@article {pmid40274667,
year = {2025},
author = {Mukherjee, A and Samanta, S and Das, S and Haque, MZ and Jana, PS and Samanta, I and Kar, I and Das, S and Nanda, PK and Thomas, P and Dandapat, P},
title = {Leveraging CRISPR-Cas-Enhanced Isothermal Amplification Tools for Quick Identification of Pathogens Causing Livestock Diseases.},
journal = {Current microbiology},
volume = {82},
number = {6},
pages = {260},
pmid = {40274667},
issn = {1432-0991},
support = {STBT-11012(27)/5/2024-ST SEC//Department of Science and Technology, Government of West Bengal/ ; },
mesh = {Animals ; *Nucleic Acid Amplification Techniques/methods ; *Livestock/microbiology/virology ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; *Animal Diseases/diagnosis/microbiology/virology ; *Bacteria/genetics/isolation & purification ; },
abstract = {Prompt and accurate diagnosis of infectious pathogens of livestock origin is of utmost importance for epidemiological surveillance and effective therapeutic strategy formulation. Among various methods, nucleic acid-based detection of pathogens is the most sensitive and specific; but the majority of these assays need expensive equipment and skilled workers. Due to the rapid advancement of clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas)-based nucleic acid detection methods, these are now being widely used for pathogen detection. CRISPR-Cas is a bacterial counterpart of "adaptive immunity", generally used for editing genome. Many CRISPR systems have been modified for nucleic acid detection due to their excellent selectivity in detecting DNA and RNA sequences. The combination of CRISPR with suitable isothermal amplification technologies has made it more sensitive, specific, versatile, and reproducible for the detection of pathogen nucleic acids at the point of care. Amplification of pathogen nucleic acid by isothermal amplification followed by CRISPR-Cas-based detection has several advantages, including short sample-to-answer times and no requirement for laboratory set-up. They are also significantly less expensive than the existing nucleic acid detection methods. This review focuses on the recent trends in the use of this precision diagnostic method for diagnosis of a wide range of animal pathogens with or without zoonotic potential, particularly various isothermal amplification strategies, and visualization methods for sensing bacteria, viruses, and parasites of veterinary and public health importance.},
}

Animals
*Nucleic Acid Amplification Techniques/methods
*Livestock/microbiology/virology
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
*Animal Diseases/diagnosis/microbiology/virology
*Bacteria/genetics/isolation & purification

@article {pmid40273916,
year = {2025},
author = {Shi, H and Al-Sayyad, N and Wasko, KM and Trinidad, MI and Doherty, EE and Vohra, K and Boger, RS and Colognori, D and Cofsky, JC and Skopintsev, P and Bryant, Z and Doudna, JA},
title = {Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2025.03.024},
pmid = {40273916},
issn = {1097-4164},
abstract = {RNA-guided CRISPR-Cas enzymes initiate programmable genome editing by recognizing a ∼20-base-pair DNA sequence next to a short protospacer-adjacent motif (PAM). To uncover the molecular determinants of high-efficiency editing, we conducted biochemical, biophysical, and cell-based assays on Streptococcus pyogenes Cas9 (SpyCas9) variants with wide-ranging genome-editing efficiencies that differ in PAM-binding specificity. Our results show that reduced PAM specificity causes persistent non-selective DNA binding and recurrent failures to engage the target sequence through stable guide RNA hybridization, leading to reduced genome-editing efficiency in cells. These findings reveal a fundamental trade-off between broad PAM recognition and genome-editing effectiveness. We propose that high-efficiency RNA-guided genome editing relies on an optimized two-step target capture process, where selective but low-affinity PAM binding precedes rapid DNA unwinding. This model provides a foundation for engineering more effective CRISPR-Cas and related RNA-guided genome editors.},
}

@article {pmid40273800,
year = {2025},
author = {Zheng, M and Bao, N and Wang, Z and Song, C and Jin, Y},
title = {Alternative splicing in autism spectrum disorder: Recent insights from mechanisms to therapy.},
journal = {Asian journal of psychiatry},
volume = {108},
number = {},
pages = {104501},
doi = {10.1016/j.ajp.2025.104501},
pmid = {40273800},
issn = {1876-2026},
abstract = {Alternative splicing (AS) is a vital and highly dynamic RNA regulatory mechanism that allows a single gene to generate multiple mRNA and protein isoforms. Dysregulation of AS has been identified as a key contributor to the pathogenesis of autism spectrum disorders (ASD). A comprehensive understanding of aberrant splicing mechanisms and their functional consequences in ASD can help uncover the molecular basis of the disorder and facilitate the development of therapeutic strategies. This review focuses on the major aberrant splicing events and key splicing regulators associated with ASD, highlighting their roles in linking defective splicing to ASD pathogenesis. In addition, a discussion of how emerging technologies, such as long-read sequencing, single-cell sequencing, spatial transcriptomics and CRISPR-Cas systems are offering novel insights into the role and mechanisms of AS in ASD is presented. Finally, the RNA splicing-based therapeutic strategies are evaluated, emphasizing their potential to address unmet clinical needs in ASD treatment.},
}

@article {pmid40273258,
year = {2025},
author = {Hayes, VM and Zhang, JT and Katz, MA and Li, Y and Kocsis, B and Brinkley, DM and Jia, N and Meeske, AJ},
title = {RNA-mediated CRISPR-Cas13 inhibition through crRNA structural mimicry.},
journal = {Science (New York, N.Y.)},
volume = {388},
number = {6745},
pages = {387-391},
doi = {10.1126/science.adr3656},
pmid = {40273258},
issn = {1095-9203},
mesh = {*CRISPR-Cas Systems ; Cryoelectron Microscopy ; *Molecular Mimicry ; *Listeria/virology/genetics ; *CRISPR-Associated Proteins/chemistry/antagonists & inhibitors ; Nucleic Acid Conformation ; *RNA, Viral/chemistry/genetics ; Bacteriophages/genetics ; Plasmids ; },
abstract = {To circumvent CRISPR-Cas immunity, phages express anti-CRISPR factors that inhibit the expression or activities of Cas proteins. Whereas most anti-CRISPRs described to date are proteins, recently described small RNAs called RNA anti-CRISPRs (rAcrs) have sequence homology to CRISPR RNAs (crRNAs) and displace them from cognate Cas nucleases. In this work, we report the discovery of rAcrVIA1-a plasmid-encoded small RNA that inhibits the RNA-targeting CRISPR-Cas13 system in its natural host, Listeria seeligeri. We solved the cryo-electron microscopy structure of the Cas13-rAcr complex, which revealed that rAcrVIA1 adopts a fold nearly identical to crRNA despite sharing negligible sequence similarity. Collectively, our findings expand the diversity of rAcrs and reveal an example of immune antagonism through RNA structural mimicry.},
}

*CRISPR-Cas Systems
Cryoelectron Microscopy
*Molecular Mimicry
*Listeria/virology/genetics
*CRISPR-Associated Proteins/chemistry/antagonists & inhibitors
Nucleic Acid Conformation
*RNA, Viral/chemistry/genetics
Bacteriophages/genetics
Plasmids

@article {pmid40273189,
year = {2025},
author = {Akiyama, C and Sakata, S and Ono, F},
title = {Normal locomotion in zebrafish lacking the sodium channel NaV1.4 suggests that the need for muscle action potentials is not universal.},
journal = {PLoS biology},
volume = {23},
number = {4},
pages = {e3003137},
pmid = {40273189},
issn = {1545-7885},
mesh = {Animals ; *Zebrafish/physiology/genetics ; *NAV1.4 Voltage-Gated Sodium Channel/genetics/metabolism/physiology ; *Action Potentials/physiology ; *Locomotion/physiology/genetics ; Muscle Contraction/physiology ; *Zebrafish Proteins/genetics/metabolism ; Muscle, Skeletal/physiology/metabolism ; Animals, Genetically Modified ; CRISPR-Cas Systems ; Tetrodotoxin/pharmacology ; Calcium/metabolism ; },
abstract = {Extensive studies over decades have firmly established the concept that action potentials (APs) in muscles are indispensable for muscle contraction. To re-examine the significance of APs, we generated zebrafish lacking APs by editing the scn4aa and scn4ab genes, which together encode NaV1.4 (NaVDKO), using the CRISPR-Cas9 system. Surprisingly, the escape response of NaVDKOs to tactile stimuli, both in the embryonic and adult stages, was indistinguishable from that of wild-type (WT) fish. Ca2+ imaging using the calcium indicator protein GCaMP revealed that myofibers isolated from WT fish could be excited by the application of acetylcholine (ACh), even in the presence of tetrodotoxin (TTX) indicating that NaVs are dispensable for skeletal muscle contraction in zebrafish. Mathematical simulations showed that the end-plate potential was able to elicit a change in membrane potential large enough to activate the dihydropyridine receptors of the entire muscle fiber owing to the small fiber size and the disseminated distribution of neuromuscular synapses in both adults and embryos. Our data demonstrate that NaVs are not essential for muscle contraction in zebrafish and that the physiological significance of NaV1.4 in muscle is not uniform across vertebrates.},
}

Animals
*Zebrafish/physiology/genetics
*NAV1.4 Voltage-Gated Sodium Channel/genetics/metabolism/physiology
*Action Potentials/physiology
*Locomotion/physiology/genetics
Muscle Contraction/physiology
*Zebrafish Proteins/genetics/metabolism
Muscle, Skeletal/physiology/metabolism
Animals, Genetically Modified
CRISPR-Cas Systems
Tetrodotoxin/pharmacology
Calcium/metabolism

@article {pmid40270959,
year = {2025},
author = {Caras, I and Ionescu, IE and Pantazica, AM and van Eerde, A and Steen, H and Heldal, I and Haugslien, S and Tucureanu, C and Chelmus, RE and Tofan, VC and Costache, A and Onu, A and Su, H and Branza-Nichita, N and Liu-Clarke, J and Stavaru, C},
title = {Humanized mouse model reveals the immunogenicity of Hepatitis B Virus vaccine candidates produced in CRISPR/Cas9-edited Nicotiana benthamiana.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1479689},
pmid = {40270959},
issn = {1664-3224},
mesh = {Animals ; *Nicotiana/genetics ; *Hepatitis B Vaccines/immunology/genetics ; Humans ; Mice ; *Hepatitis B virus/immunology ; *CRISPR-Cas Systems ; Disease Models, Animal ; *Hepatitis B/immunology/prevention & control ; Mice, Inbred NOD ; *Immunogenicity, Vaccine ; Hepatitis B Antibodies/immunology ; Antibodies, Neutralizing/immunology ; Hepatitis B Surface Antigens/immunology/genetics ; Female ; Gene Editing ; Leukocytes, Mononuclear/immunology ; Mice, Inbred BALB C ; },
abstract = {INTRODUCTION: Hepatitis B Virus (HBV) infection is still an ongoing public health issue worldwide. The most efficient tool in preventing HBV infection remains vaccination and significant efforts have been made in the last decade to improve current HBV vaccines. Owing to the strict HBV tropism for the human liver, developing animal models for preclinical screening of vaccine candidates is extremely challenging. To date, there are only a few reports regarding the use of humanized mouse models for the evaluation of the immunogenic properties of viral antigens.

METHODS: Previously we showed that a Nicotiana benthamiana-produced HBV-S/preS1[16-42] antigen elicited strong HBV-specific immune responses in BALB/c mice. In the current study, we used immunodeficient NOD.Cg-Prkdc[scid] Il2rg[tm1Wjl]/SzJ (NSG) mice as recipients of human peripheral blood mononuclear cells (hPBMCs), to evaluate the immunogenicity of the recently developed chimeric HBV immunogen produced in CRISPR/Cas9-edited N. benthamiana, under more "humanized" conditions.

RESULTS: Analysis of the immune response in NSG mice immunized with the chimeric antigen demonstrated induction of virus infection-neutralizing antibodies, indicating activation of antigen-specific B cells.

DISCUSSION: The ability of hPBMCs-engrafted NSG mice to mount specific humoral immune responses after immunization with viral antigens supports this animal model as a promising tool for pre-clinical evaluation of human vaccine antigens.},
}

Animals
*Nicotiana/genetics
*Hepatitis B Vaccines/immunology/genetics
Humans
Mice
*Hepatitis B virus/immunology
*CRISPR-Cas Systems
Disease Models, Animal
*Hepatitis B/immunology/prevention & control
Mice, Inbred NOD
*Immunogenicity, Vaccine
Hepatitis B Antibodies/immunology
Antibodies, Neutralizing/immunology
Hepatitis B Surface Antigens/immunology/genetics
Female
Gene Editing
Leukocytes, Mononuclear/immunology
Mice, Inbred BALB C

@article {pmid40270035,
year = {2025},
author = {Lin, XL and Zhou, YM and Meng, K and Yang, JY and Zhang, H and Lin, JH and Wu, HY and Wang, XY and Zhao, H and Feng, SS and Park, KS and Cai, DQ and Zheng, L and Qi, XF},
title = {CRISPR/Cas-mediated mRNA knockdown in the embryos of Xenopus tropicalis.},
journal = {Cell & bioscience},
volume = {15},
number = {1},
pages = {52},
pmid = {40270035},
issn = {2045-3701},
support = {21623110//Fundamental Research Funds for Central Universities of the Jinan University/ ; ZSYXM202303//Key Laboratory of Regenerative Medicine of Ministry of Education, Jinan University/ ; ZSYXM202402//Key Laboratory of Regenerative Medicine of Ministry of Education, Jinan University/ ; 82370247, 82070257, 81770240//National Natural Science Foundation of China/ ; 2023A1515012147//Basic and Applied Basic Research Foundation of Guangdong Province/ ; },
abstract = {The Xenopus tropicalis (Western clawed frog) is an important amphibian model for genetics, developmental and regenerative biology, due to its diploid genetic background and short generation time. CRISPR-Cas13 and CRISPR interference (CRISPRi) systems have recently been employed to suppress mRNA expression in many organisms such as yeast, plants, and mammalian cells. However, no systematic study of these two systems has been carried out in Xenopus tropicalis. Here, we show that CRISPRi rather than CRISPR-Cas13 is an effective and suitable approach to suppress specific mRNA transcription in Xenopus tropicalis embryos. We demonstrated that CRISPRi composed of dCas9 and KRAB-MeCP2 (dCas9-KM) can efficiently target exogenous and endogenous transcripts in Xenopus tropicalis embryos. Moreover, our data suggest that the new KRAB domain from ZIM3 protein (ZIM3-KRAB, ZIM3K) alone has a comparable transcript targeting capacity in Xenopus tropicalis embryos to the traditional fusion repressor KRAB-MeCP2 in which the KRAB domain from KOX1 protein. In conclusion, our results demonstrate that CRISPRi rather than CRISPR-Cas13 is an efficient knockdown platform to explore specific gene function in Xenopus tropicalis embryos.},
}

@article {pmid40269480,
year = {2025},
author = {Chen, Z and Pilehvar, E and Sadeghi, H and Pilehvar, Y},
title = {Precision Reimagined: CRISPR and Multiomics Transform Systemic Lupus Erythematosus Diagnosis and Therapy.},
journal = {International journal of rheumatic diseases},
volume = {28},
number = {4},
pages = {e70189},
doi = {10.1111/1756-185X.70189},
pmid = {40269480},
issn = {1756-185X},
mesh = {Humans ; *Lupus Erythematosus, Systemic/genetics/diagnosis/therapy/immunology ; *Precision Medicine/methods ; *Gene Editing/methods ; *Genomics/methods ; Artificial Intelligence ; *CRISPR-Cas Systems ; Predictive Value of Tests ; *Proteomics/methods ; Phenotype ; Genetic Predisposition to Disease ; *Genetic Therapy/methods ; Multiomics ; },
abstract = {Systemic lupus erythematosus (SLE) is a complex autoimmune disorder with diverse clinical manifestations and unpredictable progression, posing significant challenges to accurate diagnosis and effective treatment. Traditional biomarkers and treatments often fail to address the disease's molecular and clinical heterogeneity. Recent advancements in CRISPR gene-editing technology and multiomics approaches offer transformative opportunities for personalized SLE care by unraveling its underlying molecular complexity and enabling precise therapeutic interventions. CRISPR technology allows targeted editing of SLE-associated genetic mutations, addressing disease drivers directly, while multiomics-including genomics, transcriptomics, and proteomics-provides insights into dysregulated immune networks, identifying biomarkers and therapeutic targets. Integrating these approaches can refine patient stratification and enhance the precision of treatments. Artificial intelligence (AI) complements these technologies by synthesizing high-dimensional data, enabling personalized treatment plans, predicting disease trajectories, and optimizing therapeutic strategies. However, the integration of CRISPR and multiomics in clinical settings raises challenges, including technical limitations, ethical concerns, and economic barriers. Emerging clinical trials and case studies demonstrate the potential of these innovations to personalize care and improve outcomes. Nonetheless, the transition from experimental research to routine clinical application requires robust regulatory frameworks and strategies to address these challenges. This review aims to explore the potential of CRISPR and multiomics technologies to revolutionize SLE diagnosis and therapy, emphasizing their integration with AI to advance personalized care. By addressing existing barriers, the review envisions a future where precision medicine transforms SLE management, paving the way for individualized, patient-centered autoimmune therapy.},
}

Humans
*Lupus Erythematosus, Systemic/genetics/diagnosis/therapy/immunology
*Precision Medicine/methods
*Gene Editing/methods
*Genomics/methods
Artificial Intelligence
*CRISPR-Cas Systems
Predictive Value of Tests
*Proteomics/methods
Phenotype
Genetic Predisposition to Disease
*Genetic Therapy/methods
Multiomics

@article {pmid40268754,
year = {2025},
author = {Liu, Y and Yao, F and Zou, J and Guo, D and Jiang, W and Fan, J and Li, R and Yang, Z and Ma, Y and Deng, H and Huang, J and Tan, L},
title = {RPAD locus controls prostrate growth habit in Oryza nivara.},
journal = {The plant genome},
volume = {18},
number = {2},
pages = {e70032},
pmid = {40268754},
issn = {1940-3372},
support = {2023YFF1000401//National Key Research and Development Program of China/ ; 32125029//National Natural Science Foundation of China/ ; PC2023A01003//Pinduoduo-China Agricultural University Research Fund/ ; },
mesh = {*Oryza/genetics/growth & development ; *Quantitative Trait Loci ; Domestication ; CRISPR-Cas Systems ; Chromosomes, Plant/genetics ; Transcription Factors/genetics ; Chromosome Mapping ; Plant Breeding ; Plant Proteins/genetics ; },
abstract = {The development of ideal plant architecture is crucial for optimizing grain yield in crop breeding. The transition from prostrate growth habit in wild rice to erect growth habit in cultivated rice is one of the important events during rice domestication. Here, we identified a yield-related quantitative trait locus (QTL) cluster on the short arm of chromosome 7 using Teqing/W2014 (Oryza nivara) derived BC3F6 population. The introgression line TIL81 containing this QTL cluster exhibited significantly larger tiller angle, increased tiller numbers, and prostrate growth habit compared to the recipient parent Teqing. Using a segregating F2 population derived from a cross between TIL81 and Teqing, this yield-related QTL cluster was mapped to a similar position as the known rice plant architecture domestication (RPAD) locus controlling rice plant architecture domestication. CRISPR/Cas9-mediated genome (where CRISPR is clustered regularly interspaced short palindromic repeats) editing of four zinc finger transcription factors (OnZnF1, OnZnF6, OnZnF8, and OnZnF9) within the RPAD locus demonstrated their collective involvement in regulating plant architecture and yield-related traits. Notably, the knockout lines harboring all four zinc finger gene mutations exhibited plant architecture traits and grain yield per plant comparable to the control Teqing. These findings demonstrated that RPAD locus in O. nivara functions in prostrate growth habit and provided new insights into the molecular mechanism of plant architecture during rice domestication.},
}

