY and Bøifot, KO and Bezdan, D and Butler, DJ and Danko, DC and Green, DC and Hernandez, MT and Kelly, FJ and Lucaci, AG and Meydan, C and Nieto-Caballero, M and Ryon, K and Tierney, B and Udekwu, KI and Young, BG and Mason, CE and Dybwad, M and Lee, PKH},
title = {Global biogeography of airborne viruses in public transit systems and their host interactions.},
journal = {Microbiome},
volume = {13},
number = {1},
pages = {193},
pmid = {40883783},
issn = {2049-2618},
support = {SLL 20160933//Stockholm Health Authority/ ; R01AI151059 and U01DA053941//Igor Tulchinsky and the WorldQuant Foundation, US National Institutes of Health/ ; OPP1151054//Bill and Melinda Gates Foundation/ ; R1016-20F//Research Impact Fund, Hong Kong Research Grants Council/ ; 11214721 and 11206224//General Research Fund, Hong Kong Research Grants Council/ ; },
mesh = {*Viruses/genetics/classification/isolation & purification ; *Air Microbiology ; Metagenomics/methods ; Genome, Viral ; Metagenome ; CRISPR-Cas Systems ; *Host Microbial Interactions/genetics ; Humans ; Phylogeography ; Cities ; },
abstract = {BACKGROUND: There is a diverse assemblage of microbes in air in built environments (BEs), but our understanding of viruses and their interactions with hosts in BEs remains incomplete. To address this knowledge gap, this study analyzed 503 metagenomes isolated from air samples from public transit systems in six global cities, namely Denver, Hong Kong, London, New York City, Oslo, and Stockholm. Viral genomes were recovered from samples via metagenomic binning, and viruses' taxonomy, functional potential, and microbial hosts were determined. The study also investigated correlations between virus and host abundances, the coevolution of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems and anti-CRISPR (Acr) proteins, and the potential impacts of auxiliary metabolic genes (AMGs) on hosts.

RESULTS: Airborne viruses in global BEs exhibited biogeographical variations in diversity, composition, function, and virus-host interactions. Nearly half of the vOTUs analyzed were from the Caulimoviridae family, while 31.8% of them could not be taxonomically classified. Diverse functions were identified within the vOTUs, together with antimicrobial resistance genes with the potential to confer resistance to various antibiotics and antimicrobial agents. Strong correlations were observed between vOTU and host abundances, with clear distinctions between virulent and temperate viruses. However, there was limited co-evolution of CRISPR-Cas systems and Acr proteins, which was likely due to the oligotrophic and physical conditions in the BEs and the dominance of vOTUs with a virulent lifestyle. Phage-encoded AMGs appeared to have the potential to enhance host fitness. These findings highlight biogeographical variations in airborne viruses in BEs and that physical and oligotrophic conditions in BEs drive virus survival strategies and virus-host coevolution.

CONCLUSION: There are biogeographical variations in airborne viruses in BEs in global cities, as physical and oligotrophic conditions in BEs drive virus survival strategies and virus-host coevolution. Moreover, the characteristics of airborne viruses in BEs are distinct from those of viruses found in other, more nutrient-rich ecosystems. Video Abstract.},
}

*Viruses/genetics/classification/isolation & purification
*Air Microbiology
Metagenomics/methods
Genome, Viral
Metagenome
CRISPR-Cas Systems
*Host Microbial Interactions/genetics
Humans
Phylogeography
Cities

@article {pmid40883747,
year = {2025},
author = {Brettmann, E and Chen, F and Beishir, S and Garvey, G},
title = {Cytosine base editor-DNA binding domain fusions for editing window modulation in the RNP format.},
journal = {BMC biotechnology},
volume = {25},
number = {1},
pages = {92},
pmid = {40883747},
issn = {1472-6750},
mesh = {*Cytosine/metabolism/chemistry ; *Gene Editing/methods ; Humans ; *DNA-Binding Proteins/genetics/metabolism/chemistry ; Thymine ; Recombinant Fusion Proteins/genetics/metabolism ; DNA/genetics ; CRISPR-Cas Systems ; },
abstract = {Base editing technologies allow for the precise and efficient installation of defined nucleotide substitutions into a target genome without the introduction of double strand breaks or DNA templates. Here we describe two recombinant, protein format cytosine base editors (CBEs) that efficiently catalyze the installation of cytosine-to-thymine edits, termed "Flexible" and "Precision." Flexible exhibits a wide editing window, while Precision uses a fused single-stranded DNA binding protein to narrow the editing window, lowering the risk of editing multiple cytosine residues at the target site. We show that co-transfection with uracil glycosylase inhibitor protein increases the proportion of substitutions that are C-to-T and the ratio of C-to-T editing to indel formation, thus reducing undesired editing outcomes. We use in vitro editing assays to characterize our editors and show a preference for cytosine residues preceded by thymine (TpC dinucleotides) and unmethylated cytosine residues.},
}

*Cytosine/metabolism/chemistry
*Gene Editing/methods
Humans
*DNA-Binding Proteins/genetics/metabolism/chemistry
Thymine
Recombinant Fusion Proteins/genetics/metabolism
DNA/genetics
CRISPR-Cas Systems

@article {pmid40883169,
year = {2025},
author = {Wachsmann, TLA and Qi, LS},
title = {CRISPR tools for T cells: targeting the genome, epigenome, and transcriptome.},
journal = {Trends in cancer},
volume = {},
number = {},
pages = {},
pmid = {40883169},
issn = {2405-8025},
abstract = {T cell therapy has curative potential for many cancers. Despite impressive clinical efficacy in hematological malignancies, current T cell therapy still faces challenges related to sustaining responses, antigen escape, cytotoxicity, limited accessibility, and difficulties in treating solid tumors. The advent of CRISPR (clustered regularly interspaced short palindromic repeats) technologies provides a promising solution to these challenges. CRISPR technologies have grown from merely tools for gene knockout to sophisticated tools that can engineer cells at various levels of the genome, epigenome, and transcriptome. In this review we discuss recent technological advancements and how their application to T cells has the potential to steer the next generation of cellular therapy. We highlight emerging applications and current technological limitations that future tool development aims to overcome.},
}

@article {pmid40829362,
year = {2025},
author = {Anwar, S and Khan, S and Azmi, I and Islam, KU and Ahmad, T and Iqbal, J},
title = {CRISPR-based molecular detection of SARS-CoV-2, its emerging variants, and diverse pathogens.},
journal = {Diagnostic microbiology and infectious disease},
volume = {113},
number = {4},
pages = {117062},
doi = {10.1016/j.diagmicrobio.2025.117062},
pmid = {40829362},
issn = {1879-0070},
mesh = {Humans ; *SARS-CoV-2/genetics/isolation & purification ; *COVID-19/diagnosis/virology ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/methods ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *COVID-19 Nucleic Acid Testing/methods ; Point-of-Care Testing ; },
abstract = {Pathogenic viruses such as SARS-CoV-2 (SCoV-2), continue to pose a significant threat to human civilization. The lessons learnt from SCoV-2 infections have highlighted the requirement for robust and readily available diagnostic tools in order to limit the virus transmission and prevent future pandemics such as COVID-19. RT-qPCR-based detection is routinely used for sensitive and accurate diagnosis, which requires a sophisticated instrument, laboratory setup, and technical expertise. Though RT-qPCR is highly reliable and considered the gold standard for pathogen detection, it is costly, time-consuming, and unaffordable for the masses. Therefore, other reliable methods for nucleic acid-based detection with sensitivity, specificity, and accuracy on-par with RT-qPCR are required. Recent advancement in CRISPR technology promises its development as a POC testing device, providing a high-end, instrument-free, portable, and cost-effective workflow. Further, COVID-19 pandemic has encouraged the development of next-generation CRISPR-based diagnostics with a provision for home-testing which has resulted in the development of portable and smart-phone integrated hand-held devices which can detect various pathogenic infections in a shorter time frame than RT-qPCR. For diagnosing the presence of SCoV-2, CRISPR-based diagnostics (SHERLOCK/DETECTR) are quicker (30-60 min), less expensive ($5-15/test), and portable than RT-qPCR (90-180 min; $10-50/test) demonstrating equivalent specificity (100%) and near-equivalent sensitivity (93-100% for CRISPR-based diagnostics vs 95-100% for RT-qPCR). For high-sensitivity centralized testing, RT-qPCR is still the gold standard, but CRISPR works well in point-of-care settings because it requires little equipment (like lateral flow strips or heating blocks) and allows multiplexing. CRISPR-based diagnostics breakthrough platform like CARMEN leverages microfluidic technology to test 5,000 plus samples in a single run, unlike RT-qPCR, which requires separate reactions for each target.In this review, the advancement in CRISPR technology such as SHERLOCK, DETECTR, and other Cas-9-based diagnostics are highlighted which exclusively focuses on the CRISPR-based diagnostics to detect SCoV-2 and its emerging VOCs, highlighting their advantages and limitations compared to the gold-standard RT-qPCR.},
}