*Oryza/genetics/growth & development
*Quantitative Trait Loci
Domestication
CRISPR-Cas Systems
Chromosomes, Plant/genetics
Transcription Factors/genetics
Chromosome Mapping
Plant Breeding
Plant Proteins/genetics

@article {pmid40268745,
year = {2025},
author = {Zhang, X and Ma, D and Liu, F},
title = {CRISPR Technology and Its Emerging Applications.},
journal = {Genomics, proteomics & bioinformatics},
volume = {},
number = {},
pages = {},
doi = {10.1093/gpbjnl/qzaf034},
pmid = {40268745},
issn = {2210-3244},
abstract = {The discovery and iteration of clustered regularly interspaced short palindromic repeats (CRISPR) systems have revolutionized genome editing due to their remarkable efficiency and easy programmability, enabling precise manipulation of genomic elements. Owing to these unique advantages, CRISPR technology has the transformative potential to elucidate biological mechanisms and clinical treatments. This review provides a comprehensive overview of the development and applications of CRISPR technology. After describing the three primary CRISPR-Cas systems-CRISPR-associated protein 9 (Cas9) and Cas12a targeting DNA, and Cas13 targeting RNA-which serve as the cornerstone for technological advancements, we describe a series of novel CRISPR-Cas systems that offer new avenues for research, and then explore the applications of CRISPR technology in large-scale genetic screening, lineage tracing, genetic diagnosis, and gene therapy. As this technology evolves, it holds significant promise for studying gene functions and treating human diseases in the near future.},
}

@article {pmid40268248,
year = {2025},
author = {Biber, J and Gandor, C and Becirovic, E and Michalakis, S},
title = {Retina-directed gene therapy: Achievements and remaining challenges.},
journal = {Pharmacology & therapeutics},
volume = {},
number = {},
pages = {108862},
doi = {10.1016/j.pharmthera.2025.108862},
pmid = {40268248},
issn = {1879-016X},
abstract = {Gene therapy is an innovative medical approach that offers new treatment options for congenital and acquired diseases by transferring, correcting, inactivating or regulating genes to supplement, replace or modify a gene function. The approval of voretigene neparvovec (Luxturna), a gene therapy for RPE65-associated retinopathy, has marked a milestone for the field of retinal gene therapy, but has also helped to accelerate the development of gene therapies for genetic diseases affecting other organs. Voretigene neparvovec is a vector based on adeno-associated virus (AAV) that delivers a functional copy of RPE65 to supplement the missing function of this gene. The AAV-based gene delivery has proven to be versatile and safe for the transfer of genetic material to retinal cells. However, challenges remain in treating additional inherited as well as acquired retinopathies with this technology. Despite the high level of activity in this field, no other AAV gene therapy for retinal diseases has been approved since voretigene neparvovec. Ongoing research focuses on overcoming the current restraints through innovative strategies like AAV capsid engineering, dual-AAV vector systems, or CRISPR/Cas-mediated genome editing. Additionally, AAV gene therapy is being explored for the treatment of complex acquired diseases like age-related macular degeneration (AMD) and diabetic retinopathy (DR) by targeting molecules involved in the pathobiology of the degenerative processes. This review outlines the current state of retinal gene therapy, highlighting ongoing challenges and future directions.},
}

@article {pmid40146774,
year = {2025},
author = {Fidelito, G and Todorovski, I and Cluse, L and Vervoort, SJ and Taylor, RA and Watt, MJ},
title = {Lipid-metabolism-focused CRISPR screens identify enzymes of the mevalonate pathway as essential for prostate cancer growth.},
journal = {Cell reports},
volume = {44},
number = {4},
pages = {115470},
doi = {10.1016/j.celrep.2025.115470},
pmid = {40146774},
issn = {2211-1247},
mesh = {Male ; *Mevalonic Acid/metabolism ; *Prostatic Neoplasms/pathology/genetics/metabolism/enzymology ; Humans ; Cell Line, Tumor ; *Lipid Metabolism/genetics ; Animals ; *CRISPR-Cas Systems/genetics ; Cell Proliferation ; Mice ; *Dimethylallyltranstransferase/metabolism/genetics ; Endoplasmic Reticulum Stress ; },
abstract = {Dysregulated lipid metabolism plays an important role in prostate cancer, although the understanding of the essential regulatory processes in tumorigenesis is incomplete. We employ a CRISPR-Cas9 screen using a custom human lipid metabolism knockout library to identify essential genes for prostate cancer survival. Screening in three prostate cancer cell lines reveals 63 shared dependencies, with enrichment in terpenoid backbone synthesis and N-glycan biosynthesis. Independent knockout of key genes of the mevalonate pathway reduces cell proliferation. Further investigation focuses on NUS1, a subunit of cis-prenyltransferase required for dolichol synthesis. NUS1 knockout decreases tumor growth in vivo and viability in patient-derived xenograft (PDX)-derived organoids. Mechanistic studies reveal that loss of NUS1 promotes oxidative stress, lipid peroxidation and ferroptosis sensitivity, endoplasmic reticulum (ER) stress, and G1 cell-cycle arrest, and it dampens androgen receptor (AR) signaling, collectively leading to growth arrest. This study highlights the critical role of the mevalonate-dolichol-N-glycan biosynthesis pathway, particularly NUS1, in prostate cancer survival and growth.},
}

Male
*Mevalonic Acid/metabolism
*Prostatic Neoplasms/pathology/genetics/metabolism/enzymology
Humans
Cell Line, Tumor
*Lipid Metabolism/genetics
Animals
*CRISPR-Cas Systems/genetics
Cell Proliferation
Mice
*Dimethylallyltranstransferase/metabolism/genetics
Endoplasmic Reticulum Stress

@article {pmid40267200,
year = {2025},
author = {Brumage, L and Best, S and Hippe, DS and Grunblatt, E and Chanana, P and Wu, F and Lee, MC and Ying, Z and Ibrahim, A and Chung, JH and Vigil, A and Fatherree, J and Beronja, S and Paddison, P and Sullivan, L and Nabet, B and MacPherson, D},
title = {In vivo functional screens reveal KEAP1 loss as a driver of chemoresistance in small cell lung cancer.},
journal = {Science advances},
volume = {11},
number = {17},
pages = {eadq7084},
doi = {10.1126/sciadv.adq7084},
pmid = {40267200},
issn = {2375-2548},
mesh = {*Small Cell Lung Carcinoma/drug therapy/genetics ; Humans ; *Drug Resistance, Neoplasm ; *Kelch-Like ECH-Associated Protein 1/genetics ; DNA, Complementary/analysis ; Proto-Oncogene Proteins c-myc/metabolism ; Animals ; Mice ; CRISPR-Cas Systems ; Signal Transduction ; *Lung Neoplasms/drug therapy/genetics ; Male ; Female ; Immunologic Deficiency Syndromes ; HEK293 Cells ; },
abstract = {Exquisitely chemosensitive initially, small cell lung cancer (SCLC) exhibits dismal outcomes owing to rapid transition to chemoresistance. Elucidating the genetic underpinnings has been challenging owing to limitations with cellular models. As SCLC patient-derived xenograft (PDX) models mimic therapeutic responses, we perform genetic screens in chemosensitive PDX models to identify drivers of chemoresistance. cDNA overexpression screens identify MYC, MYCN, and MYCL, while CRISPR deletion screens identify KEAP1 loss as driving chemoresistance. Deletion of KEAP1 switched a chemosensitive SCLC PDX model to become chemoresistant and resulted in sensitivity to inhibition of glutamine metabolism. Data from the IMpower133 clinical trial revealed ~6% of patients with extensive-stage SCLC exhibit KEAP1 genetic alterations, with activation of a KEAP1/NRF2 transcriptional signature associated with reduced survival upon chemotherapy treatment. While roles for KEAP1/NRF2 have been unappreciated in SCLC, our genetic screens revealed KEAP1 loss as a driver of chemoresistance, while patient genomic analyses demonstrate clinical importance.},
}

*Small Cell Lung Carcinoma/drug therapy/genetics
Humans
*Drug Resistance, Neoplasm
*Kelch-Like ECH-Associated Protein 1/genetics
DNA, Complementary/analysis
Proto-Oncogene Proteins c-myc/metabolism
Animals
Mice
CRISPR-Cas Systems
Signal Transduction
*Lung Neoplasms/drug therapy/genetics
Male
Female
Immunologic Deficiency Syndromes
HEK293 Cells

@article {pmid40266686,
year = {2025},
author = {Zeng, Y and Tan, X and Xiao, P and Gao, P and Wang, L and Zhang, A},
title = {Natronobacterium gregoryi Argonaute inhibits class 1 integron integrase-mediated excision and integration.},
journal = {Nucleic acids research},
volume = {53},
number = {8},
pages = {},
doi = {10.1093/nar/gkaf248},
pmid = {40266686},
issn = {1362-4962},
support = {2021YFD1800402//National Key Research and Development Program of China/ ; 32172848//National Natural Science Foundation of China/ ; },
mesh = {*Argonaute Proteins/metabolism/genetics/chemistry ; *Integrases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics/chemistry ; Protein Binding ; *Integrons/genetics ; },
abstract = {Argonaute (Ago) proteins, ubiquitous in all domains of life, serve as key components in defense against foreign nucleic acids. While eukaryotic Agos (eAgos) are well characterized for guide RNA-mediated RNA targeting, prokaryotic Agos (pAgos) exhibit diverse functions, particularly in protecting bacteria from invasive DNA. The previous study identified Class 1 integron integrase (IntI-1), a tyrosine site-specific recombinase involved in horizontal transfer of antibiotic resistance genes, as a potential interaction partner of Natronobacterium gregoryi Argonaute (NgAgo), a member of pAgos. Here, we demonstrated that this interaction was direct, depended on the PIWI domain, and was independent of the catalytic activity of NgAgo. Notably, no interaction occurred between NgAgo and Cre (another tyrosine site-specific recombinase), highlighting the specificity of NgAgo-IntI-1 interaction. Furthermore, NgAgo could inhibit binding of IntI-1 to its target DNA, and then impede IntI-1-mediated integration and excision. Consistent with the above finding, few pAgos could be found in prokaryotic genomes containing IntI, whereas IntI showed significant co-occurrence with another bacterial defense system, CRISPR-Cas. In summary, our study elucidated a novel defense mechanism of pAgos through interaction with IntI-1 for inhibiting IntI-1-mediated gene excision/integration process.},
}

*Argonaute Proteins/metabolism/genetics/chemistry
*Integrases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics/chemistry
Protein Binding
*Integrons/genetics

@article {pmid40265805,
year = {2025},
author = {Han, X and Deng, Z and Liu, H and Ji, X},
title = {Current Advancement and Future Prospects in Simplified Transformation-Based Plant Genome Editing.},
journal = {Plants (Basel, Switzerland)},
volume = {14},
number = {6},
pages = {},
doi = {10.3390/plants14060889},
pmid = {40265805},
issn = {2223-7747},
support = {32370432//National Natural Science Foundation of China/ ; },
abstract = {Recent years have witnessed remarkable progress in plant biology, driven largely by the rapid evolution of CRISPR/Cas-based genome editing (GE) technologies. These tools, including versatile CRISPR/Cas systems and their derivatives, such as base editors and prime editors, have significantly enhanced the universality, efficiency, and convenience of plant functional genomics, genetics, and molecular breeding. However, traditional genetic transformation methods are essential for obtaining GE plants. These methods depend on tissue culture procedures, which are time-consuming, labor-intensive, genotype-dependent, and challenging to regenerate. Here, we systematically outline current advancements in simplifying plant GE, focusing on the optimization of tissue culture process through developmental regulators, the development of in planta transformation methods, and the establishment of nanomaterial- and viral vector-based delivery platforms. We also discuss critical challenges and future directions for achieving genotype-independent, tissue culture-free plant GE.},
}

@article {pmid40264801,
year = {2025},
author = {Zueva, AS and Shevchenko, AI and Medvedev, SP and Elisaphenko, EA and Sleptcov, AA and Nazarenko, MS and Tmoyan, NA and Zakian, SM and Zakharova, IS},
title = {Isogenic induced pluripotent stem cell line ICGi036-A-1 from a patient with familial hypercholesterolaemia, derived by correcting a pathogenic variant of the gene LDLR c.530C>T.},
journal = {Vavilovskii zhurnal genetiki i selektsii},
volume = {29},
number = {2},
pages = {189-199},
doi = {10.18699/vjgb-25-22},
pmid = {40264801},
issn = {2500-0462},
abstract = {Familial hypercholesterolaemia is a common monogenic disorder characterized by high plasma cholesterol levels leading to chronic cardiovascular disease with high risk and often early manifestation due to atherosclerotic lesions of the blood vessels. The atherosclerotic lesions in familial hypercholesterolaemia are mainly caused by pathogenic variants of the low-density lipoprotein receptor (LDLR) gene, which plays an important role in cholesterol metabolism. Normally, cholesterol-laden low-density lipoproteins bind to the LDLR receptor on the surface of liver cells to be removed from the bloodstream by internalisation with hepatocytes. In familial hypercholesterolaemia, the function of the receptor is impaired and the uptake of low-density lipoproteins is significantly reduced. As a result, cholesterol accumulates in the subendothelial space on the inner wall of blood vessels, triggering atherogenesis, the formation of atherosclerotic plaques. At present, there are no effective and universal approaches to the diagnosis and treatment of familial hypercholesterolaemia. A relevant approach to study the molecular genetic mechanisms of the disease and to obtain systems for screening chemical compounds as potential drugs is the generation of cellular models based on patient-specific induced pluripotent stem cells. The aim of our work was to derive an isogenic genetically modified induced pluripotent stem cell line by correcting the pathogenic allelic variant c.530C of the LDLR gene in the original iPSC previously obtained from a compound heterozygote patient with familial hypercholesterolaemia. The resulting isogenic iPSC line differs from the original by only one corrected nucleotide substitution, allowing us to study the direct effect of this pathogenic genetic variant on physiological changes in relevant differentiated cells. CRISPR/Cas-mediated base editing was used to correct the single nucleotide substitution. The resulting genetically modified iPSC line has pluripotency traits, a normal karyotype, a set of short tandem repeats identical to that in the original line and can be used to obtain differentiated derivatives necessary for the elaboration of relevant cell models.},
}

@article {pmid40264105,
year = {2025},
author = {Yahata, T and Toujima, S and Sasaki, I and Iwahashi, N and Fujino, M and Nishioka, K and Noguchi, T and Tanizaki-Horiuchi, Y and Kaisho, T and Ino, K},
title = {Adeno-associated virus-clustered regularly interspaced short palindromic repeats/cas9‑mediated ovarian cancer treatment targeting PD-L1.},
journal = {BMC cancer},
volume = {25},
number = {1},
pages = {749},
pmid = {40264105},
issn = {1471-2407},
mesh = {Female ; Animals ; *B7-H1 Antigen/genetics/antagonists & inhibitors/metabolism ; *Ovarian Neoplasms/therapy/genetics/immunology/pathology ; *Dependovirus/genetics ; Mice ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; Humans ; Cell Line, Tumor ; Genetic Vectors/genetics ; Immunotherapy/methods ; },
abstract = {The response rate of antibody therapy targeting immune checkpoint molecules in ovarian cancer is insufficient. This study aimed to develop a novel gene immunotherapy model targeting programmed death ligand 1 (PD-L1) in vivo in ovarian cancer using adeno-associated virus (AAV)-clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and investigate its efficacy. In vitro, we produced PD-L1-AAV particles to knock out PD-L1. PD-L1-AAV particles were transduced into the murine ovarian cancer cell line ID8. PD-L1 expression at the cellular level was significantly decreased following treatment with PD-L1-AAV particles compared with control-AAV particles. In the peritoneal dissemination model, the survival time was significantly longer in the PD-L1-AAV particles intraperitoneally injected group than that in the control group. Furthermore, intratumoral lymphocyte recruitment was analyzed by immunohistochemistry, and the number of intratumoral CD4[+] and CD8[+] T cells was significantly higher, whereas that of Foxp3[+] Treg cells was significantly lower in the PD-L1-AAV particles injected group than in the control group. No severe adverse events in normal organs, such as the lungs, spleen, liver, and kidney, were observed. These results suggest that PD-L1-targeted therapy by genome editing using AAV-CRISPR/Cas9 is a novel gene-immune therapeutic strategy for ovarian cancer.},
}

Female
Animals
*B7-H1 Antigen/genetics/antagonists & inhibitors/metabolism
*Ovarian Neoplasms/therapy/genetics/immunology/pathology
*Dependovirus/genetics
Mice
*CRISPR-Cas Systems
*Genetic Therapy/methods
Humans
Cell Line, Tumor
Genetic Vectors/genetics
Immunotherapy/methods

@article {pmid40264050,
year = {2025},
author = {Pang, KL and Li, P and Yao, XR and Xiao, WT and Ren, X and He, JY},
title = {Deciphering a proliferation-essential gene signature based on CRISPR-Cas9 screening to predict prognosis and characterize the immune microenvironment in HNSCC.},
journal = {BMC cancer},
volume = {25},
number = {1},
pages = {756},
pmid = {40264050},
issn = {1471-2407},
support = {82304083//National Natural Science Foundation of China/ ; 2023JJ40584//Natural Science Foundation of Hunan Province/ ; },
mesh = {Humans ; Prognosis ; *Squamous Cell Carcinoma of Head and Neck/genetics/immunology/pathology/mortality ; *Tumor Microenvironment/immunology/genetics ; *CRISPR-Cas Systems ; *Head and Neck Neoplasms/genetics/immunology/pathology/mortality ; Cell Proliferation/genetics ; *Biomarkers, Tumor/genetics ; Gene Expression Regulation, Neoplastic ; Female ; *Genes, Essential ; Gene Expression Profiling ; Male ; Gene Regulatory Networks ; },
abstract = {BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is a highly aggressive malignancy with a poor prognosis. Identifying reliable prognostic biomarkers and therapeutic targets is crucial for improving patient outcomes. This study aimed to systematically identify proliferation-essential genes (PEGs) associated with HNSCC prognosis using CRISPR-Cas9 screening data.

METHODS: CRISPR-Cas9 screening data from the DepMap database were used to identify PEGs in HNSCC cells. A prognostic PEGs signature was constructed using univariate Cox regression, least absolute shrinkage and selection operator (LASSO) Cox regression, and multivariate Cox regression analyses. The predictive accuracy of the signature was validated in internal and external datasets. Weighted gene co-expression network analysis (WGCNA), gene set enrichment analysis (GSEA), and immune infiltration analysis were used to investigate the underlying mechanism between high and low-risk patients. Random forest analysis and functional experiments were conducted to investigate the role of key proliferation essential genes in HNSCC progression.

RESULTS: A total of 1511 PEGs were identified. A seven-gene prognostic PEGs signature (MRPL33, NAT10, PSMC1, PSMD11, RPN2, TAF7, and ZNF335) was developed and validated, demonstrating robust prognostic performance in stratifying HNSCC patients by survival risk. WGCNA and GSEA analyses revealed a marked downregulation of immune-related pathways in high-risk patients. Immune infiltration analysis validated those high-risk patients had reduced immune scores, stromal scores, and ESTIMATE scores, as well as decreased infiltration of multiple immune cell types. Among the identified genes, PSMC1 was highlighted as a pivotal regulator of HNSCC proliferation and migration, as confirmed by functional experiments.

CONCLUSIONS: This study identifies a novel PEGs signature that effectively predicts HNSCC prognosis and stratifies patients by survival risk. PSMC1 was identified as a key gene promoting malignant progression, offering potential as a therapeutic target for HNSCC.},
}

Humans
Prognosis
*Squamous Cell Carcinoma of Head and Neck/genetics/immunology/pathology/mortality
*Tumor Microenvironment/immunology/genetics
*CRISPR-Cas Systems
*Head and Neck Neoplasms/genetics/immunology/pathology/mortality
Cell Proliferation/genetics
*Biomarkers, Tumor/genetics
Gene Expression Regulation, Neoplastic
Female
*Genes, Essential
Gene Expression Profiling
Male
Gene Regulatory Networks

@article {pmid40263874,
year = {2025},
author = {Huang, X and Li, R and Xu, J and Kang, J and Chen, X and Han, B and Xue, Y},
title = {Integrated multi-omics uncover viruses, active fermenting microbes and their metabolic profiles in the Daqu microbiome.},
journal = {Food research international (Ottawa, Ont.)},
volume = {208},
number = {},
pages = {116061},
doi = {10.1016/j.foodres.2025.116061},
pmid = {40263874},
issn = {1873-7145},
mesh = {*Fermentation ; *Microbiota ; Fungi/metabolism/genetics ; *Bacteria/metabolism/genetics ; *Viruses/genetics/classification ; Metagenomics ; Food Microbiology ; *Fermented Foods/microbiology/virology ; Bacteriophages/genetics ; *Metabolome ; Multiomics ; },
abstract = {The coexistence and coevolution of viruses and fermenting microbes have a significant impact on the structure and function of microbial communities. Although the presence of viruses in Daqu, the fermentation starter for Chinese Baijiu, has been documented, their specific effects on the community composition and metabolic functions of low, medium, and high-temperature Daqu remain unclear. In this study, we employed multi-omics technology to explore the distribution of viruses and active bacteria and fungi in various Daqu and their potential metabolic roles. Viral metagenomic sequencing showed a predominance of Parvoviridae in High-Temperature Daqu (HTQ), while Genomoviridae were dominant in Medium-Temperature Daqu (MTQ) and Low- Temperature Daqu (LTQ). Phages belonging to the Siphoviridae, Podoviridae, Herelleviridae, and Myoviridae families showed significantly different abundances across three Daqu groups. Metatranscriptomic analysis showed that fungal communities were most active in LTQ, whereas bacterial communities were dominant in MTQ and HTQ. By employing the CRISPR-Cas spacer, a higher predicted number of phage-host linkages was identified in LTQ, particularly with hosts including Lactobacillus, Staphylococcus, Acinetobacter, Enterobacter, and Bacillus. Correlation analysis showed that bacteria like Acinetobacter, Lactobacillus, and Streptococcus exhibited the strongest associations with metabolites, particularly amino acids and organic acids. The potential phage-induced metabolic differences in the three Daqu groups were mainly linked to pathways involved in the metabolism of amino acids, sugars, and organic acids. Overall, our study elucidates the impact of viruses on shaping microbial composition and influencing metabolic functions in Daqu. These results improve our comprehension of viruses and microbes in Daqu microbial communities and provide valuable insights for enhancing quality control in Daqu production.},
}

*Fermentation
*Microbiota
Fungi/metabolism/genetics
*Bacteria/metabolism/genetics
*Viruses/genetics/classification
Metagenomics
Food Microbiology
*Fermented Foods/microbiology/virology
Bacteriophages/genetics
*Metabolome
Multiomics

@article {pmid40263707,
year = {2025},
author = {Zhu, Z and Li, X and Ding, L and Wu, T},
title = {Exploring the effect of activator topology on CRISPR-Cas12a trans-cleavage activity.},
journal = {Nucleic acids research},
volume = {53},
number = {8},
pages = {},
doi = {10.1093/nar/gkaf311},
pmid = {40263707},
issn = {1362-4962},
support = {22474045//National Natural Science Foundation of China/ ; //Fundamental Research Funds for the Central Universities/ ; 2024JYCXJJ010//Huazhong University of Science and Technology/ ; },
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; *Bacterial Proteins/metabolism/chemistry/genetics ; },
abstract = {The CRISPR-Cas12a system is widely used in nucleic acid detection and biosensing due to its high sensitivity, selectivity, and simple design. However, traditional CRISPR-Cas12a sensors, which rely on linear activators, face challenges such as limited operability and low stability. This study explored the impact of three different activator topologies-linear, planar, and steric-on the trans-cleavage activity of Cas12a. We developed a Cas12a-based switch using a planar activator, which demonstrated superior operability and maintained higher activity compared to linear activators. Using this planar activator, we achieved highly sensitive detection of hypochlorous acid, with a detection limit as low as 88 nM, outperforming chemical probe-based methods. The introduction of topological activators will open new avenues for the development of CRISPR-Cas12a-based biosensors, offering broad potential for diverse applications.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism/chemistry/genetics
*Biosensing Techniques/methods
*Endodeoxyribonucleases/metabolism/chemistry/genetics
*Bacterial Proteins/metabolism/chemistry/genetics

@article {pmid40263616,
year = {2025},
author = {Wood, TWP and Henriques, WS and Cullen, HB and Romero, M and Blengini, CS and Sarathy, S and Sorkin, J and Bekele, H and Jin, C and Kim, S and Wang, X and Laureau, R and Chemiakine, A and Khondker, RC and Isola, JVV and Stout, MB and Gennarino, VA and Mogessie, B and Jain, D and Schindler, K and Suh, Y and Wiedenheft, B and Berchowitz, LE},
title = {The retrotransposon-derived capsid genes PNMA1 and PNMA4 maintain reproductive capacity.},
journal = {Nature aging},
volume = {},
number = {},
pages = {},
pmid = {40263616},
issn = {2662-8465},
support = {R35 GM124633/GM/NIGMS NIH HHS/United States ; R35 GM124633/GM/NIGMS NIH HHS/United States ; R35 GM134867/GM/NIGMS NIH HHS/United States ; R35 GM124633/GM/NIGMS NIH HHS/United States ; R35 GM136340/GM/NIGMS NIH HHS/United States ; R35 GM136340/GM/NIGMS NIH HHS/United States ; R35 GM147130/GM/NIGMS NIH HHS/United States ; R35 GM147130/GM/NIGMS NIH HHS/United States ; R35 GM146725/GM/NIGMS NIH HHS/United States ; R35 GM124633/GM/NIGMS NIH HHS/United States ; R35 GM146725/GM/NIGMS NIH HHS/United States ; R35 GM147130/GM/NIGMS NIH HHS/United States ; R35 GM136340/GM/NIGMS NIH HHS/United States ; R35 GM134867/GM/NIGMS NIH HHS/United States ; R35 GM124633/GM/NIGMS NIH HHS/United States ; NA//Irma T. Hirschl Trust (Irma T. Hirschl Charitable Trust)/ ; R01 AG069750/AG/NIA NIH HHS/United States ; R01 AG069750/AG/NIA NIH HHS/United States ; R01 AG069742/AG/NIA NIH HHS/United States ; R01 AG069742/AG/NIA NIH HHS/United States ; R01 AG069750/AG/NIA NIH HHS/United States ; R01 AG069750/AG/NIA NIH HHS/United States ; R01 NS109858/NS/NINDS NIH HHS/United States ; R01 NS109858/NS/NINDS NIH HHS/United States ; 213470/A/18/Z//Wellcome Trust (Wellcome)/ ; NA//M.J. Murdock Charitable Trust (Murdock Charitable Trust)/ ; },
abstract = {Almost half of the human genome consists of retrotransposons-'parasitic' sequences that insert themselves into the host genome via an RNA intermediate. Although most of these sequences are silenced or mutationally deactivated, they can present opportunities for evolutionary innovation: mutation of a deteriorating retrotransposon can result in a gene that provides a selective advantage to the host in a process termed 'domestication'[1-3]. The PNMA family of gag-like capsid genes was domesticated from an ancient vertebrate retrotransposon of the Metaviridae clade at least 100 million years ago[4,5]. PNMA1 and PNMA4 are positively regulated by the master germ cell transcription factors MYBL1 and STRA8, and their transcripts are bound by the translational regulator DAZL during gametogenesis[6]. This developmental regulation of PNMA1 and PNMA4 expression in gonadal tissue suggested to us that they might serve a reproductive function. Through the analysis of donated human ovaries, genome-wide association studies (GWASs) and mouse models, we found that PNMA1 and PNMA4 are necessary for the maintenance of a normal reproductive lifespan. These proteins self-assemble into capsid-like structures that exit human cells, and we observed large PNMA4 particles in mouse male gonadal tissue that contain RNA and are consistent with capsid formation.},
}

@article {pmid40263581,
year = {2025},
author = {Weiss, T and Kamalu, M and Shi, H and Li, Z and Amerasekera, J and Zhong, Z and Adler, BA and Song, MM and Vohra, K and Wirnowski, G and Chitkara, S and Ambrose, C and Steinmetz, N and Sridharan, A and Sahagun, D and Banfield, JF and Doudna, JA and Jacobsen, SE},
title = {Viral delivery of an RNA-guided genome editor for transgene-free germline editing in Arabidopsis.},
journal = {Nature plants},
volume = {},
number = {},
pages = {},
pmid = {40263581},
issn = {2055-0278},
support = {2334027//National Science Foundation (NSF)/ ; },
abstract = {Genome editing is transforming plant biology by enabling precise DNA modifications. However, delivery of editing systems into plants remains challenging, often requiring slow, genotype-specific methods such as tissue culture or transformation[1]. Plant viruses, which naturally infect and spread to most tissues, present a promising delivery system for editing reagents. However, many viruses have limited cargo capacities, restricting their ability to carry large CRISPR-Cas systems. Here we engineered tobacco rattle virus (TRV) to carry the compact RNA-guided TnpB enzyme ISYmu1 and its guide RNA. This innovation allowed transgene-free editing of Arabidopsis thaliana in a single step, with edits inherited in the subsequent generation. By overcoming traditional reagent delivery barriers, this approach offers a novel platform for genome editing, which can greatly accelerate plant biotechnology and basic research.},
}

@article {pmid40233430,
year = {2025},
author = {Li, Z and Wang, N and Wang, H and Yang, L and Li, Y and Gu, L and Fu, C and Yue, P and Yu, H},
title = {Single-cell transcriptomics reveals the mechanisms of lung injury induced by galt gene editing in mouse.},
journal = {Biochemical and biophysical research communications},
volume = {763},
number = {},
pages = {151780},
doi = {10.1016/j.bbrc.2025.151780},
pmid = {40233430},
issn = {1090-2104},
mesh = {Animals ; Mice ; *Gene Editing ; *Single-Cell Analysis ; *Lung Injury/genetics/pathology/metabolism ; *Transcriptome ; Lung/pathology/metabolism ; Mice, Inbred C57BL ; Disease Models, Animal ; CRISPR-Cas Systems ; },
abstract = {Galactosemia, caused by mutations in the GALT gene, leads to multi-organ damage. This study investigates the impact of Galt c.847 + 1G > T mutation on lung tissue using single-cell transcriptomics. We employed CRISPR/Cas9 to generate a Galt gene-edited mouse model with the Galt c. 847 + 1G > T mutation and assessed Galt expression through PCR and Western blotting. Histopathological analysis revealed significant structural lung changes, including alveolar congestion and inflammation. Single-cell RNA sequencing demonstrated a marked reduction in immune cells (NK, T, macrophages, B cells) and an increase in alveolar type II cells, vascular endothelial cells, and myofibroblasts in the GAL mouse. The increased abundance of alveolar type II cells indicated impaired differentiation and repair. Metabolic analysis revealed significant abnormalities linked to Galt c.847 + 1G > T mutation, with disruptions in TGF-β1, FGF, and Mif pathways contributing to cellular dysfunction and exacerbated lung injury. This model provides insights into the molecular mechanisms of lung injury in galactosemia, highlighting significant alterations in lung cell populations and key signaling pathways.},
}

Animals
Mice
*Gene Editing
*Single-Cell Analysis
*Lung Injury/genetics/pathology/metabolism
*Transcriptome
Lung/pathology/metabolism
Mice, Inbred C57BL
Disease Models, Animal
CRISPR-Cas Systems

@article {pmid40209323,
year = {2025},
author = {Zhang, Y and Lu, Z and Yang, H and Cheng, L and Zaklyazminskaya, E and Sokolova, O and Tan, H and Zhang, JZ},
title = {Generation of stable Cas9-EGFP expressing human induced pluripotent stem cell lines based on SeLection by Essential-gene Exon Knock-in technology.},
journal = {Stem cell research},
volume = {85},
number = {},
pages = {103710},
doi = {10.1016/j.scr.2025.103710},
pmid = {40209323},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Exons/genetics ; *Gene Knock-In Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Green Fluorescent Proteins/metabolism/genetics ; Cell Line ; Gene Editing ; *Genes, Essential ; Cell Differentiation ; *CRISPR-Associated Protein 9/metabolism/genetics ; },
abstract = {Here, we used SeLection by Essential-gene Exon Knock-in technology to generate the iPSC line with constitutive expression of Cas9-EGFP, while retaining all functions of the essential gene. Cas9-EGFP was inserted into the GAPDH exon9 via the homologous recombination, avoiding Cas9 silencing that often occurs during iPSC differentiation. The edited cell line shows precise knock-in locus with the typical characteristics and pluripotency of iPSCs. Therefore, this iPSC line is valuable for CRISPR screening or related experiments and could be widely used in the CRISPR/Cas9-based gene editing.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Exons/genetics
*Gene Knock-In Techniques/methods
*CRISPR-Cas Systems/genetics
*Green Fluorescent Proteins/metabolism/genetics
Cell Line
Gene Editing
*Genes, Essential
Cell Differentiation
*CRISPR-Associated Protein 9/metabolism/genetics

@article {pmid40205037,
year = {2025},
author = {Fielden, J and Siegner, SM and Gallagher, DN and Schröder, MS and Dello Stritto, MR and Lam, S and Kobel, L and Schlapansky, MF and Jackson, SP and Cejka, P and Jost, M and Corn, JE},
title = {Comprehensive interrogation of synthetic lethality in the DNA damage response.},
journal = {Nature},
volume = {640},
number = {8060},
pages = {1093-1102},
pmid = {40205037},
issn = {1476-4687},
mesh = {Humans ; *Synthetic Lethal Mutations/genetics ; *DNA Damage/genetics ; DNA-Binding Proteins/metabolism ; Proliferating Cell Nuclear Antigen/metabolism ; CRISPR-Cas Systems/genetics ; Endonucleases/metabolism ; Chromosome Breakage ; Flap Endonucleases/metabolism/deficiency/genetics ; Proteolysis ; Genomic Instability/genetics ; Homeostasis/genetics ; *DNA Repair/genetics ; },
abstract = {The DNA damage response (DDR) is a multifaceted network of pathways that preserves genome stability[1,2]. Unravelling the complementary interplay between these pathways remains a challenge[3,4]. Here we used CRISPR interference (CRISPRi) screening to comprehensively map the genetic interactions required for survival during normal human cell homeostasis across all core DDR genes. We captured known interactions and discovered myriad new connections that are available online. We defined the molecular mechanism of two of the strongest interactions. First, we found that WDR48 works with USP1 to restrain PCNA degradation in FEN1/LIG1-deficient cells. Second, we found that SMARCAL1 and FANCM directly unwind TA-rich DNA cruciforms, preventing catastrophic chromosome breakage by the ERCC1-ERCC4 complex. Our data yield fundamental insights into genome maintenance, provide a springboard for mechanistic investigations into new connections between DDR factors and pinpoint synthetic vulnerabilities that could be exploited in cancer therapy.},
}

Humans
*Synthetic Lethal Mutations/genetics
*DNA Damage/genetics
DNA-Binding Proteins/metabolism
Proliferating Cell Nuclear Antigen/metabolism
CRISPR-Cas Systems/genetics
Endonucleases/metabolism
Chromosome Breakage
Flap Endonucleases/metabolism/deficiency/genetics
Proteolysis
Genomic Instability/genetics
Homeostasis/genetics
*DNA Repair/genetics

@article {pmid40199100,
year = {2025},
author = {Su, M and Zhang, HS and Liu, H and Yang, K and Ying, ZM},
title = {Allosteric ribozyme-driven crRNA switch for the amplification-free detection of biomolecules.},
journal = {Biosensors & bioelectronics},
volume = {280},
number = {},
pages = {117450},
doi = {10.1016/j.bios.2025.117450},
pmid = {40199100},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *RNA, Catalytic/chemistry/genetics ; Humans ; *MicroRNAs/blood/genetics/isolation & purification/analysis ; *Adenosine Triphosphate/isolation & purification/blood/analysis ; Allosteric Regulation ; CRISPR-Cas Systems/genetics ; Limit of Detection ; CRISPR-Associated Proteins/genetics/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Currently, CRISPR-mediated biosensors are concentrating on the design of the crRNA or the activator strand to regulate the trans-cleavage activity of Cas12a. Herein, we report an allosteric ribozyme-driven crRNA switch-regulated CRISPR/Cas12a sensor for amplification-free detection of biomolecules. An allosteric ribozyme is meticulously engineered to connect the target recognition sequence with the 5' binding arm of the hammerhead ribozyme, resulting in the formation of a hairpin structure through complementary hybridization. The presence of target induces the conformational change in the allosteric module and disrupts the hairpin structure, restoring multiple-turnover cleavage RNA activity of ribozyme. Then, the activated ribozyme specifically cuts the cleavage site of the substrate-locked crRNA and releases the native crRNA to initiate CRISPR/Cas12a functions for signal reporting. The reported biosensor exhibited high sensitivity and excellent specificity for miR-155 and adenosine triphosphate (ATP) detection, giving the detection limits of 256 fM and 160 nM, respectively. For clinical validation, our proposed strategy can quantify miR-155 expression levels in cells and serum of cancer patients. Furthermore, we also demonstrate that the allosteric ribozyme-driven crRNA switch can be easily compatible with lateral flow assays, realizing visualization and the portable monitoring of target. Hence, the biosensor not only has outstanding potential in point-of-care testing, but also enables the detection of various biomolecules by flexibly substituting target recognition sequences for molecular diagnosis in the clinic.},
}