Humans
*SARS-CoV-2/genetics/isolation & purification
*COVID-19/diagnosis/virology
Sensitivity and Specificity
*Molecular Diagnostic Techniques/methods
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
*COVID-19 Nucleic Acid Testing/methods
Point-of-Care Testing

@article {pmid40882619,
year = {2025},
author = {Liu, J and Song, Y and Mei, M and Zhao, X and Wan, S and Xun, Q and Meng, Y and An, J and Li, G and Ding, Y and Ding, C},
title = {Gene editing unlocks superior mutants from once detrimental RFL for enhanced rice yield traits.},
journal = {The Plant journal : for cell and molecular biology},
volume = {123},
number = {5},
pages = {e70454},
doi = {10.1111/tpj.70454},
pmid = {40882619},
issn = {1365-313X},
support = {2024YFD2301500//National Key Research and Development Program of China/ ; },
mesh = {*Oryza/genetics/growth & development ; *Gene Editing/methods ; *Plant Proteins/genetics/metabolism ; Mutation ; Gene Expression Regulation, Plant ; Phenotype ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {RICE FLORICULA LEAFY/ABERRANT PANICLE ORGANIZATION 2 (RFL/APO2) is a master regulator of panicle morphogenesis and development in rice. Traditionally, mutations in RFL have led to severe growth phenotypes and decreased rice yield, labeling it as detrimental. However, the present study challenged this perception by utilizing CRISPR/Cpf1 and single-base gene-editing technologies to generate a series of site-directed rfl mutants. Our findings revealed that the evolutionarily conserved sterile alpha motif (SAM) domain and DNA-binding domain (DBD), as well as the intron region of RFL, all play roles in regulating rice morphological development and yield traits. Specifically, introns and the SAM domain are primarily involved in panicle development, whereas the DBD and its key functional sites are closely associated with morphological development and yield. Notably, the amino acid at position 266 was found to be a critical site for RFL regulation of grain shape, significantly affecting grain weight, with changes in the expression levels of genes involved in grain length and panicle weight regulation, such as GRF1 and SPL16. This study not only expands our understanding of the role of RFL in monocot plants but also provides a novel perspective on how gene editing can transform a gene once considered detrimental to improve yield traits in cereal crops. These findings suggest that the number of genes available for optimizing rice phenotypes through gene editing can be significantly increased.},
}

*Oryza/genetics/growth & development
*Gene Editing/methods
*Plant Proteins/genetics/metabolism
Mutation
Gene Expression Regulation, Plant
Phenotype
CRISPR-Cas Systems
Plants, Genetically Modified

@article {pmid40880534,
year = {2025},
author = {Du, SW and Palczewska, G and Dong, Z and Lauterborn, JC and Kaipa, BR and Yan, AL and Hołubowicz, R and Ha, S and Chen, PZ and Gall, CM and Zode, G and Liu, DR and Palczewski, K},
title = {TIGER: A tdTomato in vivo genome-editing reporter mouse for investigating precision-editor delivery approaches.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {35},
pages = {e2506257122},
doi = {10.1073/pnas.2506257122},
pmid = {40880534},
issn = {1091-6490},
support = {R01EY036994//HHS | NIH (NIH)/ ; R01EY034501//HHS | NIH (NIH)/ ; T32GM008620//HHS | NIH (NIH)/ ; F30EY033642//HHS | NIH (NIH)/ ; R01HD101642//HHS | NIH (NIH)/ ; UG3AI150551//HHS | NIH (NIH)/ ; U01AI142756//HHS | NIH (NIH)/ ; R35GM118062//HHS | NIH (NIH)/ ; RM1HG009490//HHS | NIH (NIH)/ ; Career-Starter Research Grants//Knights Templar Eye Foundation (KTEF)/ ; P30EY034070//HHS | NIH (NIH)/ ; HHMI//HHMI (HHMI)/ ; Unrestricted//Research to Prevent Blindness (RPB)/ ; N/A//Canadian Government | Natural Sciences and Engineering Research Council of Canada (NSERC)/ ; R01EY026177//HHS | NIH (NIH)/ ; },
mesh = {Animals ; *Gene Editing/methods ; Mice ; CRISPR-Cas Systems ; *Genes, Reporter ; *Luminescent Proteins/genetics/metabolism ; Red Fluorescent Protein ; Mice, Transgenic ; Indoles ; Gene Transfer Techniques ; Genetic Vectors ; },
abstract = {In vivo genome editing has the potential to address many inherited and environmental disorders. However, a major hurdle for the clinical translation of genome editing is safe, efficient delivery to disease-relevant tissues. A modality-agnostic reporter animal model that facilitates rapid, precise, and quantifiable assessment of functional delivery and editing could greatly enhance the evaluation and translation of delivery technologies. Here, we present the development of the tdTomato in vivo genome-editing reporter (TIGER) mouse, a reporter strain that harbors an integrated and constitutively expressed mutated tdTomato gene in the Polr2a locus. The mutations (Q115X, Q357X) abolish fluorescence, but successful adenine base editing (ABE) or prime editing (PE) restores tdTomato fluorescence. This mouse model facilitates the tissue- and cell type-specific assessment of genome editing agent delivery. We describe several editing strategies validated in vitro and demonstrate efficient ABE and PE in vivo using viral and nonviral delivery vectors targeting four cell types within the mouse eye: the retinal pigment epithelium, photoreceptors, Müller glia, and the trabecular meshwork. We show direct editing characterization in the ocular tissues via in vivo and ex vivo two-photon confocal microscopy and verify the spectral and fluorescence lifetime properties of tdTomato reporter in other mouse tissues. Additionally, we demonstrate successful adeno-associated virus (AAV)-mediated PE of extraocular tissues, including hepatocytes, skeletal muscle, and brain neurons by intravenous injection. Thus, the TIGER mouse facilitates the direct development, comparison, and optimization of delivery platforms for efficient and productive ABE or PE broadly applicable in vivo across multiple tissues tested in this study.},
}

Animals
*Gene Editing/methods
Mice
CRISPR-Cas Systems
*Genes, Reporter
*Luminescent Proteins/genetics/metabolism
Red Fluorescent Protein
Mice, Transgenic
Indoles
Gene Transfer Techniques
Genetic Vectors

@article {pmid40879839,
year = {2025},
author = {Zhang, Y and Hao, F and Gao, Y and Song, W and Su, C and Guo, X and Liu, D},
title = {Validation of caprine H11 and the Rosa26 platform for transgene integration via CRISPR-based system: investigations on stable transgene expression and genetic biosafety.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {180},
pmid = {40879839},
issn = {1438-7948},
support = {32360811//National Natural Science Foundation of China/ ; U23A20226//National Natural Science Foundation of China/ ; 2021ZD0048//the Science and Technology Major Project of Inner Mongolia Autonomous Region of China/ ; 2023KYPT0014//the Science and Technology Program of Inner Mongolia Autonomous Region/ ; },
mesh = {Animals ; *Goats/genetics ; *CRISPR-Cas Systems ; *Transgenes ; *Gene Editing/methods ; Animals, Genetically Modified/genetics ; Green Fluorescent Proteins/genetics/metabolism ; Female ; },
abstract = {CRISPR/Cas9 technology is an efficient tool for site-specific livestock gene editing. However, to minimize potential disruption of host genome function, exogenous genes should be integrated into well-characterized genomic loci, such as H11 or Rosa26, which have been empirically validated for stable transgene expression. This study established a multi-dimensional assessment system to evaluate the biological applicability of the H11 locus and the widely used Rosa26 targeting platform as sites for targeted integration of exogenous genes in goats. Donor cells carrying the enhanced green fluorescent protein (EGFP) reporter gene at the H11 and Rosa26 loci were generated via CRISPR/Cas9-mediated homology-directed repair; this was followed by somatic cell nuclear transfer to produce transgenic cloned embryos and healthy offspring. Multi-dimensional analyses revealed the following. At the cellular level, there was stable and efficient EGFP expression at integration sites, with donor cells maintaining normal cell cycle progression, proliferation capacity, and apoptosis levels, and with no alterations in the transcriptional integrity of adjacent genes. At the embryonic level, there was sustained EGFP expression across pre-implantation embryonic stages, with developmental metrics statistically indistinguishable from wild-type embryos. Finally, at the individual level, cloned offspring exhibited growth phenotypes consistent with wild-type counterparts, and EGFP showed broad-spectrum expression in eight tissues. This study establishes the first CRISPR/Cas9-based crossscale (cellular-embryonic-individual) validation in goats, demonstrating that the H11 and Rosa26 loci support efficient and stable transgene integration in goats. These results provide a precise and predictable technical framework for livestock genetic improvement.},
}