*Biosensing Techniques/methods
*RNA, Catalytic/chemistry/genetics
Humans
*MicroRNAs/blood/genetics/isolation & purification/analysis
*Adenosine Triphosphate/isolation & purification/blood/analysis
Allosteric Regulation
CRISPR-Cas Systems/genetics
Limit of Detection
CRISPR-Associated Proteins/genetics/chemistry
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40194617,
year = {2025},
author = {Wu, Z and Xu, Y and Zhou, W and Shi, L and Shi, W and Pu, L and Jiang, J},
title = {Rapid detection of Klebsiella pneumoniae based on one-tube RPA-CRISPR/Cas12a system.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {573},
number = {},
pages = {120281},
doi = {10.1016/j.cca.2025.120281},
pmid = {40194617},
issn = {1873-3492},
mesh = {*Klebsiella pneumoniae/isolation & purification/genetics ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *Recombinases/metabolism ; Limit of Detection ; Time Factors ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Klebsiella pneumoniae (KP) is a prevalent pathogen implicated in both community-acquired and nosocomial infections, often leading to severe clinical outcomes. The conventional methods for KP identification are characterized by intricacy and suboptimal efficiency. In this research, we have engineered a novel One-Tube RPA- CRISPR/Cas12a system, integrating recombinase polymerase amplification (RPA) method with the CRISPR/Cas12a diagnostic platform, to facilitate the detection of K. pneumoniae. To minimize the likelihood of aerosol-based contamination, the RPA components are positioned at the base of the tube, while the CRISPR/Cas12a components are placed at the tube's cap. The systems are combined post-RPA amplification through a brief centrifugation step, ensuring that RPA reactions are conducted independently to produce an adequate amount of target DNA before interaction with the CRISPR/Cas12a system. This method was validated using both fluorescent and lateral flow strip assays, achieving a limit of detection (LOD) of 10[0] copies/μL and 10[1] copies/μL respectively. The specificity for KP detection was found to be 100 %. Furthermore, the system demonstrated a positivity rate of 78 % (18/23) when directly extracting DNA from sputum samples, corroborated by culture and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). The simplicity and rapidity of the assay are augmented by a straightforward sample processing without extraction. The complete assay duration from specimen receipt to result is approximately 40 min, significantly reducing the turnaround time (TAT). Collectively, this system presents a streamlined, expeditious, and highly specific diagnostic approach for the detection of Klebsiella pneumoniae strains.},
}

*Klebsiella pneumoniae/isolation & purification/genetics
*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
Humans
*Recombinases/metabolism
Limit of Detection
Time Factors
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40194350,
year = {2025},
author = {Hu, H and Xue, H and Dong, K and Li, Y and Liu, P and Wang, H and Li, L and Xiao, X and Chen, H},
title = {Strand displacement-enhanced CRISPR-Cas13a system for ultra-specific detection of RNA single nucleotide variation.},
journal = {Biosensors & bioelectronics},
volume = {280},
number = {},
pages = {117445},
doi = {10.1016/j.bios.2025.117445},
pmid = {40194350},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *SARS-CoV-2/genetics/isolation & purification ; *Biosensing Techniques/methods ; *Polymorphism, Single Nucleotide/genetics ; *COVID-19/virology/diagnosis ; *MicroRNAs/genetics/analysis ; *RNA, Viral/genetics/analysis ; Limit of Detection ; },
abstract = {RNA plays a critical role in biological systems, mediating genetic information transfer and regulating gene expression. However, RNA is susceptible to variations from endogenous and exogenous sources, with potentially profound biological consequences. The CRISPR-Cas13a system has emerged as a promising tool for RNA variation detection due to its cost-effectiveness, sensitivity, and user-friendly nature. Despite this, designing a simple, universal system with high discrimination factor (DF) for single-nucleotide variations remains a challenge. Here, we present the strand displacement-enhanced Cas13a single-nucleotide variation detection assay (SECND), a sensitive, universal, and easy-to-implement method with a high DF for RNA variations. Using SECND, we detected 5 types of single-nucleotide variations, achieving a maximum DF of 1083.2. We validated the assay's effectiveness on miRNA and SARS-CoV-2 genomic RNA simulants, incorporating a 4-way strand displacement mechanism to enhance detection limits to 10 pmol/L and 50 pmol/L, and to identify variations at frequencies as low as 0.01 % and 0.1 %. Additionally, we demonstrated SECND's utility in quantifying single-nucleotide variants of miR-200b and miR-200c in ovarian cancer and retinal glioma cells. This versatile tool not only advances RNA variation detection but also has significant implications for disease research, diagnostics, and viral classification, enhancing our understanding of the CRISPR-Cas13a system and its potential applications.},
}

*CRISPR-Cas Systems/genetics
Humans
*SARS-CoV-2/genetics/isolation & purification
*Biosensing Techniques/methods
*Polymorphism, Single Nucleotide/genetics
*COVID-19/virology/diagnosis
*MicroRNAs/genetics/analysis
*RNA, Viral/genetics/analysis
Limit of Detection

@article {pmid40179699,
year = {2025},
author = {Hu, F and Zhang, Y and Yang, Y and Peng, L and Cui, S and Ma, Q and Wang, F and Wang, X},
title = {A rapid and ultrasensitive RPA-assisted CRISPR-Cas12a/Cas13a nucleic acid diagnostic platform with a smartphone-based portable device.},
journal = {Biosensors & bioelectronics},
volume = {280},
number = {},
pages = {117428},
doi = {10.1016/j.bios.2025.117428},
pmid = {40179699},
issn = {1873-4235},
mesh = {Humans ; *Smartphone/instrumentation ; *SARS-CoV-2/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; *COVID-19/diagnosis/virology ; *Nucleic Acid Amplification Techniques/instrumentation ; *Biosensing Techniques/instrumentation ; *COVID-19 Nucleic Acid Testing/instrumentation ; Equipment Design ; Sensitivity and Specificity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The spread of infectious diseases can be controlled by early identification of the source of infection and timely diagnosis to stop transmission. Real-time fluorescence quantitative polymerase chain reaction (PCR) is the current gold standard for pathogen diagnosis, with high detection sensitivity and accuracy. However, due to the need for specialized equipment, laboratories, and personnel, it is difficult to achieve rapid and immediate diagnosis during large-scale infectious disease outbreaks. Herein, an optimized CRISPR-based nucleic acid detection method was developed that reduces the CRISPR detection time to 15 min while maintaining high sensitivity. By using nucleic acid extraction-free and lyophilization techniques, the 'sample-in-result-out' detection of the two target genes of SARS-CoV-2, the human internal reference gene, and the negative quality control sample can be completed in 20 min, with a sensitivity of 0.5 copies/μL. Additionally, to facilitate the application, a smartphone-based reverse transcription-recombinase polymerase amplification (RT-RPA)-assisted CRISPR-rapid, portable nucleic acid detection device was developed, integrating functions such as heating, centrifugation, mixing, optical detection and result output. Process control, output, and uploading of detection results were conducted through smartphones. The device is not dependent on a power supply and can perform on-site rapid virus detection in resource-limited settings. Real-time uploading of results helps to rapidly implement epidemic prevention and control measures, providing an innovative means of detection, control, and prevention of virus-based infectious diseases. This important work provides a new and effective tool to manage potential future outbreaks of infectious diseases.},
}

Humans
*Smartphone/instrumentation
*SARS-CoV-2/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
*COVID-19/diagnosis/virology
*Nucleic Acid Amplification Techniques/instrumentation
*Biosensing Techniques/instrumentation
*COVID-19 Nucleic Acid Testing/instrumentation
Equipment Design
Sensitivity and Specificity
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40157320,
year = {2025},
author = {Li, J and Yin, L and Wang, C and Xu, Y},
title = {Generation of a homozygous ABCA7 knockout cell line (AHMUCNi002-A) in human iPSCs using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {85},
number = {},
pages = {103700},
doi = {10.1016/j.scr.2025.103700},
pmid = {40157320},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *ATP-Binding Cassette Transporters/genetics/metabolism/deficiency ; Homozygote ; Cell Line ; *Gene Knockout Techniques ; Gene Editing ; },
abstract = {ABCA7, located on chromosome 19, encodes an ATP-binding cassette transporter. Loss-of-function variants of ABCA7 are associated with an increased risk of Alzheimer's disease. To explore the role of ABCA7 deficiency in the pathogenesis of Alzheimer's disease, CRISPR/Cas9 genome-editing technology was utilized to generate a homozygous ABCA7 knockout in human induced pluripotent stem cells (hiPSCs). The resulting ABCA7 knockout cell line exhibited normal pluripotency, a stable karyotype, and the ability to differentiate into all three germ layers.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*ATP-Binding Cassette Transporters/genetics/metabolism/deficiency
Homozygote
Cell Line
*Gene Knockout Techniques
Gene Editing

@article {pmid40147061,
year = {2025},
author = {Zhou, H and Liu, Y and Chen, Q and Huang, K and Ren, PG},
title = {Generation of TP53 knock out induced pluripotent stem cell using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {85},
number = {},
pages = {103699},
doi = {10.1016/j.scr.2025.103699},
pmid = {40147061},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Tumor Suppressor Protein p53/genetics/deficiency/metabolism ; Animals ; Mice ; Humans ; *Gene Knockout Techniques ; Cell Line ; Teratoma/pathology ; },
abstract = {The TP53 gene is an important tumor suppressor gene. Through CRISPR/Cas9 technology, we have established a TP53 gene knockout cell line in iPSCs (SIIBRi001-A). This cell line maintains normal stem cell-like morphology, karyotype, expresses markers of pluripotency, and is capable of generating teratomas in immunodeficient mice. Quantitative analysis of pluripotency gene expression remains normal. This cell line can be utilized for studying the mechanisms underlying tumorigenesis.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Tumor Suppressor Protein p53/genetics/deficiency/metabolism
Animals
Mice
Humans
*Gene Knockout Techniques
Cell Line
Teratoma/pathology

@article {pmid40120557,
year = {2025},
author = {Ludwik, KA and Opitz, R and Jyrch, S and Megges, M and Kühnen, P and Stachelscheid, H},
title = {Correction of the Allan-Herndon-Dudley syndrome-causing SLC16A2 mutation G401R in a patient derived hiPSC line.},
journal = {Stem cell research},
volume = {85},
number = {},
pages = {103698},
doi = {10.1016/j.scr.2025.103698},
pmid = {40120557},
issn = {1876-7753},
mesh = {Humans ; *X-Linked Intellectual Disability/genetics/pathology/therapy ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Monocarboxylic Acid Transporters/genetics/metabolism ; *Muscle Hypotonia/genetics/pathology/therapy ; *Muscular Atrophy/genetics/pathology/therapy ; Cell Line ; *Mutation ; Symporters/genetics ; Male ; CRISPR-Cas Systems ; Gene Editing ; Mutation, Missense ; },
abstract = {The X-linked Allan-Herndon-Dudley syndrome (AHDS) is a genetic disorder characterized by severe psychomotor impairment, resulting from mutations in the SLC16A2 gene, which encodes the thyroid hormone transporter MCT8 (monocarboxylate transporter 8). Previously, we established a hiPSC line from a patient carrying the SLC16A2:R401G mutation (BIHi045-A). Using CRISPR/Cas9-mediated gene editing, we targeted exon 3 of SLC16A2 and used single-stranded oligodeoxynucleotides as homology-directed repair templates to correct the R401G missense mutation, generating an isogenic control cell line.},
}

Humans
*X-Linked Intellectual Disability/genetics/pathology/therapy
*Induced Pluripotent Stem Cells/metabolism/cytology
*Monocarboxylic Acid Transporters/genetics/metabolism
*Muscle Hypotonia/genetics/pathology/therapy
*Muscular Atrophy/genetics/pathology/therapy
Cell Line
*Mutation
Symporters/genetics
Male
CRISPR-Cas Systems
Gene Editing
Mutation, Missense

@article {pmid40108474,
year = {2025},
author = {Yuan, S and Sun, R and Shi, H and Chapman, NM and Hu, H and Guy, C and Rankin, S and Kc, A and Palacios, G and Meng, X and Sun, X and Zhou, P and Yang, X and Gottschalk, S and Chi, H},
title = {VDAC2 loss elicits tumour destruction and inflammation for cancer therapy.},
journal = {Nature},
volume = {640},
number = {8060},
pages = {1062-1071},
pmid = {40108474},
issn = {1476-4687},
mesh = {Animals ; *Voltage-Dependent Anion Channel 2/deficiency/genetics/metabolism ; Mice ; Interferon-gamma/immunology/pharmacology ; *Neoplasms/immunology/therapy/pathology/genetics ; Membrane Proteins/metabolism ; *Inflammation/immunology/pathology/genetics ; Humans ; Mitochondria/metabolism/pathology ; Signal Transduction ; Female ; *Immunotherapy ; Tumor Microenvironment/immunology ; bcl-2 Homologous Antagonist-Killer Protein/metabolism ; Nucleotidyltransferases/metabolism ; Cell Line, Tumor ; Male ; CRISPR-Cas Systems/genetics ; CD8-Positive T-Lymphocytes/immunology ; BH3 Interacting Domain Death Agonist Protein/metabolism ; Mice, Inbred C57BL ; },
abstract = {Tumour cells often evade immune pressure exerted by CD8[+] T cells or immunotherapies through mechanisms that are largely unclear[1,2]. Here, using complementary in vivo and in vitro CRISPR-Cas9 genetic screens to target metabolic factors, we established voltage-dependent anion channel 2 (VDAC2) as an immune signal-dependent checkpoint that curtails interferon-γ (IFNγ)-mediated tumour destruction and inflammatory reprogramming of the tumour microenvironment. Targeting VDAC2 in tumour cells enabled IFNγ-induced cell death and cGAS-STING activation, and markedly improved anti-tumour effects and immunotherapeutic responses. Using a genome-scale genetic interaction screen, we identified BAK as the mediator of VDAC2-deficiency-induced effects. Mechanistically, IFNγ stimulation increased BIM, BID and BAK expression, with VDAC2 deficiency eliciting uncontrolled IFNγ-induced BAK activation and mitochondrial damage. Consequently, mitochondrial DNA was aberrantly released into the cytosol and triggered robust activation of cGAS-STING signalling and type I IFN response. Importantly, co-deletion of STING signalling components dampened the therapeutic effects of VDAC2 depletion in tumour cells, suggesting that targeting VDAC2 integrates CD8[+] T cell- and IFNγ-mediated adaptive immunity with a tumour-intrinsic innate immune-like response. Together, our findings reveal VDAC2 as a dual-action target to overcome tumour immune evasion and establish the importance of coordinately destructing and inflaming tumours to enable efficacious cancer immunotherapy.},
}

Animals
*Voltage-Dependent Anion Channel 2/deficiency/genetics/metabolism
Mice
Interferon-gamma/immunology/pharmacology
*Neoplasms/immunology/therapy/pathology/genetics
Membrane Proteins/metabolism
*Inflammation/immunology/pathology/genetics
Humans
Mitochondria/metabolism/pathology
Signal Transduction
Female
*Immunotherapy
Tumor Microenvironment/immunology
bcl-2 Homologous Antagonist-Killer Protein/metabolism
Nucleotidyltransferases/metabolism
Cell Line, Tumor
Male
CRISPR-Cas Systems/genetics
CD8-Positive T-Lymphocytes/immunology
BH3 Interacting Domain Death Agonist Protein/metabolism
Mice, Inbred C57BL

@article {pmid40088038,
year = {2025},
author = {Blomme, J and Arraiza Ribera, J and De Clerck, O and Jacobs, TB},
title = {Consolidating Ulva functional genomics: gene editing and new selection systems.},
journal = {The New phytologist},
volume = {246},
number = {4},
pages = {1710-1723},
doi = {10.1111/nph.70068},
pmid = {40088038},
issn = {1469-8137},
support = {BOF/STA/202209/016//Bijzonder Onderzoeksfonds UGent/ ; BOF20/PDO/016//Bijzonder Onderzoeksfonds UGent/ ; 9329//Gordon and Betty Moore Foundation/ ; 12T3418N//Fonds Wetenschappelijk Onderzoek/ ; G015623N//Fonds Wetenschappelijk Onderzoek/ ; G0AHR24N//Fonds Wetenschappelijk Onderzoek/ ; GOH3817N//Fonds Wetenschappelijk Onderzoek/ ; I001621N//Fonds Wetenschappelijk Onderzoek/ ; },
mesh = {*Ulva/genetics/microbiology ; *Gene Editing/methods ; *Genomics/methods ; *Selection, Genetic ; CRISPR-Cas Systems/genetics ; Mutation/genetics ; },
abstract = {The green seaweed Ulva compressa is a promising model for functional biology. In addition to historical research on growth and development, -omics data and molecular tools for stable transformation are available. However, more efficient tools are needed to study gene function. Here, we expand the molecular toolkit for Ulva. We screened the survival of Ulva and its mutualistic bacteria on 14 selective agents and established that Blasticidin deaminases (BSD or bsr) can be used as selectable markers to generate stable transgenic lines. We show that Cas9 and Cas12a RNPs are suitable for targeted mutagenesis and can generate genomic deletions of up to 20 kb using the marker gene ADENINE PHOSPHORIBOSYLTRANSFERASE (APT). We demonstrate that the targeted insertion of a selectable marker via homology-directed repair or co-editing with APT is possible for nonmarker genes. We evaluated 31 vector configurations and found that the bicistronic fusion of Cas9 to a resistance marker or the incorporation of introns in Cas9 led to the most mutants. We used this to generate mutants in three nonmarker genes using a co-editing strategy. This expanded molecular toolkit now enables us to reliably make gain- and loss-of-function mutants; additional optimizations will be necessary to allow for vector-based multiplex genome editing in Ulva.},
}

*Ulva/genetics/microbiology
*Gene Editing/methods
*Genomics/methods
*Selection, Genetic
CRISPR-Cas Systems/genetics
Mutation/genetics

@article {pmid40035150,
year = {2025},
author = {Zhu, H and Zhou, T and Guan, J and Li, Z and Yang, X and Li, Y and Sun, J and Xu, Q and Xuan, YH},
title = {Precise genome editing of Dense and Erect Panicle 1 promotes rice sheath blight resistance and yield production in japonica rice.},
journal = {Plant biotechnology journal},
volume = {23},
number = {5},
pages = {1832-1846},
doi = {10.1111/pbi.70010},
pmid = {40035150},
issn = {1467-7652},
support = {32372482//National Natural Science Foundation of China/ ; },
mesh = {*Oryza/genetics/microbiology/growth & development ; *Disease Resistance/genetics ; *Gene Editing ; *Plant Diseases/microbiology/genetics/immunology ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Rhizoctonia/physiology ; Plants, Genetically Modified ; Gene Expression Regulation, Plant ; },
abstract = {The primary goals of crop breeding are to enhance yield and improve disease resistance. However, the "trade-off" mechanism, in which signalling pathways for resistance and yield are antagonistically regulated, poses challenges for achieving both simultaneously. Previously, we demonstrated that knock-out mutants of the Dense and Erect Panicle 1 (DEP1) gene can significantly enhance rice resistance to sheath blight (ShB), and we mapped DEP1's association with panicle length. In this study, we discovered that dep1 mutants significantly reduced rice yield. Nonetheless, truncated DEP1 was able to achieve both ShB resistance and yield increase in japonica rice. To further explore the function of truncated DEP1 in promoting yield and ShB resistance, we generated CRISPR/Cas9-mediated genome editing mutants, including a full-length deletion mutant of DEP1, named dep1, and a truncated version, dep1-cys. Upon inoculation with Rhizoctonia solani, the dep1-cys mutant demonstrated stronger ShB resistance than the dep1 mutant. Additionally, dep1-cys increased yield per plant, whereas dep1 reduced it. Compared to the full DEP1 protein, the truncated DEP1 (dep1-cys) demonstrated a decreased interaction affinity with IDD14 and increased affinity with IDD10, which are known to positively and negatively regulate ShB resistance through the activation of PIN1a and ETR2, respectively. The dep1-cys mutant exhibited higher PIN1a and lower ETR2 expression than wild-type plants, suggesting that dep1-cys modulated IDD14 and IDD10 interactions to regulate PIN1a and ETR2, thereby enhancing ShB resistance. Overall, these data indicate that precise genome editing of DEP1 could simultaneously improve both ShB resistance and yield, effectively mitigating trade-off regulation in rice.},
}

*Oryza/genetics/microbiology/growth & development
*Disease Resistance/genetics
*Gene Editing
*Plant Diseases/microbiology/genetics/immunology
*Plant Proteins/genetics/metabolism
CRISPR-Cas Systems
Rhizoctonia/physiology
Plants, Genetically Modified
Gene Expression Regulation, Plant

@article {pmid40011774,
year = {2025},
author = {Cui, X and Yang, H and Cai, C and Beaman, C and Yang, X and Liu, H and Ren, X and Amador, Z and Jones, IR and Keough, KC and Zhang, M and Fair, T and Abnousi, A and Mishra, S and Ye, Z and Hu, M and Pollen, AA and Pollard, KS and Shen, Y},
title = {Comparative characterization of human accelerated regions in neurons.},
journal = {Nature},
volume = {640},
number = {8060},
pages = {991-999},
pmid = {40011774},
issn = {1476-4687},
mesh = {Humans ; *Neurons/metabolism/cytology ; Animals ; Pan troglodytes/genetics ; Induced Pluripotent Stem Cells/cytology/metabolism ; Cell Differentiation/genetics ; Nerve Tissue Proteins/genetics/metabolism ; Enhancer Elements, Genetic/genetics ; Transcription Factors/metabolism ; *Evolution, Molecular ; Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism ; Species Specificity ; CRISPR-Cas Systems/genetics ; *Regulatory Sequences, Nucleic Acid/genetics ; Gene Expression Regulation ; },
abstract = {Human accelerated regions (HARs) are conserved genomic loci that have experienced rapid nucleotide substitutions following the divergence from chimpanzees[1,2]. HARs are enriched in candidate regulatory regions near neurodevelopmental genes, suggesting their roles in gene regulation[3]. However, their target genes and functional contributions to human brain development remain largely uncharacterized. Here we elucidate the cis-regulatory functions of HARs in human and chimpanzee induced pluripotent stem (iPS) cell-induced excitatory neurons. Using genomic[4] and chromatin looping information, we prioritized 20 HARs and their chimpanzee orthologues for functional characterization via single-cell CRISPR interference, and demonstrated their species-specific gene regulatory functions. Our findings reveal diverse functional outcomes of HAR-mediated cis-regulation in human neurons, including attenuated NPAS3 expression by altering the binding affinities of multiple transcription factors in HAR202 and maintaining iPS cell pluripotency and neuronal differentiation capacities through the upregulation of PUM2 by 2xHAR.319. Finally, we used prime editing to demonstrate differential enhancer activity caused by several HAR26;2xHAR.178 variants. In particular, we link one variant in HAR26;2xHAR.178 to elevated SOCS2 expression and increased neurite outgrowth in human neurons. Thus, our study sheds new light on the endogenous gene regulatory functions of HARs and their potential contribution to human brain evolution.},
}

Humans
*Neurons/metabolism/cytology
Animals
Pan troglodytes/genetics
Induced Pluripotent Stem Cells/cytology/metabolism
Cell Differentiation/genetics
Nerve Tissue Proteins/genetics/metabolism
Enhancer Elements, Genetic/genetics
Transcription Factors/metabolism
*Evolution, Molecular
Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism
Species Specificity
CRISPR-Cas Systems/genetics
*Regulatory Sequences, Nucleic Acid/genetics
Gene Expression Regulation

@article {pmid39936495,
year = {2025},
author = {Li, L and Fu, X and Qi, X and Xiao, B and Liu, C and Wu, Q and Zhu, J and Xie, C},
title = {Harnessing haploid-inducer mediated genome editing for accelerated maize variety development.},
journal = {Plant biotechnology journal},
volume = {23},
number = {5},
pages = {1604-1614},
doi = {10.1111/pbi.14608},
pmid = {39936495},
issn = {1467-7652},
support = {2023YFD1202901//National Key Research and Development Program of China/ ; 32201872//National Science Foundation of China/ ; 32301914//National Science Foundation of China/ ; YBXM2429//Nanfan special project, CAAS/ ; CARS-02-06//China Agriculture Research System/ ; },
mesh = {*Zea mays/genetics/growth & development ; *Gene Editing/methods ; *Haploidy ; CRISPR-Cas Systems/genetics ; Plant Breeding/methods ; Genome, Plant/genetics ; Plants, Genetically Modified ; Mutation ; },
abstract = {The integration of haploid induction and genome editing, termed HI-Edit/IMGE, is a promising tool for generating targeted mutations for crop breeding. However, the technical components and stacking suitable for the maize seed industry have yet to be fully characterised and tested. Here, we developed and assessed three HI-Edit/IMGE maize lines: Edit[Wx], Edit[Sh], and Edit[Wx&Sh], using the haploid inducer CHOI3 and lines engineered using the CRISPR-Cas9 system targeting the Waxy1 (Wx1) and Shrunken2 (Sh2) genes. We meticulously characterised the HI-Edit/IMGE systems, focusing on copy numbers and the mutant alleles mtl and dmp, which facilitate haploid induction. Using B73 and six other parental lines of major commercial varieties as recipients, HI-Edit/IMGE demonstrated maternal haploid induction efficiencies ranging from 8.55% to 20.89% and targeted mutation rates between 0.38% and 1.46%. Comprehensive assessment verified the haploid identification, target gene editing accuracy, genome background integrity, and related agronomic traits. Notably, Edit[Wx&Sh] successfully combined distinct CRISPR-Cas9 systems to induce multiple desired mutations, highlighting the potential of HI-Edit/IMGE in accelerating the integration of edited traits into commercial maize varieties. Our findings underscore the importance of meticulous Cas9 copy number characterisation and highlight potential challenges related to somatic chimerism. We also validated the performance of single-cross haploids derived using the HI-Edit/IMGE process. Our results confirm the industrial applicability of generating targeted mutations through pollination and provide critical insights for further optimising this technology.},
}

*Zea mays/genetics/growth & development
*Gene Editing/methods
*Haploidy
CRISPR-Cas Systems/genetics
Plant Breeding/methods
Genome, Plant/genetics
Plants, Genetically Modified
Mutation

@article {pmid39865897,
year = {2025},
author = {van Belle, J and Schaart, JG and Dechesne, AC and Fei, D and Cisneros, AO and Serafini, M and Visser, RGF and van Loo, EN},
title = {Direct and indirect effects of multiplex genome editing of F5H and FAD2 in oil crop camelina.},
journal = {Plant biotechnology journal},
volume = {23},
number = {5},
pages = {1399-1412},
doi = {10.1111/pbi.14593},
pmid = {39865897},
issn = {1467-7652},
support = {6635405//European Commission/ ; },
mesh = {*Gene Editing/methods ; *Fatty Acid Desaturases/genetics/metabolism ; *Brassicaceae/genetics/metabolism ; *Plant Oils/metabolism ; *Plant Proteins/genetics/metabolism ; Seeds/genetics/metabolism ; Mutation ; Fatty Acids/metabolism ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {Mutants with simultaneous germline mutations were obtained in all three F5H genes and all three FAD2 genes (one to eleven mutated alleles) in order to improve the feed value of the seed meal and the fatty acid composition of the seed oil. In mutants with multiple mutated F5H alleles, sinapine in seed meal was reduced by up to 100%, accompanied by a sharp reduction in the S-monolignol content of lignin without causing lodging or stem break. A lower S-lignin monomer content in stems can contribute to improved stem degradability allowing new uses of stems. Mutants in all six FAD2 alleles showed an expected increase in MUFA from 8.7% to 74% and a reduction in PUFA from 53% to 13% in the fatty acids in seed oil. Remarkably, some full FAD2 mutants showed normal growth and seed production and not the dwarfing phenotype reported in previous studies. The relation between germline mutation allele dosage and phenotype was influenced by the still ongoing activity of the CRISPR/Cas9 system, leading to new somatic mutations in the leaves of flowering plants. The correlations between the total mutation frequency (germline plus new somatic mutations) for F5H with sinapine content, and FAD2 with fatty acid composition were higher than the correlations between germline mutation count and phenotypes. This shows the importance of quantifying both the germline mutations and somatic mutations when studying CRISPR/Cas9 effects in situations where the CRISPR/Cas9 system is not yet segregated out.},
}

*Gene Editing/methods
*Fatty Acid Desaturases/genetics/metabolism
*Brassicaceae/genetics/metabolism
*Plant Oils/metabolism
*Plant Proteins/genetics/metabolism
Seeds/genetics/metabolism
Mutation
Fatty Acids/metabolism
CRISPR-Cas Systems
Plants, Genetically Modified

@article {pmid39632651,
year = {2025},
author = {Xiao, L and Qin, B and Zhang, X and Yao, Y and Dou, X and Liu, Y and Duan, X},
title = {Precision Control of Cell Type-Specific Behavior via RNA Sensing and Editing.},
journal = {Small methods},
volume = {9},
number = {4},
pages = {e2400952},
doi = {10.1002/smtd.202400952},
pmid = {39632651},
issn = {2366-9608},
support = {2021YFA0911600//National Key R&D Program of China/ ; 82471079//National Natural Science Foundation of China/ ; 2023TP2225//Hunan Engineering Research Center for Glaucoma with Artificial Intelligence in Diagnosis and Application of New Materials/ ; 2023JJ70014//Natural Science Foundation of Hunan Province, China/ ; kq2208495//Changsha Municipal Natural Science Foundation/ ; AR2206D5//Science and Technology Foundation of Aier Eye Hospital Group, China/ ; AIG2305D03//Science and Technology Foundation of Aier Eye Hospital Group, China/ ; RCJC20221008092723011//Shenzhen Science and Technology Innovation Program/ ; JCYJ20220818102001002//Shenzhen Science and Technology Innovation Program/ ; 2023yjlcyj006//Shenzhen High-level Hospital Construction Fund/ ; },
mesh = {Humans ; *Retinoblastoma/genetics/therapy/pathology ; *CRISPR-Cas Systems/genetics ; Animals ; *RNA/genetics/metabolism ; Cell Line, Tumor ; *Gene Editing/methods ; *RNA Editing ; Cell Proliferation/genetics ; Apoptosis/genetics ; Mice ; },
abstract = {In the realms of bioengineering and biopharmaceuticals, there exists a critical demand for advanced genetic tools that can interact with specific cell signaling pathways to accurately identify and target various cell types. This research introduces the innovative CRISPR-ADAReader system, which enables precise manipulation of cell activity through sensing target RNA. Featuring both positive and negative feedback loops, the system allows for tailored regulation across different cell types in response to various internal signals, showcasing exceptional programmability, specificity, and sensitivity. By choosing distinct RNAs as activation signals, the CRISPR-ADAReader efficiently monitors and alters targeted cell behaviors. In a case study focusing on retinoblastoma treatment, the system distinctively initiates positive feedback and self-silencing actions by detecting MCYN and Rb transcripts, thus safeguarding normal retinal pigment epithelial cells while promoting apoptosis in cancer cells. Moreover, the CRISPR-ADAReader demonstrates significant anti-tumor effectiveness in live models, markedly reducing retinoblastoma cell proliferation through the activation of several cancer-suppression pathways, outperforming the capabilities of the ADAR-sensor. Notably, the system also shows an excellent in vivo safety profile. In conclusion, the CRISPR-ADAReader system represents a groundbreaking method for the detection and editing of RNA, offering a potent instrument for the customized and precise governance of cell behavior.},
}

Humans
*Retinoblastoma/genetics/therapy/pathology
*CRISPR-Cas Systems/genetics
Animals
*RNA/genetics/metabolism
Cell Line, Tumor
*Gene Editing/methods
*RNA Editing
Cell Proliferation/genetics
Apoptosis/genetics
Mice

@article {pmid40263339,
year = {2025},
author = {Zhao, C and Cao, Y and Ibrahim, N and Wang, Y and Martemyanov, KA},
title = {Efficient in vivo labeling of endogenous proteins with SMART delineates retina cellular and synaptic organization.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3768},
pmid = {40263339},
issn = {2041-1723},
support = {EY018139//U.S. Department of Health & Human Services | NIH | National Eye Institute (NEI)/ ; EY028033//U.S. Department of Health & Human Services | NIH | National Eye Institute (NEI)/ ; EY030554//U.S. Department of Health & Human Services | NIH | National Eye Institute (NEI)/ ; },
mesh = {Animals ; *Retina/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Synapses/metabolism ; Mice ; },
abstract = {A key application of CRISPR/Cas9-based genomic editing is modification of genes to introduce engineered sequences. However, the editing flexibility is severely constrained by the requirement for targeting sites in proximity to the desired modification site, which makes many modifications intractable. Here, we develop a strategy that overcomes this key limitation to allow CRISPR-based editing at any position with high efficiency. It relies on reconstructing the targeted gene using Silently Mutate And Repair Template (SMART) where we mutate the gap sequence in the repair template to prevent its base pairing with the target DNA while maintaining the same amino acid coding. Using vertebrate retina as a neuronal model system we document the application of SMART editing for labeling endogenous proteins in vivo with high efficiency. We show that SMART editing allows us to access numerous cell types in the retina and address fundamental cell biological questions pertaining to its organization. We propose that this approach will facilitate functional genomic studies in a wide range of systems and increase the precision of corrective gene therapies.},
}

Animals
*Retina/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Synapses/metabolism
Mice

@article {pmid40263274,
year = {2025},
author = {Rengifo-Gonzalez, M and Mazzuoli, MV and Janssen, AB and Rueff, AS and Burnier, J and Liu, X and Veening, JW},
title = {Make-or-break prime editing for genome engineering in Streptococcus pneumoniae.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3796},
pmid = {40263274},
issn = {2041-1723},
support = {310030_200792//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; NCCR AntiResist 51NF40_180541//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; TMPFP3_210202//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 771534-PneumoCaTChER//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; },
mesh = {*Streptococcus pneumoniae/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Bacterial ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; *Genetic Engineering/methods ; CRISPR-Associated Protein 9/metabolism/genetics ; },
abstract = {CRISPR-Cas9 has revolutionized genome engineering by allowing precise introductions of DNA double-strand breaks (DSBs). However, genome engineering in bacteria is still a complex, multi-step process requiring a donor DNA template for repair of DSBs. Prime editing circumvents this need as the repair template is indirectly provided within the prime editing guide RNA (pegRNA). Here, we developed make-or-break Prime Editing (mbPE) that allows for precise and effective genetic engineering in the opportunistic human pathogen Streptococcus pneumoniae. In contrast to traditional prime editing in which a nicking Cas9 is employed, mbPE harnesses wild type Cas9 in combination with a pegRNA that destroys the seed region or protospacer adjacent motif. Since most bacteria poorly perform template-independent end joining, correctly genome-edited clones are selectively enriched during mbPE. We show that mbPE is RecA-independent and can be used to introduce point mutations, deletions and targeted insertions, including protein tags such as a split luciferase, at selection efficiencies of over 93%. mbPE enables sequential genome editing, is scalable, and can be used to generate pools of mutants in a high-throughput manner. The mbPE system and pegRNA design guidelines described here will ameliorate future bacterial genome editing endeavors.},
}

*Streptococcus pneumoniae/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Genome, Bacterial
RNA, Guide, CRISPR-Cas Systems/genetics
DNA Breaks, Double-Stranded
*Genetic Engineering/methods
CRISPR-Associated Protein 9/metabolism/genetics

@article {pmid40262634,
year = {2025},
author = {Silverstein, RA and Kim, N and Kroell, AS and Walton, RT and Delano, J and Butcher, RM and Pacesa, M and Smith, BK and Christie, KA and Ha, LL and Meis, RJ and Clark, AB and Spinner, AD and Lazzarotto, CR and Li, Y and Matsubara, A and Urbina, EO and Dahl, GA and Correia, BE and Marks, DS and Tsai, SQ and Pinello, L and De Ravin, SS and Liu, Q and Kleinstiver, BP},
title = {Custom CRISPR-Cas9 PAM variants via scalable engineering and machine learning.},
journal = {Nature},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41586-025-09021-y},
pmid = {40262634},
issn = {1476-4687},
abstract = {Engineering and characterizing proteins can be time-consuming and cumbersome, motivating the development of generalist CRISPR-Cas enzymes[1-4] to enable diverse genome editing applications. However, such enzymes have caveats such as an increased risk of off-target editing[3,5,6]. To enable scalable reprogramming of Cas9 enzymes, here we combined high-throughput protein engineering with machine learning (ML) to derive bespoke editors more uniquely suited to specific targets. Via structure/function-informed saturation mutagenesis and bacterial selections, we obtained nearly 1,000 engineered SpCas9 enzymes and characterized their protospacer-adjacent motif[7] (PAM) requirements to train a neural network that relates amino acid sequence to PAM specificity. By utilizing the resulting PAM ML algorithm (PAMmla) to predict the PAMs of 64 million SpCas9 enzymes, we identified efficacious and specific enzymes that outperform evolution-based and engineered SpCas9 enzymes as nucleases and base editors in human cells while reducing off-targets. An in silico directed evolution method enables user-directed Cas9 enzyme design, including for allele-selective targeting of the RHO P23H allele in human cells and mice. Together, PAMmla integrates ML and protein engineering to curate a catalog of SpCas9 enzymes with distinct PAM requirements, and motivates the use of efficient and safe bespoke Cas9 enzymes instead of generalist enzymes for various applications.},
}