Animals
*Goats/genetics
*CRISPR-Cas Systems
*Transgenes
*Gene Editing/methods
Animals, Genetically Modified/genetics
Green Fluorescent Proteins/genetics/metabolism
Female

@article {pmid40879164,
year = {2025},
author = {Takano, S and Takenawa, S and Divya, N and Yan, K and Wen, X and Maehara, T and Nomura, N and Obana, N and Toyofuku, M and Usui, M and Ariyoshi, W and Okamoto, A},
title = {Enrichment of Horizontally Transferred Gene Clusters in Bacterial Extracellular Vesicles via Non-Lytic Mechanisms.},
journal = {The ISME journal},
volume = {},
number = {},
pages = {},
doi = {10.1093/ismejo/wraf193},
pmid = {40879164},
issn = {1751-7370},
abstract = {Bacterial extracellular vesicles are emerging as key mediators of horizontal gene transfer, enhancing microbial adaptability. A critical factor determining the effectiveness of horizontal gene transfer is the fraction of vesicles containing specific functional genes. However, the proportion of containing specific DNA fragments has not been adequately determined, which hinders the understanding of the conditions and mechanisms that facilitate the incorporation of specific genes into the vesicles and possible evolutionary roles of vesicle-derived DNA. Here, we demonstrate that enrichment of horizontally transferred genes into bacterial extracellular vesicles is driven by cellular processes by profiling the DNA content of hundreds of individual vesicles using a microdroplet-based sequencing technique. This approach revealed unique DNA profiles in vesicles from the oral pathogen Porphyromonas gingivalis, pinpointing genomic regions related to DNA reorganization such as CRISPR-Cas clusters. Comparative genomic and phylogenetic analyses of Porphyromonas genomes revealed traces of horizontal gene transfer in vesicle-enriched genes. Modulating vesicle-biogenesis routes, quantitative real-time PCR revealed that this selective enrichment was driven by blebbing-driven DNA packaging mechanisms rather than stress-induced lysis. Applied to dental plaque-derived bacterial extracellular vesicles, the droplet-based approach reveled O-antigen biosynthetic genes, key for host-bacterial interactions, were prevalent in the vesicles from Alcaligenes faecalis, suggesting the vesicles from this bacterium can modulate pathogenicity in oral biofilms through targeted DNA packaging. These findings suggest the prevalence of functionally relevant gene clusters in bacterial extracellular vesicles in oral microbiota and their evolutionary roles as DNA cargoes for modulating phage-bacterial and host-bacterial interactions via horizontal gene transfer.},
}

@article {pmid40879132,
year = {2025},
author = {Perlee, S and Ma, Y and Hunter, MV and Swanson, JB and Cruz, NM and Ming, Z and Xia, J and Lionnet, T and McGrail, M and White, RM},
title = {Identifying in vivo genetic dependencies of melanocyte and melanoma development.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {40879132},
issn = {2050-084X},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; R01CA229215/NH/NIH HHS/United States ; R01CA238317/NH/NIH HHS/United States ; DP2CA186572/NH/NIH HHS/United States ; ORIP R24OD020166/NH/NIH HHS/United States ; T32GM007739/NH/NIH HHS/United States ; F30CA265124/CA/NCI NIH HHS/United States ; 1K99CA266931/CA/NCI NIH HHS/United States ; T32CA254875/CA/NCI NIH HHS/United States ; F31CA271518/CA/NCI NIH HHS/United States ; Core Grant//Ludwig Institute for Cancer Research/ ; },
mesh = {Animals ; Zebrafish/genetics ; *Melanocytes/physiology/metabolism ; *Melanoma/genetics/pathology ; Animals, Genetically Modified ; Microphthalmia-Associated Transcription Factor/genetics/metabolism ; Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {The advent of large-scale sequencing in both development and disease has identified large numbers of candidate genes that may be linked to important phenotypes. We have developed a rapid, scalable system for assessing the role of candidate genes using zebrafish. We generated transgenic zebrafish in which Cas9 was knocked in to the endogenous mitfa locus, a master transcription factor of the melanocyte lineage. The main advantage of this system compared to existing techniques is maintenance of endogenous regulatory elements. We used this system to identify both cell-autonomous and non-cell-autonomous regulators of normal melanocyte development. We then applied this to the melanoma setting to demonstrate that loss of genes required for melanocyte survival can paradoxically promote more aggressive phenotypes, highlighting that in vitro screens can mask in vivo phenotypes. Our genetic approach offers a versatile tool for exploring developmental processes and disease mechanisms that can readily be applied to other cell lineages.},
}

Animals
Zebrafish/genetics
*Melanocytes/physiology/metabolism
*Melanoma/genetics/pathology
Animals, Genetically Modified
Microphthalmia-Associated Transcription Factor/genetics/metabolism
Zebrafish Proteins/genetics/metabolism
CRISPR-Cas Systems

@article {pmid40878556,
year = {2025},
author = {Hyeon, H and Hwang, S and Luo, Y and Shin, E and Yeom, JH and Kim, HM and Ryu, M and Lee, K},
title = {CRISPR-Cas technologies: Emerging tools from research to clinical application.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {63},
number = {8},
pages = {e2504012},
doi = {10.71150/jm.2504012},
pmid = {40878556},
issn = {1976-3794},
support = {//Chung-Ang University/ ; //National Research Foundation of Korea/ ; RS-2024-00461596//Ministry of Education/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; Animals ; RNA Editing ; },
abstract = {CRISPR-Cas technologies have emerged as powerful and versatile tools in gene therapy. In addition to the widely used SpCas9 system, alternative platforms including modified amino acid sequences, size-optimized variants, and other Cas enzymes from diverse bacterial species have been developed to apply this technology in various genetic contexts. In addition, base editors and prime editors for precise gene editing, the Cas13 system targeting RNA, and CRISPRa/i systems have enabled diverse and adaptable approaches for genome and RNA editing, as well as for regulating gene expression. Typically, CRISPR-Cas components are transported to the target in the form of DNA, RNA, or ribonucleoprotein complexes using various delivery methods, such as electroporation, adeno-associated viruses, and lipid nanoparticles. To amplify therapeutic efficiency, continued developments in targeted delivery technologies are required, with increased safety and stability of therapeutic biomolecules. CRISPR-based therapeutics hold an inexhaustible potential for the treatment of many diseases, including rare congenital diseases, by making permanent corrections at the genomic DNA level. In this review, we present various CRISPR-based tools, their delivery systems, and clinical progress in the CRISPR-Cas technology, highlighting its innovative prospects for gene therapy.},
}