@article {pmid40261967,
year = {2025},
author = {Liu, X and Wang, P and Wang, S and Liao, W and Ouyang, M and Lin, S and Lin, R and Sarris, PF and Michalopoulou, V and Feng, X and Zhang, Z and Xu, Z and Chen, G and Zhu, B},
title = {The circular RNA circANK suppresses rice resistance to bacterial blight by inhibiting microRNA398b-mediated defense.},
journal = {The Plant cell},
volume = {37},
number = {4},
pages = {},
doi = {10.1093/plcell/koaf082},
pmid = {40261967},
issn = {1532-298X},
support = {32272479//National Natural Science Foundation of China/ ; CSTB2022NSCQ-MSX0524//Chongqing Natural Science Foundation/ ; 2025YFHZ0043//Science & Technology Department of Sichuan Province/ ; },
mesh = {*Oryza/genetics/microbiology/immunology ; *MicroRNAs/genetics/metabolism ; *RNA, Circular/genetics/metabolism ; *Plant Diseases/microbiology/genetics/immunology ; *Disease Resistance/genetics ; *Xanthomonas/pathogenicity/physiology ; Gene Expression Regulation, Plant ; *RNA, Plant/genetics/metabolism ; Reactive Oxygen Species/metabolism ; CRISPR-Cas Systems/genetics ; Plant Proteins/genetics/metabolism ; },
abstract = {Circular RNAs (circRNAs) are prevalent in eukaryotic cells and have been linked to disease progressions. Their unique circular structure and stability make them potential biomarkers and therapeutic targets. Compared with animal models, plant circRNA research is still in its infancy. The lack of effective tools to specifically knock down circRNAs without affecting host gene expression has slowed the progress of plant circRNA research. Here, we have developed a CRISPR-Cas13d tool that can specifically knock down circRNAs in plant systems, successfully achieving the targeted knockdown of circRNAs in rice (Oryza sativa). We further focused on Os-circANK (a circRNA derived from Ankyrin repeat-containing protein), a circRNA differentially expressed in rice upon pathogen infection. Physiological and biochemical experiments revealed that Os-circANK functions as a sponge for miR398b, suppressing the cleavage of Cu/Zn-superoxidase dismutase (CSD)1/CSD2/copper chaperone for superoxide dismutase/superoxidase dismutaseX through competing endogenous RNA, leading to reduced reactive oxygen species levels following Xanthomonas oryzae pv. oryzae (Xoo) infection and a negative regulation of rice resistance to bacterial blight. Our findings indicate Os-circANK inhibits rice resistance to bacterial blight via the microRNA398b(miR398b)/CSD/SOD pathway.},
}

*Oryza/genetics/microbiology/immunology
*MicroRNAs/genetics/metabolism
*RNA, Circular/genetics/metabolism
*Plant Diseases/microbiology/genetics/immunology
*Disease Resistance/genetics
*Xanthomonas/pathogenicity/physiology
Gene Expression Regulation, Plant
*RNA, Plant/genetics/metabolism
Reactive Oxygen Species/metabolism
CRISPR-Cas Systems/genetics
Plant Proteins/genetics/metabolism

@article {pmid40261966,
year = {2025},
author = {Cheng, Y and Li, G and Qi, A and Mandlik, R and Pan, C and Wang, D and Ge, S and Qi, Y},
title = {A comprehensive all-in-one CRISPR toolbox for large-scale screens in plants.},
journal = {The Plant cell},
volume = {37},
number = {4},
pages = {},
doi = {10.1093/plcell/koaf081},
pmid = {40261966},
issn = {1532-298X},
support = {IOS-2029889//NSF Plant Genome Research Program/ ; 2021-67013-34554//USDA-NIFA Agricultural Innovation through Gene Editing/ ; 2020-33522-32274//USDA-NIFA Biotechnology Risk Assessment Research/ ; 21010111//Foundation for Food and Agriculture Research/ ; },
mesh = {Oryza/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Solanum lycopersicum/genetics ; Protoplasts/metabolism ; Plants, Genetically Modified ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome, Plant/genetics ; Acetolactate Synthase/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas) technologies facilitate routine genome engineering of one or a few genes at a time. However, large-scale CRISPR screens with guide RNA libraries remain challenging in plants. Here, we have developed a comprehensive all-in-one CRISPR toolbox for Cas9-based genome editing, cytosine base editing, adenine base editing (ABE), Cas12a-based genome editing and ABE, and CRISPR-Act3.0-based gene activation in both monocot and dicot plants. We evaluated all-in-one T-DNA expression vectors in rice (Oryza sativa, monocot) and tomato (Solanum lycopersicum, dicot) protoplasts, demonstrating their broad and reliable applicability. To showcase the applications of these vectors in CRISPR screens, we constructed guide RNA (gRNA) pools for testing in rice protoplasts, establishing a high-throughput approach to select high-activity gRNAs. Additionally, we demonstrated the efficacy of sgRNA library screening for targeted mutagenesis of ACETOLACTATE SYNTHASE in rice, recovering novel candidate alleles for herbicide resistance. Furthermore, we carried out a CRISPR activation screen in Arabidopsis thaliana, rapidly identifying potent gRNAs for FLOWERING LOCUS T activation that confer an early-flowering phenotype. This toolbox contains 61 versatile all-in-one vectors encompassing nearly all commonly used CRISPR technologies. It will facilitate large-scale genetic screens for loss-of-function or gain-of-function studies, presenting numerous promising applications in plants.},
}

Oryza/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Solanum lycopersicum/genetics
Protoplasts/metabolism
Plants, Genetically Modified
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Genome, Plant/genetics
Acetolactate Synthase/genetics

@article {pmid40261268,
year = {2025},
author = {Liu, Y and Wu, Y and Liu, Y and Zhang, Q and Yuan, H and Li, S and Li, Z and Wang, B and Chang, Y and Liu, M},
title = {Arrest of CRISPR-Cas12a by Nonspecific Single-Stranded DNA for Biosensing.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.4c07081},
pmid = {40261268},
issn = {1520-6882},
abstract = {CRISPR-Cas technologies have emerged as powerful biosensing tools for the sensitive and specific detection of non-nucleic acid targets. However, existing biosensing strategies suffer from poor compatibility across diverse targets due to the complicated engineering of crRNA and DNA activator required for the CRISPR-Cas activity regulation. Herein, we report a novel and straightforward strategy for designing CRISPR-Cas12a-based biosensors that function by switching structures from single-stranded (ss)DNA/CRISPR-Cas12a assembly to DNA activator/CRISPR-Cas12a complex in the presence of target bacterium. The strategy begins with a ssDNA assembly made of a trans-acting RNA-cleaving DNAzyme (tRCD) and an RNA/DNA chimeric substrate (RCS). The ssDNA assembly has the ability to bind Cas12a nonspecifically, thus indeed blocking the CRISPR-Cas12a activity. By exploiting the specific recognition and cleavage capacities of tRCD for RCS in the presence of a target, the target-bound tRCD and the cleaved RCS are released from Cas12a, thus restoring the CRISPR-Cas12a activity. This method has been successfully applied for the sensitive (detection limit: 10[2] CFU/mL) detection of Escherichia coli (E. coli, EC) and Burkholderia gladioli (B. gladioli, BG). For the blind testing of 30 clinical urine samples, it exhibited 100% sensitivity and 100% specificity in identifying E. coli-associated urinary tract infections (UTIs).},
}

@article {pmid40261207,
year = {2025},
author = {Liao, X and Li, Y and Wu, Y and Li, X and Shang, X},
title = {Deep Learning-Based Classification of CRISPR Loci Using Repeat Sequences.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00174},
pmid = {40261207},
issn = {2161-5063},
abstract = {With the widespread application of the CRISPR-Cas system in gene editing and related fields, along with the increasing availability of metagenomic data, the demand for detecting and classifying CRISPR-Cas systems in metagenomic data sets has grown significantly. Traditional classification methods for CRISPR-Cas systems primarily rely on identifying cas genes near CRISPR arrays. However, in cases where cas gene information is absent, such as in metagenomes or fragmented genome assemblies, traditional methods may fail. Here, we present a deep learning-based method, CRISPRclassify-CNN-Att, which classifies CRISPR loci solely based on repeat sequences. CRISPRclassify-CNN-Att utilizes convolutional neural networks (CNNs) and self-attention mechanisms to extract features from repeat sequences. It employs a stacking strategy to address the imbalance of samples across different subtypes and uses transfer learning to improve classification accuracy for subtypes with fewer samples. CRISPRclassify-CNN-Att demonstrates outstanding performance in classifying multiple subtypes, particularly those with larger sample sizes. Although CRISPR loci classification traditionally depends on cas genes, CRISPRclassify-CNN-Att offers a novel approach that serves as a significant complement to cas-based methods, enabling the classification of orphan or distant CRISPR loci. The proposed tool is freely accessible via https://github.com/Xingyu-Liao/CRISPRclassify-CNN-Att.},
}

@article {pmid40259370,
year = {2025},
author = {Wang, T and Brown, C and Doherty, N and Byrne, NM and Islam, R and Doherty, M and Feng, J and Yin, C and Chambers, S and McQuoid, L and Mohamed-Smith, L and Butterworth, KT and Kerr, EM and Coulter, JA},
title = {Mannose and PMI depletion overcomes radiation resistance in HPV-negative head and neck cancer.},
journal = {Cell communication and signaling : CCS},
volume = {23},
number = {1},
pages = {189},
pmid = {40259370},
issn = {1478-811X},
support = {EP/X525625/1//Engineering and Physical Sciences Research Council/ ; MR/X502881/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Humans ; *Radiation Tolerance/drug effects ; *Mannose/metabolism/pharmacology ; *Head and Neck Neoplasms/radiotherapy/pathology/metabolism ; *Mannose-6-Phosphate Isomerase/metabolism/deficiency/genetics ; Animals ; Cell Line, Tumor ; Mice ; Glycolysis ; CRISPR-Cas Systems ; },
abstract = {Radiotherapy is critical component of multidisciplinary cancer care, used as a primary and adjuvant treatment for patients with head and neck squamous cell carcinoma. This study investigates how mannose, a naturally occurring monosaccharide, combined with phosphomannose isomerase (PMI) depletion, enhances the sensitivity of HPV-negative head and neck tumour models to radiation. Isogenic PMI knockout models were generated by CRISPR/Cas9 gene editing, yielding a 20-fold increase in sensitivity to mannose in vitro, and causing significant tumour growth delay in vivo. This effect is driven by metabolic reprogramming, resulting in potent glycolytic suppression coupled with consistent depletion of ATP and glycolytic intermediates in PMI-depleted models. Functionally, these changes impede DNA damage repair following radiation, resulting in a significant increase in radiation sensitivity. Mannose and PMI ablation supressed both oxygen consumption rate and extracellular acidification, pushing cells towards a state of metabolic quiescence, effects contributing to increased radiation sensitivity under both normoxic and hypoxic conditions. In 3D-tumoursphere models, metabolic suppression by mannose and PMI depletion was shown to elevate intra-tumoursphere oxygen levels, contributing to significant in vitro oxygen-mediated radiosensitisation. These findings position PMI as a promising anti-tumour target, highlighting the potential of mannose as a metabolic radiosensitiser enhancing cancer treatment efficacy.},
}

Humans
*Radiation Tolerance/drug effects
*Mannose/metabolism/pharmacology
*Head and Neck Neoplasms/radiotherapy/pathology/metabolism
*Mannose-6-Phosphate Isomerase/metabolism/deficiency/genetics
Animals
Cell Line, Tumor
Mice
Glycolysis
CRISPR-Cas Systems

@article {pmid40259121,
year = {2025},
author = {Potlapalli, BP and Dassau, F and Fuchs, J and Sushmoy, DR and Houben, A},
title = {CRISPR-CISH: an in situ chromogenic DNA repeat detection system for research and life science education.},
journal = {Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology},
volume = {33},
number = {1},
pages = {7},
pmid = {40259121},
issn = {1573-6849},
support = {HO1779/33-1//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *DNA/genetics ; *In Situ Hybridization/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Repetitive Sequences, Nucleic Acid ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {In situ hybridization is a technique to visualize specific DNA sequences within nuclei and chromosomes. Various DNA in situ fluorescent labeling methods have been developed, which typically involve global DNA denaturation prior to the probe hybridization and often require fluorescence microscopes for visualization. Here, we report the development of a CRISPR/dCas9-mediated chromogenic in situ DNA detection (CRISPR-CISH) method that combines chromogenic signal detection with CRISPR imaging. This non-fluorescent approach uses 3' biotin-labeled tracrRNA and target-specific crRNA to form mature gRNA, which activates dCas9 to bind to target sequences. The subsequent application of streptavidin alkaline phosphatase or horseradish peroxidase generates chromogenic, target-specific signals that can be analyzed using conventional bright-field microscopes. Additionally, chromatin counterstains were identified to aid in the interpretation of CRISPR-CISH-generated target signals. This advancement makes in situ DNA detection techniques more accessible to researchers, diagnostic applications, and educational institutions in resource-limited settings.},
}

*CRISPR-Cas Systems
Humans
*DNA/genetics
*In Situ Hybridization/methods
RNA, Guide, CRISPR-Cas Systems/genetics
*Repetitive Sequences, Nucleic Acid
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40259107,
year = {2025},
author = {Yang, J and Song, J and Feng, Z and Ma, Y},
title = {Application of CRISPR-Cas9 in microbial cell factories.},
journal = {Biotechnology letters},
volume = {47},
number = {3},
pages = {46},
pmid = {40259107},
issn = {1573-6776},
support = {BY2021KYQD02//Binzhou Medical University/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Metabolic Engineering/methods ; *Bacteria/genetics/metabolism ; },
abstract = {Metabolically engineered bacterial strains are rapidly emerging as a pivotal focus in the biosynthesis of an array of bio-based ingredients. Presently, CRISPR (clustered regularly interspaced short palindromic repeats) and its associated RNA-guided endonuclease (Cas9) are regarded as the most promising tool, having ushered in a transformative advancement in genome editing. Because of CRISPR-Cas9's accuracy and adaptability, metabolic engineers are now able to create novel regulatory systems, optimize pathways more effectively, and make extensive genome-scale alterations. Nevertheless, there are still obstacles to overcome in the application of CRISPR-Cas9 in novel microorganisms, particularly those industrial microorganism hosts that are resistant to traditional genetic manipulation techniques. How to further extend CRISPR-Cas9 to these microorganisms is an urgent problem to be solved. This article first introduces the mechanism and application of CRISPR-Cas9, and then discusses how to optimize CRISPR-Cas9 as a genome editing tool, including how to reduce off-target effects and how to improve targeting efficiency by optimizing design. Through offering a comprehensive perspective on the revolutionary effects of CRISPR-Cas9 in microbial cell factories, we hope to stimulate additional research and development in this exciting area.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Metabolic Engineering/methods
*Bacteria/genetics/metabolism

@article {pmid40258142,
year = {2025},
author = {Filsinger, GT and Mychack, A and Lyerly, E and Henriksen, C and Bartlett, TM and Kuchwara, H and Eitzinger, S and Bernhardt, TG and Walker, S and Church, GM and Wannier, TM},
title = {A diverse single-stranded DNA-annealing protein library enables efficient genome editing across bacterial phyla.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {17},
pages = {e2414342122},
doi = {10.1073/pnas.2414342122},
pmid = {40258142},
issn = {1091-6490},
mesh = {*Gene Editing/methods ; *DNA, Single-Stranded/genetics/metabolism ; *Genome, Bacterial ; Bacteriophages/genetics ; CRISPR-Cas Systems ; Gene Library ; *Bacteria/genetics/classification ; *Viral Proteins/genetics/metabolism ; *DNA-Binding Proteins/genetics/metabolism ; Phylogeny ; Homologous Recombination ; Staphylococcus aureus/genetics ; },
abstract = {Genome modification is essential for studying and engineering bacteria, yet making efficient modifications to most species remains challenging. Bacteriophage-encoded single-stranded DNA-annealing proteins (SSAPs) can facilitate efficient genome editing by homologous recombination, but their typically narrow host range limits broad application. Here, we demonstrate that a single library of 227 SSAPs enables efficient genome-editing across six diverse bacteria from three divergent classes: Actinomycetia (Mycobacterium smegmatis and Corynebacterium glutamicum), Alphaproteobacteria (Agrobacterium tumefaciens and Caulobacter crescentus), and Bacilli (Lactococcus lactis and Staphylococcus aureus). Surprisingly, the most effective SSAPs frequently originated from phyla distinct from their bacterial hosts, challenging the assumption that phylogenetic relatedness is necessary for recombination efficiency, and supporting the value of a large unbiased library. Across these hosts, the identified SSAPs enable genome modifications requiring efficient homologous recombination, demonstrated through three examples. First, we use SSAPs with Cas9 in C. crescentus to introduce single amino acid mutations with >70% efficiency. Second, we adapt SSAPs for dsDNA editing in C. glutamicum and S. aureus, enabling one-step gene knockouts using PCR products. Finally, we apply SSAPs for multiplexed editing in S. aureus to precisely map the interaction between a conserved protein and a small-molecule inhibitor. Overall, this library-based SSAP screen expands engineering capabilities across diverse, previously recalcitrant microbes, enabling efficient genetic manipulation for both fundamental research and biotechnological applications.},
}

*Gene Editing/methods
*DNA, Single-Stranded/genetics/metabolism
*Genome, Bacterial
Bacteriophages/genetics
CRISPR-Cas Systems
Gene Library
*Bacteria/genetics/classification
*Viral Proteins/genetics/metabolism
*DNA-Binding Proteins/genetics/metabolism
Phylogeny
Homologous Recombination
Staphylococcus aureus/genetics

@article {pmid40120517,
year = {2025},
author = {Wang, Q and Chen, X and Li, Y and Yang, S and Fan, S and Xia, J and Wu, H},
title = {Protein-inorganic hybrid flowers with a two-stage accelerated strategy for stimulated activation of CRISPR/Cas12a enhance polynucleotide kinase biosensing.},
journal = {Talanta},
volume = {292},
number = {},
pages = {127981},
doi = {10.1016/j.talanta.2025.127981},
pmid = {40120517},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Polynucleotide 5'-Hydroxyl-Kinase/metabolism/analysis ; Humans ; DNA Nucleotidylexotransferase/metabolism/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Polynucleotide kinases (PNK) play a crucial role in DNA damage repair and are closely associated with specific diseases, making them promising targets for therapeutic intervention. In this study, we propose a two-stage accelerated strategy that utilizes protein-inorganic hybrid flowers (PHFs) to enhance the performance of the terminal deoxynucleotidyl transferase (TdT)-combined CRISPR/Cas12a system (TCS) for efficient detection of PNK activity. In TCS, the participation of PHFs confines the substrate probes (SPs) to a limited space, thereby significantly enhancing the local concentration of phosphorylated 3' termini of SPs and effectively promoting the enzymatic reaction kinetics as the first step in the accelerated strategy. Upon encountering the target PNK, the phosphorylated 3' termini were promptly recognized and dephosphorylated to 3'-OH termini. Subsequently, TdT catalyzed the assembly of deoxyadenosine triphosphates (dATPs) without a template, rapidly activating the CRISPR/Cas12a system by forming multiple polyadenine (poly-A) chains. PHF-fixed poly-A chains then substantially boosted the localized concentration of CRISPR/Cas12a systems and vastly enhanced their efficacy in cleaving reporter nucleic acids. Our findings indicated that the spatial confinement effect facilitated by PHFs promoted frequent molecular collisions and accelerated multiple enzymatic reactions. The developed sensing strategy allows for the detection of PNK activity within a linear range of 0.001-1 U/mL, with a detection limit of 1.82 × 10[-4] U/mL. Additionally, this strategy has been successfully applied to detect PNK activity in cell extracts and to screen for PNK inhibitors. Owing to these advantages, PNK can be rapidly and accurately detected with a high sensitivity, specificity, and biostability.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems
*Polynucleotide 5'-Hydroxyl-Kinase/metabolism/analysis
Humans
DNA Nucleotidylexotransferase/metabolism/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid39930103,
year = {2025},
author = {Ling, S and Zhang, X and Dai, Y and Jiang, Z and Zhou, X and Lu, S and Qian, X and Liu, J and Selfjord, N and Satir, TM and Lundin, A and Touza, JL and Firth, M and Van Zuydam, N and Bilican, B and Akcakaya, P and Hong, J and Cai, Y},
title = {Customizable virus-like particles deliver CRISPR-Cas9 ribonucleoprotein for effective ocular neovascular and Huntington's disease gene therapy.},
journal = {Nature nanotechnology},
volume = {20},
number = {4},
pages = {543-553},
pmid = {39930103},
issn = {1748-3395},
support = {no. 31971364//National Natural Science Foundation of China (National Science Foundation of China)/ ; no. 32370148//National Natural Science Foundation of China (National Science Foundation of China)/ ; no. 81970766//National Natural Science Foundation of China (National Science Foundation of China)/ ; no. 82171102//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Huntington Disease/therapy/genetics ; *CRISPR-Cas Systems/genetics ; Animals ; *Genetic Therapy/methods ; Humans ; Gene Editing/methods ; *Ribonucleoproteins/genetics ; Induced Pluripotent Stem Cells/metabolism ; Huntingtin Protein/genetics ; HEK293 Cells ; *Virion/genetics ; Mice ; Disease Models, Animal ; },
abstract = {In vivo CRISPR gene editing holds enormous potential for various diseases. Ideally, CRISPR delivery should be cell type-specific and time-restricted for optimal efficacy and safety, but customizable methods are lacking. Here we develop a cell-tropism programmable CRISPR-Cas9 ribonucleoprotein delivery system (RIDE) based on virus-like particles. The efficiency of RIDE was comparable to that of adeno-associated virus and lentiviral vectors and higher than lipid nanoparticles. RIDE could be readily reprogrammed to target dendritic cells, T cells and neurons, and significantly ameliorated the disease symptoms in both ocular neovascular and Huntington's disease models via cell-specific gene editing. In addition, RIDE could efficiently edit the huntingtin gene in patients' induced pluripotent stem cell-derived neurons and was tolerated in non-human primates. This study is expected to facilitate the development of in vivo CRISPR therapeutics.},
}

*Huntington Disease/therapy/genetics
*CRISPR-Cas Systems/genetics
Animals
*Genetic Therapy/methods
Humans
Gene Editing/methods
*Ribonucleoproteins/genetics
Induced Pluripotent Stem Cells/metabolism
Huntingtin Protein/genetics
HEK293 Cells
*Virion/genetics
Mice
Disease Models, Animal

@article {pmid39742661,
year = {2025},
author = {Huang, X and Feng, X and Yan, YH and Xu, D and Wang, K and Zhu, C and Dong, MQ and Huang, X and Guang, S and Chen, X},
title = {Compartmentalized localization of perinuclear proteins within germ granules in C. elegans.},
journal = {Developmental cell},
volume = {60},
number = {8},
pages = {1251-1270.e3},
doi = {10.1016/j.devcel.2024.12.016},
pmid = {39742661},
issn = {1878-1551},
mesh = {Animals ; *Caenorhabditis elegans/metabolism/genetics ; *Caenorhabditis elegans Proteins/metabolism/genetics ; *Germ Cells/metabolism ; *Cytoplasmic Granules/metabolism ; Cell Nucleus/metabolism ; CRISPR-Cas Systems ; },
abstract = {Germ granules, or nuage, are RNA-rich condensates that are often docked on the cytoplasmic surface of germline nuclei. C. elegans perinuclear germ granules are composed of multiple subcompartments, including P granules, Mutator foci, Z granules, SIMR foci, P -bodies, and E granules. Although many perinuclear proteins have been identified, their precise localization within the subcompartments of the germ granule is still unclear. Here, we systematically labeled perinuclear proteins with fluorescent tags via CRISPR-Cas9 technology. Using this nematode strain library, we identified a series of proteins localized in Z or E granules and extended the characterization of the D granule. Finally, we found that the LOTUS domain protein MIP-1/EGGD-1 regulated the multiphase organization of the germ granule. Overall, our work identified the germ-granule architecture and redefined the compartmental localization of perinuclear proteins. Additionally, the library of genetically modified nematode strains will facilitate research on C. elegans germ granules.},
}

Animals
*Caenorhabditis elegans/metabolism/genetics
*Caenorhabditis elegans Proteins/metabolism/genetics
*Germ Cells/metabolism
*Cytoplasmic Granules/metabolism
Cell Nucleus/metabolism
CRISPR-Cas Systems

@article {pmid40255449,
year = {2025},
author = {Harshini, P and Varghese, R and Pachamuthu, K and Ramamoorthy, S},
title = {Enhanced pigment production from plants and microbes: a genome editing approach.},
journal = {3 Biotech},
volume = {15},
number = {5},
pages = {129},
pmid = {40255449},
issn = {2190-572X},
abstract = {Pigments are known for their vital roles in the growth and development of plants and microbes. In addition, they are also an imperative component of several industries, including textiles, foods, and pharmaceuticals, owing to their immense colours and therapeutic potential. Conventionally, pigments are obtained from plant resources, and the advent of in-vitro propagation techniques boosted the massive production. However, it could not meet the booming demand, leading to the incorporation of new genetic engineering tools. This review focuses on the role of various genetic engineering techniques in enhancing pigment production in plants and microorganisms. It also critically analyzes the efficacy and bottlenecks of these techniques in augmenting pigment biosynthesis. Furthermore, the use of microbes as pigment biofactories and the prospects in the field of genome editing to augment pigment synthesis are discussed. The limitations in the existing techniques underline the need for advanced genome editing strategies to broaden the mass production of pigments to meet the surging needs.},
}

@article {pmid40253348,
year = {2025},
author = {Hurtado, JE and Schieferecke, AJ and Halperin, SO and Guan, J and Aidlen, D and Schaffer, DV and Dueber, JE},
title = {Nickase fidelity drives EvolvR-mediated diversification in mammalian cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3723},
pmid = {40253348},
issn = {2041-1723},
support = {T32 GM139780/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; Animals ; CRISPR-Cas Systems/genetics ; *DNA-Directed DNA Polymerase/genetics/metabolism ; *Deoxyribonuclease I/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; HEK293 Cells ; *Directed Molecular Evolution/methods ; Mutation ; Mutagenesis ; },
abstract = {In vivo genetic diversifiers have previously enabled efficient searches of genetic variant fitness landscapes for continuous directed evolution. However, existing genomic diversification modalities for mammalian genomic loci exclusively rely on deaminases to generate transition mutations within target loci, forfeiting access to most missense mutations. Here, we engineer CRISPR-guided error-prone DNA polymerases (EvolvR) to diversify all four nucleotides within genomic loci in mammalian cells. We demonstrate that EvolvR generates both transition and transversion mutations throughout a mutation window of at least 40 bp and implement EvolvR to evolve previously unreported drug-resistant MAP2K1 variants via substitutions not achievable with deaminases. Moreover, we discover that the nickase's mismatch tolerance limits EvolvR's mutation window and substitution biases in a gRNA-specific fashion. To compensate for gRNA-to-gRNA variability in mutagenesis, we maximize the number of gRNA target sequences by incorporating a PAM-flexible nickase into EvolvR. Finally, we find a strong correlation between predicted free energy changes underlying R-loop formation and EvolvR's performance using a given gRNA. The EvolvR system diversifies all four nucleotides to enable the evolution of mammalian cells, while nuclease and gRNA-specific properties underlying nickase fidelity can be engineered to further enhance EvolvR's mutation rates.},
}

Humans
Animals
CRISPR-Cas Systems/genetics
*DNA-Directed DNA Polymerase/genetics/metabolism
*Deoxyribonuclease I/metabolism/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
HEK293 Cells
*Directed Molecular Evolution/methods
Mutation
Mutagenesis

@article {pmid40253208,
year = {2025},
author = {Xin, Y and Guo, T and Qiao, M},
title = {Current application and future prospects of CRISPR-Cas in lactic acid Bacteria: A review.},
journal = {Food research international (Ottawa, Ont.)},
volume = {209},
number = {},
pages = {116315},
doi = {10.1016/j.foodres.2025.116315},
pmid = {40253208},
issn = {1873-7145},
mesh = {*CRISPR-Cas Systems ; *Lactobacillales/genetics/metabolism ; Gene Editing/methods ; *Food Microbiology ; Probiotics ; Fermentation ; Metabolic Engineering ; },
abstract = {Lactic acid bacteria (LABs) have a long history of use in food and beverages fermentation. Recently, several LABs have gained attention as starter or non-starter cultures and probiotics for making functional fermented foods, which have the potential to enhance human health. In addition, certain LABs show great potential as microbial cell factories for producing food-related chemicals. However, enhancing the outcomes of starter and non-starter cultures, exploring the complicated probiotic mechanism of LABs, and engineering strains to enhance the yields of high-value compounds for precision fermentation remains challenging due to the time-consuming and labor-intensive current genome editing tools. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) system, originally an adaptive immune system in bacteria, has revolutionized genome editing, metabolic engineering and synthetic biology. Its versatility has resulted in extensive applications across diverse organisms. The widespread distribution of CRISPR-Cas systems and the diversity of CRISPR arrays in LAB genomes highlight their potential for studying the evolution of LABs. This review discusses the current advancement of CRISPR-Cas systems in engineering LABs for food application. Moreover, it outlines future research directions aimed at harnessing CRISPR-Cas systems to advance lactic acid bacterial research and drive innovation in food science.},
}

*CRISPR-Cas Systems
*Lactobacillales/genetics/metabolism
Gene Editing/methods
*Food Microbiology
Probiotics
Fermentation
Metabolic Engineering

@article {pmid40252747,
year = {2025},
author = {Zou, W and Huo, B and Tu, Y and Zhu, Y and Hu, Y and Li, Q and Yu, X and Liu, B and Tang, W and Tan, S and Xiao, H},
title = {Metabolic reprogramming by chemo-gene co-delivery nanoparticles for chemo-immunotherapy in head and neck squamous cell carcinoma.},
journal = {Acta biomaterialia},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.actbio.2025.04.031},
pmid = {40252747},
issn = {1878-7568},
abstract = {The therapeutic effects of platinum-based drugs are closely linked to the dysregulation of tumor metabolic-immune microenvironment, particularly aberrant lactate accumulation. Herein, we engineered multifunctional nanoparticles (PPPt[IV] NPs) through electrostatic self-assembly of poly(β-amino ester) to co-encapsulate the cisplatin prodrug (Pt[IV]) and CRISPR/Cas9-PKM2 plasmids. Mechanistically, PPPt[IV] NPs efficiently entered cells via endocytosis, followed by escape from lysosomal degradation and cargo release. The reduction of Pt[IV] prodrug to the active Pt[II] via GSH depletion induced DNA damage and ROS upregulation, thereby triggering apoptosis. Concurrently, CRISPR/Cas9-mediated PKM2 knockdown suppressed the Warburg effect, resulting in reduced lactate production and downregulated expression of HIF-1α and PD-L1. These alterations drove immune microenvironment remodeling through enhanced dendritic cell maturation, polarized M1 macrophages, and altered cytokine profiles (characterized by upregulation of IFN-γ, TNF-α, and IL-12 alongside suppression of IL-10), ultimately activating T cell-mediated antitumor immunity. Compared to conventional cisplatin, PPPt[IV] NPs demonstrated superior efficacy against both primary and recurrent tumors while reducing nephrotoxicity through synergistic chemo-immunotherapeutic effects, offering a valuable strategy for HNSCC treatment. STATEMENT OF SIGNIFICANCE: This study engineered an innovative nanoplatform (PPPt[IV]) that synergistically integrates a Pt[IV] prodrug with a CRISPR/Cas-PKM2 plasmid for treating head and neck squamous cell carcinoma. By simultaneously enhancing DNA damage and reversing lactate-mediated immunosuppression, PPPt[IV] nanoplatform achieved chemo-immunotherapy that showed greater suppression of primary and recurrent tumors with reduced renal toxicity compared to cisplatin. This nanotechnology-driven strategy provides valuable insights for the combination of platinum-based drugs with immunometabolic interventions.},
}

@article {pmid40251402,
year = {2025},
author = {Gonzalez, E and Anderson, MAE and Ang, JXD and Nevard, K and Shackleford, L and Larrosa-Godall, M and Leftwich, PT and Alphey, L},
title = {Optimization of SgRNA expression with RNA pol III regulatory elements in Anopheles stephensi.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {13408},
pmid = {40251402},
issn = {2045-2322},
support = {INV-008549/GATES/Gates Foundation/United States ; BBS/E/I/00007033, BBS/E/I/00007038, and BBS/E/I/00007039/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Anopheles/genetics ; Animals ; *RNA Polymerase III/genetics ; CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic ; Mosquito Vectors/genetics ; },
abstract = {Anopheles stephensi, a major Asian malaria vector, is invading Africa and has been implicated in recent outbreaks of urban malaria. Control of this species is key to eliminating malaria in Africa. Genetic control strategies, and CRISPR/Cas9-based gene drives are emerging as promising species-specific, environmentally friendly, scalable, affordable methods for pest control. To implement these strategies, a key parameter to optimize for high efficiency is the spatiotemporal control of Cas9 and the gRNA. Here, we assessed the ability of four RNA Pol III promoters to bias the inheritance of a gene drive element inserted into the cd gene of An. stephensi. We determined the homing efficiency and examined eye phenotype as a proxy for non-homologous end joining (NHEJ) events in somatic tissue. We found all four promoters to be active, with mean inheritance rates up to 99.8%. We found a strong effect of the Cas9-bearing grandparent (grandparent genotype), likely due to maternally deposited Cas9.},
}

*Anopheles/genetics
Animals
*RNA Polymerase III/genetics
CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
Promoter Regions, Genetic
Mosquito Vectors/genetics

@article {pmid40251060,
year = {2025},
author = {Huang, W and Ruyechan, MC and Ralston, KS},
title = {Work with me here: variations in genome content and emerging genetic tools in Entamoeba histolytica.},
journal = {Trends in parasitology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.pt.2025.03.010},
pmid = {40251060},
issn = {1471-5007},
abstract = {Entamoeba histolytica is the causative agent of amoebiasis, a significant source of morbidity and mortality in developing nations. Despite this, E. histolytica is understudied, leading to few treatment options and a poor understanding of pathogenesis. Genetic tools have historically been limited. By applying modern approaches, it was recently revealed that the genome is aneuploid. Interestingly, gene expression levels do not correlate with ploidy, potentially highlighting the importance of RNAi in gene regulation. Characterization of the RNAi pathway has led to potent tools for targeted gene knockdown, and the advent of RNAi-based forward genetics. CRISPR/Cas tools for editing the endogenous genome are an exciting possibility on the horizon. We celebrate the gains that have made E. histolytica tractable and anticipate continued advances.},
}

@article {pmid40250956,
year = {2025},
author = {Eidelman, M and Eisenberg, E and Levanon, EY},
title = {Global quantification of off-target activity by base editors.},
journal = {Methods in enzymology},
volume = {713},
number = {},
pages = {255-270},
doi = {10.1016/bs.mie.2024.11.036},
pmid = {40250956},
issn = {1557-7988},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Adenosine Deaminase/genetics/metabolism ; },
abstract = {Base editors are engineered deaminases combined with CRISPR components. These engineered deaminases are designed to target specific sites within DNA or RNA to make a precise change in the molecule. In therapeutics, they hold promise for correcting mutations associated with genetic diseases. However, a key challenge is minimizing unintended edits at off-target sites, which could lead to harmful mutations. Researchers are actively addressing this concern through a variety of optimization efforts that aim to improve the precision of base editors and minimize off-target activity. Here, we examine the various types of off-target activity, and the methods used to evaluate them. Current methods for finding off-target activity focus on identifying similar sequences in the genome or in the transcriptome, assuming the guide RNA misdirects the editor. The main method presented here, that was originally developed to quantify editing levels mediated by the ADAR enzyme, takes a different approach, investigating the inherent activity of base editors themselves, which might lead to off-target edits beyond sequence similarity. The editing index tool quantifies global off-target editing, eliminates the need to detect individual off-target sites, and allows for assessment of the global load of mutations.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
Adenosine Deaminase/genetics/metabolism

@article {pmid40195937,
year = {2025},
author = {Wu, Q and Li, S and Long, X and Liu, L and Zhao, Q and Cui, Y and Zhang, Y and He, Y},
title = {β-Galactosidase-Mediated, Mn[2+]-Activated CRISPR/Cas12a Cascade Reaction for Immunosorbent Assay of Carbendazim.},
journal = {Analytical chemistry},
volume = {97},
number = {15},
pages = {8402-8410},
doi = {10.1021/acs.analchem.4c07110},
pmid = {40195937},
issn = {1520-6882},
mesh = {*Benzimidazoles/analysis/metabolism ; *CRISPR-Cas Systems ; *beta-Galactosidase/metabolism/chemistry ; *Carbamates/analysis ; Enzyme-Linked Immunosorbent Assay/methods ; *Manganese/chemistry/metabolism ; Manganese Compounds/chemistry ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The CRISPR/Cas12a system is an emerging enzymatic tool for the development of enzyme-linked immunosorbent assay (ELISA) methods, owing to its robust signal amplification capability. Currently, most CRISPR/Cas12a-based ELISA approaches rely on strategies that convert target detection into nucleic acid analysis. This report presents a novel enzymatic cascade reaction for signal transduction and amplification in the development of a CRISPR/Cas12a-based ELISA method, utilizing β-galactosidase (β-gal)-mediated activation of the CRISPR/Cas12a system. Carbendazim (CBD), a widely used and versatile broad-spectrum benzimidazole fungicide, was chosen as the model analyte. In the absence of CBD, streptavidin-labeled β-gal is captured by a biotinylated secondary antibody immobilized on the microplate. The captured β-gal catalyzes the hydrolysis of p-aminophenyl β-D-galactopyranoside to generate p-aminophenol. This compound subsequently facilitates the decomposition of MnO2 nanosheets, leading to the generation of Mn[2+] ions. The Mn[2+] ions modulate the activity of the CRISPR/Cas12a system, thus producing high fluorescence in the detection solution. In the presence of CBD, the amount of β-gal captured on the microplate is reduced, thereby preventing effective cleavage of the reporter molecule by Cas12a, which results in a low fluorescence signal. After systematically optimizing experimental conditions, the developed method successfully detected CBD, demonstrating high sensitivity, selectivity, and applicability in complex food matrices. In comparison to the traditional nucleic acid-activated CRISPR/Cas12a-based ELISA method, our approach, which integrates β-gal-mediated, Mn[2+]-activated CRISPR/Cas12a cascade reactions into ELISA, exhibits superior analytical performance, thereby broadening the applicability of CRISPR/Cas12a for sensitive and convenient small-molecule analysis.},
}