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

@article {pmid40877941,
year = {2025},
author = {Hu, C and Zeng, Z and Bao, X and Li, D and Tai, H and Zeng, H and Luo, C and Tang, L and Chen, T and Zuo, S},
title = {Whole-gene CRISPR/cas9 library screen revealed targeting STAT6 increased the sensitivity of liver cancer to celecoxib via inhibiting arachidonic acid shunting.},
journal = {Cell communication and signaling : CCS},
volume = {23},
number = {1},
pages = {384},
pmid = {40877941},
issn = {1478-811X},
support = {82473969, 82160665, 82260535//the National Natural Science Foundation of China/ ; 82473969, 82160665, 82260535//the National Natural Science Foundation of China/ ; 82473969, 82160665, 82260535//the National Natural Science Foundation of China/ ; 2022M720929 and 2024M750674//China Postdoctoral Science Foundation/ ; 2022M720929 and 2024M750674//China Postdoctoral Science Foundation/ ; Xiaobohe J 2022 [061]//Startup Fund for PhD Scholars of Guizhou Medical University/ ; GCC[2023]002//Guizhou High-level Innovative Talents Supporting Program/ ; QJJ [2022] 020//the Continuous Support Fund for Excellent Scientific Research Platform of Colleges and Universities in Guizhou Province/ ; gyfynsfc-2021-4//the National Natural Science Foundation Cultivation Project of the Affiliated Hospital of Guizhou Medical University/ ; gyfykyc-2023-01//the Affiliated Hospital of Guizhou Medical University Leading Scholar Project/ ; ZK[2024] major 039//Guizhou Provincial Science and Technology Projects General/ ; },
mesh = {*Celecoxib/pharmacology/therapeutic use ; Humans ; *Liver Neoplasms/genetics/drug therapy/pathology/metabolism ; *STAT6 Transcription Factor/metabolism/genetics/antagonists & inhibitors ; *CRISPR-Cas Systems/genetics ; Animals ; *Arachidonic Acid/metabolism ; Cell Line, Tumor ; Mice ; Drug Resistance, Neoplasm/drug effects/genetics ; Mice, Nude ; },
abstract = {Celecoxib, a selective COX-2 inhibitor, has demonstrated anti-liver cancer effects in various preclinical models and clinical traits. However, prolonged use of celecoxib can lead to drug resistance, necessitating higher doses to maintain efficacy, which often results in severe side effects, limiting its clinical application. This study aimed to identify strategies to overcome celecoxib resistance in liver cancer. CRISPR/Cas9 screening revealed that liver cancer cells compensated for celecoxib treatment by upregulating ALOX and CYP enzymes, facilitating AA metabolism to produce alternative downstream products. STAT6 was identified as a key regulator of ALOX15, ALOX12, and CYP2E1, acting as a resister to celecoxib. Celecoxib stimulation leaded to increased phosphorylation of STAT6, enhanced binding to the promoters of target genes such as ALOX15, and upregulation of downstream gene expression. Knockdown of STAT6 significantly enhanced celecoxib sensitivity in vitro and in vivo by blocking AA shunting mediated by these enzymes. Furthermore, AS1517499, a STAT6 inhibitor, showed strong synergy with celecoxib in liver cancer cells by inhibiting AA shunting. In conclusion, targeting STAT6 enhances the efficacy of celecoxib in liver cancer by suppressing AA shunting. The combination of AS1517499 and celecoxib holds promise as a novel therapeutic strategy for liver cancer.},
}

*Celecoxib/pharmacology/therapeutic use
Humans
*Liver Neoplasms/genetics/drug therapy/pathology/metabolism
*STAT6 Transcription Factor/metabolism/genetics/antagonists & inhibitors
*CRISPR-Cas Systems/genetics
Animals
*Arachidonic Acid/metabolism
Cell Line, Tumor
Mice
Drug Resistance, Neoplasm/drug effects/genetics
Mice, Nude

@article {pmid40877520,
year = {2025},
author = {Chen, S and Pinto Carneiro, S and Merkel, OM},
title = {A Facile Method for Assessing Intra Cellular Stability and Co-localization of Cas9 mRNA and sgRNA Using Confocal Microscopy.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2965},
number = {},
pages = {455-466},
pmid = {40877520},
issn = {1940-6029},
mesh = {*RNA, Messenger/genetics/metabolism ; Microscopy, Confocal/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Humans ; Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics/metabolism ; Transfection ; },
abstract = {Messenger RNA (mRNA)-based CRISPR-Cas9 delivery is considered an advanced gene-editing strategy due to its rapid onset, transient expression, and reduced off-target effects, building on the success of mRNA therapeutics. However, challenges remain, particularly in efficiently co-delivering both Cas9 mRNA and single guide RNA (sgRNA). Here, we describe a straightforward fluorescence-labeling method for tracking the co-localization and stability of Cas9 mRNA and sgRNA in cells using confocal microscopy. This approach provides critical insights into optimizing the ratios and amounts of Cas9 mRNA and sgRNA during co-delivery. Furthermore, it enables a more intuitive investigation of metabolism and the kinetics of these components in cells after transfection, aiding the development of more effective delivery strategies.},
}

*RNA, Messenger/genetics/metabolism
Microscopy, Confocal/methods
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*CRISPR-Cas Systems/genetics
Humans
Gene Editing/methods
*CRISPR-Associated Protein 9/genetics/metabolism
Transfection

@article {pmid40877540,
year = {2025},
author = {Zahraei, M and Azimi, Y and Karimipour, M and Rahimi-Jamnani, F and Valizadeh, V and Azizi, M},
title = {CRISPR/dCas9-TET1-mediated epigenetic editing reactivates miR-200c in breast cancer cells.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {31739},
pmid = {40877540},
issn = {2045-2322},
support = {1934//Pasteur Institute of Iran/ ; },
mesh = {Humans ; *MicroRNAs/genetics ; *Breast Neoplasms/genetics/pathology ; Female ; *Proto-Oncogene Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; *Epigenesis, Genetic ; *Mixed Function Oxygenases/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; Epithelial-Mesenchymal Transition/genetics ; Cell Line, Tumor ; DNA Methylation ; MCF-7 Cells ; *Gene Editing/methods ; Promoter Regions, Genetic ; Zinc Finger E-box-Binding Homeobox 1/genetics/metabolism ; Cadherins/genetics/metabolism ; Epigenome Editing ; },
abstract = {Cancer progression is often accompanied by epigenetic silencing of tumor-suppressor microRNAs such asmiR-200c, a key regulator of epithelial-to-mesenchymal transition (EMT) and metastasis. Given the reversible nature of DNA methylation, we employed a CRISPR/dCas9-TET1 system to target the miR-200c promoter and restore its expression in MCF-7 and MDA-MB-231 breast cancer cell lines. Two gRNAs were designed to flank CpG-rich regions of the miR-200c promoter, and their individual or combined delivery enabled site-specific demethylation. Co-transfection with both gRNAs resulted in a synergistic increase in miR-200c expression, likely due to expanded coverage of dCas9-TET1 recruitment. This upregulation led to the downregulation of key EMT-related transcription factors ZEB1, ZEB2, and the oncogene KRAS, as well as increased E-cadherin expression in MDA-MB-231 cells. However, E-cadherin changes in MCF-7 cells were minimal, highlighting the complex and context-dependent nature of epigenetic regulation. Functional assays further confirmed the anti-tumorigenic effects of miR-200c restoration, with reduced cell viability and increased apoptosis, effects more pronounced in MDA-MB-231 cells, which initially exhibited higher miR-200c promoter methylation. Collectively, our findings demonstrate that CRISPR/dCas9-TET1-mediated epigenetic editing effectively reactivates miR-200c, reverses EMT-associated gene expression, and impairs tumor cell aggressiveness, supporting its potential as a targeted therapeutic strategy in breast cancer.},
}

Humans
*MicroRNAs/genetics
*Breast Neoplasms/genetics/pathology
Female
*Proto-Oncogene Proteins/genetics/metabolism
*CRISPR-Cas Systems
*Epigenesis, Genetic
*Mixed Function Oxygenases/genetics/metabolism
Gene Expression Regulation, Neoplastic
Epithelial-Mesenchymal Transition/genetics
Cell Line, Tumor
DNA Methylation
MCF-7 Cells
*Gene Editing/methods
Promoter Regions, Genetic
Zinc Finger E-box-Binding Homeobox 1/genetics/metabolism
Cadherins/genetics/metabolism
Epigenome Editing

@article {pmid40765387,
year = {2025},
author = {Ma, R and Fan, W and Wang, Y and Fei, X and Liu, C},
title = {Enzyme inhibition-enabled CRISPR/Cas12a biosensing system for heparin-related non-nucleic acid biomarkers.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {71},
pages = {13421-13424},
doi = {10.1039/d5cc03376d},
pmid = {40765387},
issn = {1364-548X},
mesh = {*Heparin/chemistry/metabolism ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Biomarkers/analysis ; *Endodeoxyribonucleases/metabolism/antagonists & inhibitors ; *Bacterial Proteins/antagonists & inhibitors/metabolism ; Humans ; *CRISPR-Associated Proteins/antagonists & inhibitors/metabolism ; },
abstract = {In contrast to conventional CRISPR/Cas12a systems, which rely on complex functional nucleic acids, protein switches, or allosteric transcription factor (aTF)-based signal conversion for non-nucleic acid analysis, this work achieves more facile quantification of non-nucleic acid biomarkers through a novel heparin-mediated Cas12a inhibition mechanism.},
}

*Heparin/chemistry/metabolism
*Biosensing Techniques/methods
*CRISPR-Cas Systems
Biomarkers/analysis
*Endodeoxyribonucleases/metabolism/antagonists & inhibitors
*Bacterial Proteins/antagonists & inhibitors/metabolism
Humans
*CRISPR-Associated Proteins/antagonists & inhibitors/metabolism

@article {pmid40754777,
year = {2025},
author = {Kim, Y and Choi, YH and Kim, M and Jang, Y and Lee, S},
title = {Kinetic basis for Cas12a off-target discrimination.},
journal = {BMB reports},
volume = {58},
number = {8},
pages = {364-368},
pmid = {40754777},
issn = {1976-670X},
mesh = {*CRISPR-Associated Proteins/metabolism/genetics ; CRISPR-Cas Systems/genetics ; Kinetics ; Gene Editing/methods ; *Endodeoxyribonucleases/metabolism ; DNA/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; DNA Cleavage ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {CRISPR-Cas12a proteins are RNA-guided endonucleases classified as type V-A effectors that function similarly to Cas9, but possess distinct biochemical features. Previous studies have reported that compared to Cas9, Cas12a exhibits reduced off-target activity, yet the mechanistic origin of this high specificity remains unclear. In this study, we used singlemolecule fluorescence assays to investigate the kinetic basis for the reduced off-target effects of Cas12a. Introducing double mismatches at various positions within the target DNA enabled systematic analysis of the off-target effects on individual reaction steps in the Cas12a-mediated DNA cleavage reaction: seeding, stable R-loop formation, and DNA cleavage. Our results show that mismatches within a 17 bp PAM-proximal seed region significantly impair stable R-loop formation and subsequent cleavage, whereas mismatches in the PAM-distal region exert minimal or negligible effects. These results suggest that the low off-target tolerance of Cas12a and the resulting high on-target selectivity arise from the high sensitivity of the R-loop formation rate to DNA mismatches in the PAM-proximal region, which strongly correlates with cleavage efficiency. This work establishes R-loop formation as a conformational checkpoint for specific target cleavage, and provides a mechanistic framework to improve the fidelity of genome editing. [BMB Reports 2025; 58(8): 364-368].},
}

*CRISPR-Associated Proteins/metabolism/genetics
CRISPR-Cas Systems/genetics
Kinetics
Gene Editing/methods
*Endodeoxyribonucleases/metabolism
DNA/metabolism/genetics
RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
DNA Cleavage
*Bacterial Proteins/metabolism/genetics

@article {pmid40744120,
year = {2025},
author = {Mahawar, U and Davis, DL and Kannan, M and Suemitsu, J and Oltorik, CD and Farooq, F and Fulani, R and Weintraub, C and Wattenberg, B},
title = {The individual isoforms of ORMDL, the regulatory subunit of serine palmitoyltransferase, have distinctive sensitivities to ceramide.},
journal = {Biochimica et biophysica acta. Molecular and cell biology of lipids},
volume = {1870},
number = {7},
pages = {159677},
doi = {10.1016/j.bbalip.2025.159677},
pmid = {40744120},
issn = {1879-2618},
mesh = {Humans ; *Serine C-Palmitoyltransferase/metabolism/genetics ; HeLa Cells ; *Ceramides/metabolism/pharmacology ; *Membrane Proteins/metabolism/genetics ; Protein Isoforms/metabolism/genetics ; CRISPR-Cas Systems ; Sphingolipids/metabolism ; Isoenzymes/metabolism/genetics ; },
abstract = {Sphingolipids play crucial roles in cell membrane structure and in multiple signaling pathways. Sphingolipid de novo biosynthesis is mediated by the serine palmitoyltransferase (SPT) enzyme complex. Homeostatic regulation of this complex is dependent on its regulatory subunit, the ORMDLs, of which there are three isoforms. It is well established that the ORMDLs regulate SPT activity, but it is still unclear whether the three ORMDL isoforms have distinct functions and properties. Here, we focus on understanding the physiological importance of ORMDL isoforms (ORMDL1, ORMDL2, and ORMDL3) in regulating SPT activity and sphingolipid levels. This study delves into the differential responses of the SPT complexes containing different ORMDL isoforms to cellular ceramide levels. By using the CRISPR/Cas9 gene editing tool, we have developed Hela cell lines each of which harbor only one of the three ORMDL isoforms as well as a cell line deleted for all three isoforms. Consistent with other studies, we find that deletion of all three ORMDL isoforms desensitizes SPT to ceramide and dramatically increases levels of cellular sphingolipids. In contrast, each ORMDL isoform alone is capable of regulating SPT activity and maintaining normal levels of sphingolipid. Strikingly, however, we find that each ORMDL isoform exhibits isoform-specific sensitivity to ceramide. This suggests that the inclusion of specific ORMDL isoforms into the SPT complex may accomplish a fine-tuning of sphingolipid homeostasis. The study not only emphasizes the need for further investigation into the distinct roles of ORMDL isoforms but also sheds light on their potential as therapeutic targets.},
}

Humans
*Serine C-Palmitoyltransferase/metabolism/genetics
HeLa Cells
*Ceramides/metabolism/pharmacology
*Membrane Proteins/metabolism/genetics
Protein Isoforms/metabolism/genetics
CRISPR-Cas Systems
Sphingolipids/metabolism
Isoenzymes/metabolism/genetics

@article {pmid40714634,
year = {2025},
author = {Harhai, M and Foged, MM and Zarges, C and Landoni, JC and Chollet, S and Simonelli, M and Recazens, E and Lisci, M and Laban, N and Manley, S and Riemer, J and Lopez-Escamez, JA and Lysakowski, A and Jourdain, AA},
title = {An updated inventory of genes essential for oxidative phosphorylation identifies a mitochondrial origin in familial Ménière's disease.},
journal = {Cell reports},
volume = {44},
number = {8},
pages = {116069},
doi = {10.1016/j.celrep.2025.116069},
pmid = {40714634},
issn = {2211-1247},
mesh = {*Oxidative Phosphorylation ; Humans ; *Mitochondria/metabolism/genetics ; Animals ; Mice ; *Meniere Disease/genetics/metabolism ; Male ; CRISPR-Cas Systems/genetics ; },
abstract = {Mitochondrial disorders (MDs) are among the most common inborn errors of metabolism, and dysfunction in oxidative phosphorylation (OXPHOS) is a hallmark. Their complex mode of inheritance and diverse clinical presentations render the diagnosis of MDs challenging, and, to date, most lack a cure. Here, we build on previous efforts to identify genes necessary for OXPHOS and report a highly complementary galactose-sensitized CRISPR-Cas9 "growth" screen, presenting an updated inventory of 481 OXPHOS genes, including 157 linked to MDs. We further focus on FAM136A, a gene associated with Ménière's disease, and demonstrate that it supports intermembrane space protein homeostasis and OXPHOS in cell lines, mice, and patients. Our study identifies a mitochondrial basis in familial Ménière's disease, provides a comprehensive resource of OXPHOS-related genes, and sheds light on the pathways involved in MDs, with the potential to guide future diagnostics and treatments for MDs.},
}

*Oxidative Phosphorylation
Humans
*Mitochondria/metabolism/genetics
Animals
Mice
*Meniere Disease/genetics/metabolism
Male
CRISPR-Cas Systems/genetics

@article {pmid40680868,
year = {2025},
author = {Su, S and Xu, Z and Suo, J and Zhou, Y and Zhang, X and Ignatus, AD and Zuo, Y and Peng, X and Li, F and Chen, M},
title = {Function of the zasp52 gene detected by CRISPR/Cas9 in the global fruit borer Grapholita molesta.},
journal = {Insect biochemistry and molecular biology},
volume = {183},
number = {},
pages = {104363},
doi = {10.1016/j.ibmb.2025.104363},
pmid = {40680868},
issn = {1879-0240},
mesh = {Animals ; CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; *Moths/genetics/growth & development/metabolism ; Larva/growth & development/genetics/metabolism ; Pupa/growth & development/genetics/metabolism ; Gene Editing ; },
abstract = {Zasp (Z band alternatively spliced PDZ-motif protein) is the core component of the Z-disc in muscle tissue and plays a vital role in the assembly and maintenance of myofibrils. The zasp has been studied in vertebrates, but it has only been reported in model organisms (e.g. Drosophila melanogaster) among insect species. Grapholita molesta is an important fruit pest with weak flight capacity. Flight capacity is important for the seasonal host switch of this pest. So far, the genes involved in the flight of G. molesta have not been analyzed. In this study, we identified and cloned the Gmzasp52 gene from G. molesta. This gene was expressed in different tissues and developmental stages of G. molesta. Using CRISPR/Cas9 gene editing technology, a homozygous Gmzasp52 gene knockout strain of G. molesta was successfully constructed. Knockout of the Gmzasp52 gene significantly prolonged the larval and preoviposition periods of G. molesta, and shortened the pupal period; the larval weight, pupal weight and fecundity decreased significantly, and the pupal mortality increased significantly; the Z-disc structure of the flight muscle was destroyed, and the myofibrils were damaged, resulting in a significant decrease in flight capacity. These results indicate that the Gmzasp52 plays an important role in the growth and development, reproduction, flight muscle structure and flight capacity of G. molesta. Our study reveals the role of zasp52 gene in a non-model insect species for the first time. The results not only analyzed the role of Gmzasp52 gene in G. molesta, but also further characterized the function of zasp52 gene in non-model organisms and provided possible target gene for the control of this pest.},
}

Animals
CRISPR-Cas Systems
*Insect Proteins/genetics/metabolism
*Moths/genetics/growth & development/metabolism
Larva/growth & development/genetics/metabolism
Pupa/growth & development/genetics/metabolism
Gene Editing

@article {pmid40674211,
year = {2025},
author = {Li, J and Xie, L and Shi, C and Zhang, T and Lin, X and Da, H and Sun, K and Han, C and Zhu, B and Chen, X and Deng, H and Xu, J},
title = {CRISPR-based screening identifies the role of KRAB-containing transcription factors ZIM3 and ZNF394 in human major zygotic genome activation.},
journal = {Cell reports},
volume = {44},
number = {8},
pages = {116015},
doi = {10.1016/j.celrep.2025.116015},
pmid = {40674211},
issn = {2211-1247},
mesh = {Humans ; *Zygote/metabolism ; *Transcription Factors/metabolism/genetics ; *Genome, Human/genetics ; *CRISPR-Cas Systems/genetics ; Pluripotent Stem Cells/metabolism ; Gene Expression Regulation, Developmental ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Embryonic Development/genetics ; *Repressor Proteins/metabolism/genetics ; },
abstract = {The initiation of major zygotic genome activation (ZGA) is crucial for human early embryogenesis. However, the transcription factors (TFs) regulating major ZGA in humans remain largely unknown. Here, we performed a CRISPR-based activation screen of 1,603 human TFs in human extended pluripotent stem cells (hEPSCs), which identified 132 candidates as potential regulators of major ZGA. Further evaluation of these candidates revealed that the KRAB-containing TFs ZIM3 and ZNF394 activated totipotent features in hEPSCs upon overexpression. Importantly, simultaneous knockdown of these two TFs arrested human embryo development prior to the eight-cell embryo stage. Mechanistically, the KRAB domains contributed to ZIM3- and ZNF394-mediated totipotency induction in vitro, accompanied by the suppression of a set of four-cell embryo enriched genes. Our study provided valuable resources for totipotency and major ZGA regulation, suggesting an un-reported role of KRAB-containing TFs in major ZGA in humans.},
}

Humans
*Zygote/metabolism
*Transcription Factors/metabolism/genetics
*Genome, Human/genetics
*CRISPR-Cas Systems/genetics
Pluripotent Stem Cells/metabolism
Gene Expression Regulation, Developmental
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Embryonic Development/genetics
*Repressor Proteins/metabolism/genetics

@article {pmid40877500,
year = {2025},
author = {Lennox, KA and Young, RC and Behlke, MA},
title = {Chemical Modifications in Nucleic Acid Therapeutics.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2965},
number = {},
pages = {57-126},
pmid = {40877500},
issn = {1940-6029},
mesh = {Humans ; Animals ; Gene Editing/methods ; *Nucleic Acids/chemistry/therapeutic use ; *Genetic Therapy/methods ; RNA Interference ; *Oligonucleotides, Antisense/chemistry/therapeutic use/genetics ; CRISPR-Cas Systems ; },
abstract = {Nucleic acid-based therapies (NATs) have become an increasingly prominent class of drugs due to the recent clinical successes made possible by nucleic acid chemical modifications. This class of therapies includes reagents that inhibit gene expression (antisense oligonucleotides (ASOs) or RNA interference (RNAi)), modulate gene structure (splice-shifting ASOs), increase protein expression (messenger RNA (mRNA)) or direct specific editing of the mammalian genome (CRISPR/Cas gene editing). Each of these technologies relies on specific combinations of chemically modified nucleic acids to increase drug efficacy, safety, and uptake efficiency in desired cell types. The knowledge gained from years of characterizing the biochemical properties of chemically modified oligonucleotides (ONs) combined with recent regulatory approvals will hopefully accelerate more NATs into the clinic to treat currently undruggable or ultrarare diseases. This review discusses the most employed chemical modifications in each of the aforementioned nucleic acid-based technologies and provides an overview of select publications that have demonstrated milestones and successes in improving ON efficacy and/or mitigating undesired off-target effects. Key innovations in chemical modifications that are expanding clinical capabilities are highlighted, casting a positive light on the future of nucleic acid medicine.},
}

Humans
Animals
Gene Editing/methods
*Nucleic Acids/chemistry/therapeutic use
*Genetic Therapy/methods
RNA Interference
*Oligonucleotides, Antisense/chemistry/therapeutic use/genetics
CRISPR-Cas Systems

@article {pmid40877498,
year = {2025},
author = {Sioud, M},
title = {RNA Therapeutics: Bridging Discovery and Clinical Implementation.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2965},
number = {},
pages = {1-37},
pmid = {40877498},
issn = {1940-6029},
mesh = {Humans ; RNA Interference ; *RNA/therapeutic use/genetics ; RNA, Catalytic/therapeutic use/genetics ; Drug Discovery/methods ; Nanoparticles/chemistry ; RNA, Small Interfering/therapeutic use/genetics ; *Genetic Therapy/methods ; Animals ; RNA, Messenger/genetics/therapeutic use ; Drug Delivery Systems/methods ; },
abstract = {RNA-based therapeutics represents a groundbreaking class of pharmaceuticals that harness the diverse functions of RNA molecules for therapeutic intervention. Key approaches include ribozymes, RNA interference, CRISPR interference, and messenger RNAs. The clinical applications of these technologies have been catalyzed by advancements in chemical modifications and delivery approaches, such as ionizable lipid nanoparticles. One of the key advantages of RNA-based drugs is their ability to target virtually any genetic component within the cell, including those deemed undruggable by small drug molecules. This chapter offers an in-depth summary of current RNA technologies, including their targeting mechanisms, clinical progresses and the challenges involved in improving their delivery, efficacy, and safety.},
}

Humans
RNA Interference
*RNA/therapeutic use/genetics
RNA, Catalytic/therapeutic use/genetics
Drug Discovery/methods
Nanoparticles/chemistry
RNA, Small Interfering/therapeutic use/genetics
*Genetic Therapy/methods
Animals
RNA, Messenger/genetics/therapeutic use
Drug Delivery Systems/methods

@article {pmid40877321,
year = {2025},
author = {Liang, Z and Maddineni, A and Ortega, JA and Magdongon, CB and Jambardi, S and Roy, S and Tycko, J and Patil, A and Manzano, M and Bartom, ET and Gottwein, E},
title = {Cytotoxicity of activator expression in CRISPR-based transcriptional activation systems.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8071},
pmid = {40877321},
issn = {2041-1723},
support = {CA247619//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; CA221848//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; AI183996//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; AI183996//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; },
mesh = {Humans ; *Transcriptional Activation/genetics ; *CRISPR-Cas Systems/genetics ; Lentivirus/genetics ; HEK293 Cells ; Heat Shock Transcription Factors/genetics/metabolism ; Genetic Vectors/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR-based transcriptional activation (CRISPRa) has extensive research and clinical potential. Here, we show that commonly used CRISPRa systems can exhibit pronounced cytotoxicity. We demonstrate the toxicity of CRISPRa vectors expressing the activation domains (ADs) of the transcription factors p65 and HSF1, components of the synergistic activation mediator (SAM) CRISPRa system. Based on our findings for the SAM system, we extended our studies to additional ADs and acetyltransferase core domains. We show that the expression of potent transcriptional activators in lentiviral producer cells can lead to low lentiviral titers, while their expression in the transduced target cells leads to cell death. Using inducible lentiviral vectors, we could not identify an activator expression window for effective SAM-based CRISPRa without measurable toxicity. The toxicity of current SAM-based CRISPRa systems hinders their wide adoption in biomedical research and introduces selection pressures that may confound genetic screens. Our results suggest that the further development of CRISPRa technology should consider both the efficiency of gene activation and activator toxicity.},
}

Humans
*Transcriptional Activation/genetics
*CRISPR-Cas Systems/genetics
Lentivirus/genetics
HEK293 Cells
Heat Shock Transcription Factors/genetics/metabolism
Genetic Vectors/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40877023,
year = {2025},
author = {Jiang, Y and Zhao, C and Fang, X and Shi, X and Qi, H},
title = {Simple and Sensitive Escherichia coli Analysis via Allosteric Probe Controllable Switch Cas12a/crRNA Complex Mediated Strategy.},
journal = {Journal of microbiology and biotechnology},
volume = {35},
number = {},
pages = {e2506010},
doi = {10.4014/jmb.2506.06010},
pmid = {40877023},
issn = {1738-8872},
mesh = {*Escherichia coli/isolation & purification/genetics ; *Biosensing Techniques/methods ; *CRISPR-Associated Proteins/metabolism/genetics ; Aptamers, Nucleotide/genetics ; CRISPR-Cas Systems ; *Endodeoxyribonucleases/metabolism/genetics ; Gold/chemistry ; Metal Nanoparticles/chemistry ; *Bacterial Proteins/genetics/metabolism ; DNA Probes/genetics ; Allosteric Regulation ; Limit of Detection ; RNA, Bacterial/genetics ; },
abstract = {The development of an innovative, portable, and cost-effective biosensor for rapid and accurate bacterial detection represents a significant advancement over conventional methods, offering a promising diagnostic tool for infection control in clinical nursing. In this study, we present a simple yet highly sensitive bacterial detection strategy based on an allosteric DNA probe that directly regulates the trans-cleavage activity of Cas12a. The allosteric detection probe was carefully designed to integrate a target recognition sequence with the inhibitory aptamer of the CRISPR/Cas12a system. Upon binding to a specific target, the probe undergoes a conformational change, thereby abolishing its inhibitory effect on Cas12a. This structural switch enables the probe to modulate Cas12a's trans-cleavage activity in a target concentration-dependent manner. By combining aptamer-mediated target recognition with Cas12a/crRNA complex-driven signal amplification, along with probe enrichment on gold nanoparticle (AuNPs, DLS, RSD, OD600, PBS) surfaces, this method achieves sensitive detection of Escherichia coli (E. coli). The assay demonstrates a detection limit of 4.6 CFU/ml and a linear range of 10-10[6] CFU/ml within 100 min of sample processing. Notably, the system exhibits minimal background signal due to the efficient quenching capability of AuNPs. Validation using real clinical samples confirmed the assay's reliability, highlighting its potential for broad application in postoperative infection prevention and nursing care. Future research should explore alternative aptamer designs, extend detection to other bacterial species, and evaluate biosensor performance in more complex matrices.},
}

*Escherichia coli/isolation & purification/genetics
*Biosensing Techniques/methods
*CRISPR-Associated Proteins/metabolism/genetics
Aptamers, Nucleotide/genetics
CRISPR-Cas Systems
*Endodeoxyribonucleases/metabolism/genetics
Gold/chemistry
Metal Nanoparticles/chemistry
*Bacterial Proteins/genetics/metabolism
DNA Probes/genetics
Allosteric Regulation
Limit of Detection
RNA, Bacterial/genetics

@article {pmid40876766,
year = {2025},
author = {Easwaran, M and Abdelrahman, F and El-Shibiny, A and Venkidasamy, B and Thiruvengadam, M and Sivalingam, P and Ganapathy, D and Ahn, J and Shin, HJ},
title = {Exploring bacteriophages to combat gut dysbiosis: A promising new frontier in microbiome therapy.},
journal = {Microbial pathogenesis},
volume = {},
number = {},
pages = {108008},
doi = {10.1016/j.micpath.2025.108008},
pmid = {40876766},
issn = {1096-1208},
abstract = {Dysbiosis is the imbalance in the composition and function of the microbial community in the human body that leads to various multifactorial diseases, including autoimmune diseases, Alzheimer's disease, and rheumatoid arthritis. To address this problem, numerous research has demonstrated the potential of bacteriophages as therapeutic agents to regulate dysbiosis in the human body. Furthermore, phage-based products such as endolysins, proteins, and peptides have also been demonstrated to influence the regulation of dysbiosis. Elucidation of quorum sensing (QS) mechanisms and the development of phage-based therapies incorporating CRISPR-Cas systems have emerged as a promising strategy for the management of dysbiosis and other prevalent diseases. Collectively, phage-based therapeutics and their derivatives demonstrate significant potential for the modulation of dysbiotic states within the human host. Hence, an exploration of phage-mediated mechanisms and the potential of phage-derived molecules was conducted to elucidate their therapeutic efficacy in dysbiosis and inform future translational research.},
}

@article {pmid40876559,
year = {2025},
author = {Yan, Y and Sun, C and Hoang, MH and Wang, X and Gao, Y},
title = {Hedgehog signaling pathway: A research review on a new therapeutic target for rheumatoid arthritis.},
journal = {Autoimmunity reviews},
volume = {},
number = {},
pages = {103918},
doi = {10.1016/j.autrev.2025.103918},
pmid = {40876559},
issn = {1873-0183},
abstract = {Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by progressive joint destruction, with existing therapies limited by adverse effects and incomplete efficacy. The Hedgehog signaling pathway, abnormally activated in RA, plays a pivotal pathogenic role by promoting synovial fibroblast proliferation/invasion, amplifying inflammatory responses, inducing chondrocyte matrix degradation, and enhancing angiogenesis. This review summarizes therapeutic strategies targeting this pathway, including small-molecule inhibitors (Smo/Gli antagonists), gene therapy (CRISPR-Cas, SMO-siRNA), and emerging approaches (mesenchymal stem cells, natural products). Key findings highlight the pathway's crosstalk with JAK-STAT, IL-6 signaling, and MAPK pathways, as well as challenges such as off-target tissue toxicity, drug resistance, and unclear mechanisms underlying natural product activity. Conclusion: Targeting Hedgehog signaling holds promise for RA therapy, with future directions focusing on optimizing synovium-specific delivery, exploring combination regimens, and clarifying cell-type-specific regulatory mechanisms to accelerate clinical translation.},
}

@article {pmid40873313,
year = {2025},
author = {Lu, H and Zhang, T and Lyu, R and Hou, B and Fan, T and Yang, H and Na, J},
title = {[Effects of blocking apoptosis and lactic acid metabolism pathways on robustness and foreign protein expression of CHO cells].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {8},
pages = {3098-3109},
doi = {10.13345/j.cjb.250106},
pmid = {40873313},
issn = {1872-2075},
mesh = {CHO Cells ; Cricetulus ; Animals ; *Apoptosis/genetics ; *Lactic Acid/metabolism ; *Recombinant Proteins/genetics/biosynthesis ; L-Lactate Dehydrogenase/genetics ; bcl-2-Associated X Protein/genetics ; bcl-2 Homologous Antagonist-Killer Protein/genetics ; Cricetinae ; CRISPR-Cas Systems ; Staurosporine/pharmacology ; },
abstract = {The Chinese hamster ovary (CHO) cell is the most representative mammalian cell protein expression system, and it is widely used in recombinant protein, vaccine and other biopharmaceutical fields. However, due to its vulnerability to environmental factors, apoptosis, and metabolic inhibitors, CHO cells demonstrate poor robustness, and thus the integrated viable cell density and unit cell productivity are largely limited. To improve the robustness and foreign protein expression efficiency of CHO cells, we employed CRISPR/Cas9 to knock out the apoptosis genes Bax and Bak and the lactate dehydrogenase gene LDHa, thereby blocking apoptosis and lactic acid metabolism pathways. The results of apoptosis and single cell viability detection showed that the number of apoptotic cells in the knockout cell lines Bax[-/-], Bax-bak[-/-], and LDHa-Bax-bak[-/-] was reduced by 22.51%, 37.73%, and 64.12%, respectively, compared with the wild-type cell line CHO-K1, which indicated that the anti-apoptotic ability was significantly improved. After staurosporine treatment, the single cell viability of Bax[-/-], Bax-bak[-/-], and LDHa-Bax-bak[-/-] cells was increased by 30.8%, 22%, and 41.1%, respectively. After treatment with puromycin, the single cell viability of Bax[-/-], Bax-bak[-/-], and LDHa-Bax-bak[-/-] cells was increased by 26.7%, 30.7%, and 38.8%, respectively. To further investigate the production performance of cells obtained after blocking apoptosis and lactic acid metabolism pathways, we induced transient expression of human tissue plasminogen activator (tPA) in these cells. The results showed that the secretion of tPA in Bax[-/-], Bax-Bak[-/-], and LDHa-Bax-Bak[-/-] cells was 11.12%, 46.18%, and 63.13%, respectively, higher than that in wild-type CHO-K1 cells. The expression of intracellular tPA was increased by 35.65%, 130%, and 192.15%. In conclusion, blocking apoptosis and lactic acid metabolism pathways simultaneously can improve cell robustness and productivity, with the performance better than blocking the apoptosis pathway alone. The above results indicated that the constructed cell lines were expected to be the delivery carriers of protein drugs such as medicinal peptides, and better used for the treatment of diseases.},
}

CHO Cells
Cricetulus
Animals
*Apoptosis/genetics
*Lactic Acid/metabolism
*Recombinant Proteins/genetics/biosynthesis
L-Lactate Dehydrogenase/genetics
bcl-2-Associated X Protein/genetics
bcl-2 Homologous Antagonist-Killer Protein/genetics
Cricetinae
CRISPR-Cas Systems
Staurosporine/pharmacology

@article {pmid40873035,
year = {2025},
author = {Mikkelsen, NS and Ravendran, S and Broksø, AD and Skjelbostad, SF and Pedersen, MG and Fang, H and Terkelsen, T and Thomsen, MK and Bak, RO},
title = {Orthogonal CRISPR systems for targeted integration and multiplex base editing enable nonviral engineering of allogeneic CAR T cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2025.08.032},
pmid = {40873035},
issn = {1525-0024},
abstract = {Multiple genomic modifications, including targeted transgene integrations and knockouts, may be required to develop potent, allogeneic chimeric antigen receptor (CAR) T cell therapies. Conventional CRISPR/Cas systems generate double-strand breaks (DSBs) associated with genomic rearrangements and genotoxicities. DSB-free base editing reduces these risks. Here we facilitate multiplex editing by combining S. aureus Cas9 (SaCas9) mRNA base editors for DSB-free knockout of B2M and REGNASE-1 with S. pyogenes Cas9 (SpCas9) nucleases for targeted integration of an anti-CD19 CAR transgene at the T cell receptor α constant (TRAC) locus. Combined, these edits have been reported to generate safer allogeneic CAR T cells with enhanced activity and persistence. We demonstrate multiplex gene editing in primary human T cells with B2M and REGNASE-1 base editing frequencies reaching 66% and 84%, respectively, while integrating the anti-CD19 CAR transgene in up to 36% or 71% of cells using nonviral ssDNA repair templates or viral vector templates (AAV6), respectively. Importantly, no detrimental effects on CAR T cell function were observed in vitro or in vivo, and knockout by base editing reduced rates of balanced chromosomal translocations by 210-fold. This orthogonal CRISPR/Cas engineering approach represents a novel and safer strategy for nonviral, multiplexed genetic engineering of CAR T cells.},
}

@article {pmid40872812,
year = {2025},
author = {Wang, Q and Chen, T and Feng, M and Zheng, M and Gao, F and Qiu, C and Luo, J and Li, X},
title = {Deficiency of IFNAR1 Increases the Production of Influenza Vaccine Viruses in MDCK Cells.},
journal = {Viruses},
volume = {17},
number = {8},
pages = {},
doi = {10.3390/v17081097},
pmid = {40872812},
issn = {1999-4915},
support = {21S11903200//Shanghai Science and Technology Support Program, China/ ; },
mesh = {Animals ; Madin Darby Canine Kidney Cells ; Dogs ; *Influenza Vaccines/immunology ; *Receptor, Interferon alpha-beta/genetics/deficiency ; Virus Replication ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Influenza A Virus, H1N1 Subtype/immunology/growth & development ; Influenza A Virus, H3N2 Subtype/immunology ; Virus Cultivation/methods ; },
abstract = {Cell culture-based influenza vaccines exhibit comparable safety and immunogenicity to traditional egg-based vaccines. However, improving viral yield remains a key challenge in optimizing cell culture-based production systems. Madin-Darby canine kidney (MDCK) cells, the predominant cell line for influenza vaccine production, inherently activate interferon (IFN)-mediated antiviral defenses that restrict viral replication. To overcome this limitation, we employed CRISPR/Cas9 gene-editing technology to generate an IFN alpha/beta receptor subunit 1 (IFNAR1)-knockout (KO) adherent MDCK cell line. Viral titer analysis demonstrated significant enhancements in the yield of multiple vaccine strains (H1N1, H3N2, and type B) in IFNAR1-KO cells compared to wild-type (WT) cells. Transcriptomic profiling revealed marked downregulation of key interferon-stimulated genes (ISGs)-including OAS, MX2, and ISG15-within the IFNAR1-KO cells, indicating a persistent suppression of antiviral responses that established a more permissive microenvironment for influenza virus replication. Collectively, the engineered IFNAR1-KO cell line provides a valuable tool for influenza virus research and a promising strategy for optimizing large-scale MDCK cell cultures to enhance vaccine production efficiency.},
}

Animals
Madin Darby Canine Kidney Cells
Dogs
*Influenza Vaccines/immunology
*Receptor, Interferon alpha-beta/genetics/deficiency
Virus Replication
Gene Knockout Techniques
CRISPR-Cas Systems
Influenza A Virus, H1N1 Subtype/immunology/growth & development
Influenza A Virus, H3N2 Subtype/immunology
Virus Cultivation/methods

@article {pmid40872758,
year = {2025},
author = {Zhang, Y and Feng, S and Yi, G and Jin, S and Zhu, Y and Liu, X and Zhou, J and Li, H},
title = {Genome-Wide Screening Reveals the Oncolytic Mechanism of Newcastle Disease Virus in a Human Colonic Carcinoma Cell Line.},
journal = {Viruses},
volume = {17},
number = {8},
pages = {},
doi = {10.3390/v17081043},
pmid = {40872758},
issn = {1999-4915},
support = {32072853//National Natural Science Foundation of China/ ; 2025SF-YBXM-506//Key Research and Development Project of Shaanxi Province/ ; },
mesh = {*Newcastle disease virus/genetics/physiology ; Humans ; *Oncolytic Viruses/genetics/physiology ; *Colonic Neoplasms/therapy/virology/genetics ; *Oncolytic Virotherapy/methods ; Genome, Viral ; HCT116 Cells ; CRISPR-Cas Systems ; Virus Replication ; Cell Line, Tumor ; },
abstract = {Viral oncolysis is considered a promising cancer treatment method because of its good tolerability and durable anti-tumor effects. Compared with other oncolytic viruses, Newcastle disease virus (NDV) has some distinct advantages. As an RNA virus, NDV does not recombine with the host genome, making it safer compared with DNA viruses and retroviruses; NDV can induce syncytium formation, allowing the virus to spread among cells without exposure to host neutralizing antibodies; and its genome adheres to the hexamer genetic code rule (genome length as a multiple of six nucleotides), ensuring accurate replication, low recombination rates, and high genetic stability. Although wild-type NDV has a killing effect on various tumor cells, its oncolytic effect and working mechanism are diverse, increasing the complexity of generating engineered oncolytic viruses with NDV. This study aims to employ whole-genome CRISPR-Cas9 knockout screening and RNA sequencing to identify putative key regulatory factors involved in the interaction between NDV and human colon cancer HCT116 cells and map their global interaction networks. The results suggests that NDV infection disrupts cellular homeostasis, thereby exerting oncolytic effects by inhibiting cell metabolism and proliferation. Meanwhile, the antiviral immune response triggered by NDV infection, along with the activation of anti-apoptotic signaling pathways, may be responsible for the limited oncolytic efficacy of NDV against HCT116 cells. These findings not only enhance our understanding of the oncolytic mechanism of NDV against colonic carcinoma but also provide potential strategies and targets for the development of NDV-based engineered oncolytic viruses.},
}

*Newcastle disease virus/genetics/physiology
Humans
*Oncolytic Viruses/genetics/physiology
*Colonic Neoplasms/therapy/virology/genetics
*Oncolytic Virotherapy/methods
Genome, Viral
HCT116 Cells
CRISPR-Cas Systems
Virus Replication
Cell Line, Tumor