*Benzimidazoles/analysis/metabolism
*CRISPR-Cas Systems
*beta-Galactosidase/metabolism/chemistry
*Carbamates/analysis
Enzyme-Linked Immunosorbent Assay/methods
*Manganese/chemistry/metabolism
Manganese Compounds/chemistry
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40172020,
year = {2025},
author = {Cheng, HJ and Liu, Y and Li, HD and Yin, BC and Ma, PQ and Ye, BC},
title = {Catalytic hairpin assembly-coupled CRISPR/Cas12a biosensor for sensitive detection of melamine in dairy products.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {34},
pages = {6300-6303},
doi = {10.1039/d5cc00114e},
pmid = {40172020},
issn = {1364-548X},
mesh = {*Triazines/analysis ; *Biosensing Techniques/methods ; *Dairy Products/analysis ; *CRISPR-Cas Systems ; Catalysis ; *Food Contamination/analysis ; Limit of Detection ; },
abstract = {We combined catalytic hairpin assembly (CHA) with the Cas12a system for detecting melamine adulteration. This system involved two-step signal conversion and two-level amplification, boosting the sensor's versatility and sensitivity. The sensor showed excellent specificity and applicability for melamine detection in dairy products, and was broadened to viral nucleic acid detection.},
}

*Triazines/analysis
*Biosensing Techniques/methods
*Dairy Products/analysis
*CRISPR-Cas Systems
Catalysis
*Food Contamination/analysis
Limit of Detection

@article {pmid40160100,
year = {2025},
author = {Yang, S and Ren, L and Fan, N and Wang, S and Shen, B and Liu, Z and Li, X and Ding, S},
title = {CRISPR-Cas12a with split crRNA for the direct and sensitive detection of microRNA.},
journal = {The Analyst},
volume = {150},
number = {9},
pages = {1884-1890},
doi = {10.1039/d5an00142k},
pmid = {40160100},
issn = {1364-5528},
mesh = {*MicroRNAs/genetics/blood/analysis ; *CRISPR-Cas Systems/genetics ; Humans ; Limit of Detection ; *Endodeoxyribonucleases/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; DNA/genetics/chemistry ; },
abstract = {microRNAs (miRNAs) have been identified as potential biomarkers. Despite the prevalence of quantitative PCR in the field of miRNA detection, this technology is encumbered by the complexity of its methodology. This study presents a novel CRISPR/Cas12a-based method for the direct and sensitive detection of miRNA-21 using split crRNA. The system comprises Cas12a protein, crRNA-handle, and activator DNA complementary to the target miRNA. In the presence of the target miRNA, it binds to the activator DNA, forming a duplex. The formed duplex, in conjunction with the crRNA-handle, activates Cas12a's trans-cleavage activity. This leads to cleavage of a fluorescent reporter, generating an enhanced signal. The method enables direct RNA detection without reverse transcription or sample amplification, offering simplicity and efficiency. This method demonstrates high sensitivity with a minimum detectable limit of 5 pM. Furthermore, the method's specificity is substantiated by its capacity to discern point mutations in miRNA. This system has been shown to quantitatively analyse miRNA-21 levels present within serum, as evidenced by the recovery experiment. Therefore, the method's simplicity, stability, and cost-effectiveness render it a powerful tool for nucleic acid detection, with potential for clinical applications.},
}

*MicroRNAs/genetics/blood/analysis
*CRISPR-Cas Systems/genetics
Humans
Limit of Detection
*Endodeoxyribonucleases/genetics/metabolism
*CRISPR-Associated Proteins/genetics/metabolism
*Bacterial Proteins/genetics/metabolism
DNA/genetics/chemistry

@article {pmid40135711,
year = {2025},
author = {He, S and Lin, W and Liu, X and Li, F and Liang, H and Xu, H and Lu, C and Xing, C},
title = {A DNA concatemer-encoded CRISPR/Cas12a fluorescence sensor for sensitive detection of Pb[2+] based on DNAzymes.},
journal = {The Analyst},
volume = {150},
number = {9},
pages = {1778-1784},
doi = {10.1039/d5an00189g},
pmid = {40135711},
issn = {1364-5528},
mesh = {*Lead/analysis ; *DNA, Catalytic/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; *DNA/chemistry/genetics ; Spectrometry, Fluorescence/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/metabolism/genetics ; *Water Pollutants, Chemical/analysis ; Fluorescence ; Animals ; Milk/chemistry ; Bacterial Proteins ; },
abstract = {Lead pollution presents a significant threat to ecological systems and human health, underscoring the urgent need for highly sensitive detection methods. Herein, we introduce a novel DNA concatemer-encoded CRISPR/Cas12a fluorescence sensor (MDD-Cas12a) for sensitive detection of Pb[2+] based on DNAzymes. To accomplish this, we designed a substrate strand containing a long DNA concatemer encoding multiple protospacer adjacent motifs (PAMs) and protospacer sequences for activation of the CRISPR/Cas12a system. The DNA concatemer was subsequently anchored to the surface of magnetic beads (MBs) to fabricate a MBs-DNA concatemer nanoprobe. In the presence of Pb[2+], the DNAzyme structure catalyzes the cleavage of the substrate strand, leading to the release of DNA concatemers. Following magnetic separation, the released DNA concatemers significantly activate the non-specific trans-cleavage activity of the Cas12a/crRNA complex. The fluorescence reporter DNA is then completely cleaved by the activated Cas12a/crRNA complex, and the Pb[2+] concentration in the sample can be quantified by measuring the fluorescence signal. By harnessing the specific recognition capability of DNAzymes for Pb[2+], the programmability of DNA concatemers, and the self-amplification features of the CRISPR/Cas12a system, the MDD-Cas12a platform demonstrates high sensitivity and specificity for detecting Pb[2+] in milk and lake water samples.},
}

*Lead/analysis
*DNA, Catalytic/chemistry/genetics/metabolism
*CRISPR-Cas Systems
*Biosensing Techniques/methods
Limit of Detection
*DNA/chemistry/genetics
Spectrometry, Fluorescence/methods
*CRISPR-Associated Proteins/genetics/metabolism
*Endodeoxyribonucleases/metabolism/genetics
*Water Pollutants, Chemical/analysis
Fluorescence
Animals
Milk/chemistry
Bacterial Proteins

@article {pmid39302534,
year = {2025},
author = {Wiley, L and Cheek, M and LaFar, E and Ma, X and Sekowski, J and Tanguturi, N and Iltis, A},
title = {The Ethics of Human Embryo Editing via CRISPR-Cas9 Technology: A Systematic Review of Ethical Arguments, Reasons, and Concerns.},
journal = {HEC forum : an interdisciplinary journal on hospitals' ethical and legal issues},
volume = {37},
number = {2},
pages = {267-303},
pmid = {39302534},
issn = {1572-8498},
mesh = {Humans ; *Gene Editing/ethics/methods ; *CRISPR-Cas Systems ; },
abstract = {The possibility of editing the genomes of human embryos has generated significant discussion and interest as a matter of science and ethics. While it holds significant promise to prevent or treat disease, research on and potential clinical applications of human embryo editing also raise ethical, regulatory, and safety concerns. This systematic review included 223 publications to identify the ethical arguments, reasons, and concerns that have been offered for and against the editing of human embryos using CRISPR-Cas9 technology. We identified six major themes: risk/harm; potential benefit; oversight; informed consent; justice, equity, and other social considerations; and eugenics. We explore these themes and provide an overview and analysis of the critical points in the current literature.},
}

Humans
*Gene Editing/ethics/methods
*CRISPR-Cas Systems

@article {pmid38858586,
year = {2025},
author = {Pandey, S and Gao, XD and Krasnow, NA and McElroy, A and Tao, YA and Duby, JE and Steinbeck, BJ and McCreary, J and Pierce, SE and Tolar, J and Meissner, TB and Chaikof, EL and Osborn, MJ and Liu, DR},
title = {Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing.},
journal = {Nature biomedical engineering},
volume = {9},
number = {1},
pages = {22-39},
pmid = {38858586},
issn = {2157-846X},
support = {P01 CA065493/CA/NCI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R01 HL160970/HL/NHLBI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; Liu investigatorship//Howard Hughes Medical Institute (HHMI)/ ; U01 AI142756/AI/NIAID NIH HHS/United States ; R01 EB031172/EB/NIBIB NIH HHS/United States ; R01 AR063070/AR/NIAMS NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; *Recombinases/genetics/metabolism ; HEK293 Cells ; Integrases/genetics/metabolism ; Animals ; CRISPR-Cas Systems ; },
abstract = {Methods for the targeted integration of genes in mammalian genomes suffer from low programmability, low efficiencies or low specificities. Here we show that phage-assisted continuous evolution enhances prime-editing-assisted site-specific integrase gene editing (PASSIGE), which couples the programmability of prime editing with the ability of recombinases to precisely integrate large DNA cargoes exceeding 10 kilobases. Evolved and engineered Bxb1 recombinase variants (evoBxb1 and eeBxb1) mediated up to 60% donor integration (3.2-fold that of wild-type Bxb1) in human cell lines with pre-installed recombinase landing sites. In single-transfection experiments at safe-harbour and therapeutically relevant sites, PASSIGE with eeBxb1 led to an average targeted-gene-integration efficiencies of 23% (4.2-fold that of wild-type Bxb1). Notably, integration efficiencies exceeded 30% at multiple sites in primary human fibroblasts. PASSIGE with evoBxb1 or eeBxb1 outperformed PASTE (for 'programmable addition via site-specific targeting elements', a method that uses prime editors fused to recombinases) on average by 9.1-fold and 16-fold, respectively. PASSIGE with continuously evolved recombinases is an unusually efficient method for the targeted integration of genes in mammalian cells.},
}

Humans
*Gene Editing/methods
*Recombinases/genetics/metabolism
HEK293 Cells
Integrases/genetics/metabolism
Animals
CRISPR-Cas Systems

@article {pmid40250676,
year = {2025},
author = {Gaba, S and Sahu, M and Chauhan, N and Jain, U},
title = {Transforming growth factor alpha: Key insights into physiological role, cancer therapeutics, and biomarker potential (A review).},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {143212},
doi = {10.1016/j.ijbiomac.2025.143212},
pmid = {40250676},
issn = {1879-0003},
abstract = {Transforming Growth Factor Alpha (TGF-α) is a critical member of the epidermal growth factor (EGF) family and a key regulator of various physiological processes, including cellular proliferation, survival, differentiation, wound repair, and tissue regeneration. Deficiencies or mutations in TGF-α have been associated with impaired tissue development and organ growth, underscoring its critical role in maintaining normal and healthy physiology. Alterations in its levels are frequently implicated in the neoplastic transformation of cells, contributing to cancer development. Several strategies for targeting TGF-α in cancer therapy have been explored, such as the use of antibodies, recombinant proteins, oligonucleotide-mediated interference in ligand synthesis, ligand sequestration via binding proteins, and modulation of the signal transduction pathway. Furthermore, there is growing interest in the potential of TGF-α as a diagnostic or prognostic biomarker for cancer. This review delves into the role of TGF-α in normal physiology and its involvement in carcinogenesis. It highlights therapies targeting TGF-α and explores future directions in targeting TGF-α /EGFR signaling using advancing approaches, including nanoparticle-based drug delivery systems, CRISPR-Cas genome editing tool, PROTAC, and combination therapies. By bringing attention to this molecule, we aim to explore its untapped potential in cancer treatment and inspire further research into its promising applications across related fields. While recent studies highlight the promise of TGF-α as a clinical biomarker, further research is needed to validate its specificity and integration into personalized medicine. By providing a comprehensive overview of TGF-α in both normal and pathological contexts, this review aims to offer new insights into its translational applications in cancer therapeutics and biomarker discovery.},
}

@article {pmid40249536,
year = {2025},
author = {Wang, F and Chen, Y and Huang, R and Lu, D and Zhang, J and Yang, Y and Dang, H and Liu, M and Chen, Z and Sun, X and Wang, Z},
title = {Identification of SURF4 and RALGAPA1 as promising therapeutic targets in glioblastoma and pan-cancer through integrative multi-omics, CRISPR-Cas9 screening and prognostic meta-analysis.},
journal = {Cancer immunology, immunotherapy : CII},
volume = {74},
number = {6},
pages = {175},
pmid = {40249536},
issn = {1432-0851},
support = {No. 82171309//National Natural Science Foundation of China/ ; No. 82201445//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Glioblastoma/genetics/pathology/mortality ; Prognosis ; *Brain Neoplasms/genetics/pathology ; *Membrane Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Biomarkers, Tumor/genetics ; Tumor Microenvironment ; Gene Expression Regulation, Neoplastic ; Multiomics ; },
abstract = {Glioblastoma (GBM) is the most aggressive and malignant type of primary brain tumor, with a median survival time of less than two years and a uniformly poor prognosis, despite multimodal therapeutic approaches, which highlights an urgent need for novel therapeutic targets. In this study, by integrative multi-omics analysis from CPTAC database, DepMap database and seven independent GBM cohorts, four hub genes (CD44, SURF4, IGSF3 and RALGAPA1) were identified as essential genes regulated by cancer driver genes with robust prognostic value. GBM multi-omics data from public and in-house cohorts validated that CD44 and SURF4 might be synthetic lethal partners of loss-of-function tumor suppressor genes. Analysis for immune-related pathway activity revealed complex regulation relationships of the four hub genes in tumor microenvironment (TME). Further investigation on SURF4 in pathway activity, immune therapy response and drug sensitivity proposed that SURF4 emerged as a promising therapeutic target for GBM, even for pan-cancer. Pan-cancer multi-omics exploration suggested that RALGAPA1 may be a tumor suppressor gene. By screening the first-generation and second-generation DepMap database, four genes (CCDC106, GAL3ST1, GDI2 and HSF1) might be considered as synthetic targets after mutation of RALGAPA1 as a tumor suppressor gene.},
}

Humans
*Glioblastoma/genetics/pathology/mortality
Prognosis
*Brain Neoplasms/genetics/pathology
*Membrane Proteins/genetics/metabolism
CRISPR-Cas Systems
*Biomarkers, Tumor/genetics
Tumor Microenvironment
Gene Expression Regulation, Neoplastic
Multiomics

@article {pmid40247031,
year = {2025},
author = {Loughran, AJ and Narina, S and Klein, J and Siwak, JF and Connelly, JP and Pruett-Miller, SM},
title = {Rapid and robust validation of pooled CRISPR knockout screens using CelFi.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {13358},
pmid = {40247031},
issn = {2045-2322},
support = {P30 CA021765/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Knockout Techniques/methods ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; INDEL Mutation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Pooled CRISPR screens are vital in the unbiased interrogation of gene function and are instrumental in uncovering therapeutic targets and biological processes. However, follow-up hit validation is critical to confirm observed results. Researchers need a simple and robust approach to rapidly verify putative hits and test resulting observations. Thus, we developed a CRISPR-based method for hit validation that tests the effect of a genetic perturbation on cell fitness. By editing target loci and monitoring the indel profiles over time, we have created a Cellular Fitness (CelFi) assay that can elucidate cellular vulnerabilities and verify hits from pooled CRISPR knockout screens. Unlike traditional cellular fitness assays that evaluate viability over time, the CelFi assay correlates changes in the indel profile at the target gene with a selective growth advantage or disadvantage in individual cells over time. Moreover, the CelFi assay can be utilized to evaluate gene dependencies and test new hypotheses, regardless of variations in single guide RNA optimization, ribonucleoprotein concentration, and gene copy number.},
}

*CRISPR-Cas Systems
Humans
*Gene Knockout Techniques/methods
Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
INDEL Mutation
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40246839,
year = {2025},
author = {Nakamura, K and Aoyama-Ishiwatari, S and Nagao, T and Paaran, M and Obara, CJ and Sakurai-Saito, Y and Johnston, J and Du, Y and Suga, S and Tsuboi, M and Nakakido, M and Tsumoto, K and Kishi, Y and Gotoh, Y and Kwak, C and Rhee, HW and Seo, JK and Kosako, H and Potter, C and Carragher, B and Lippincott-Schwartz, J and Polleux, F and Hirabayashi, Y},
title = {Mitochondrial complexity is regulated at ER-mitochondria contact sites via PDZD8-FKBP8 tethering.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3401},
pmid = {40246839},
issn = {2041-1723},
support = {R35 NS127232/NS/NINDS NIH HHS/United States ; JP19dm0207082//Japan Agency for Medical Research and Development (AMED)/ ; JP20H04898//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; N/A//Secom Science and Technology Foundation (SSTF)/ ; JP22H05532//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP24H01348//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP24H01269//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP21wm0525015//Japan Agency for Medical Research and Development (AMED)/ ; N/A//Uehara Memorial Foundation/ ; },
mesh = {*Mitochondria/metabolism/ultrastructure ; *Endoplasmic Reticulum/metabolism/ultrastructure ; Humans ; *Tacrolimus Binding Proteins/metabolism/genetics ; *Mitochondrial Membranes/metabolism/ultrastructure ; HeLa Cells ; HEK293 Cells ; Proteomics ; Membrane Proteins/metabolism ; Animals ; Cryoelectron Microscopy ; CRISPR-Cas Systems ; },
abstract = {Mitochondria-ER membrane contact sites (MERCS) represent a fundamental ultrastructural feature underlying unique biochemistry and physiology in eukaryotic cells. The ER protein PDZD8 is required for the formation of MERCS in many cell types, however, its tethering partner on the outer mitochondrial membrane (OMM) is currently unknown. Here we identify the OMM protein FKBP8 as the tethering partner of PDZD8 using a combination of unbiased proximity proteomics, CRISPR-Cas9 endogenous protein tagging, Cryo-electron tomography, and correlative light-electron microscopy. Single molecule tracking reveals highly dynamic diffusion properties of PDZD8 along the ER membrane with significant pauses and captures at MERCS. Overexpression of FKBP8 is sufficient to narrow the ER-OMM distance, whereas independent versus combined deletions of these two proteins demonstrate their interdependence for MERCS formation. Furthermore, PDZD8 enhances mitochondrial complexity in a FKBP8-dependent manner. Our results identify a novel ER-mitochondria tethering complex that regulates mitochondrial morphology in mammalian cells.},
}

*Mitochondria/metabolism/ultrastructure
*Endoplasmic Reticulum/metabolism/ultrastructure
Humans
*Tacrolimus Binding Proteins/metabolism/genetics
*Mitochondrial Membranes/metabolism/ultrastructure
HeLa Cells
HEK293 Cells
Proteomics
Membrane Proteins/metabolism
Animals
Cryoelectron Microscopy
CRISPR-Cas Systems

@article {pmid40244677,
year = {2025},
author = {Wang, X and Yue, Y and Zhai, Y and Wang, F and Zhuang, X and Wu, S and Yang, Y and Tabashnik, BE and Wu, Y},
title = {Functional redundancy in the toxic pathway of Bt protein Cry1Ab, but not Cry1Fa, against the Asian corn borer.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {16},
pages = {e2503674122},
doi = {10.1073/pnas.2503674122},
pmid = {40244677},
issn = {1091-6490},
support = {2022YFD1400901//Ministry of Science and Technology of China, National Key R&D Program/ ; 32372587)//National Natural Science Foundation of China/ ; 2020-67013-31924//USDA National Institute of Food and Agriculture, Agriculture and Food Research Initiative/ ; },
mesh = {Animals ; *Endotoxins/genetics/toxicity/metabolism ; Bacillus thuringiensis Toxins ; *Bacterial Proteins/toxicity/genetics/metabolism/pharmacology ; *Hemolysin Proteins/genetics/toxicity/metabolism/pharmacology ; *Moths/genetics/drug effects ; Multidrug Resistance-Associated Protein 2 ; Insecticide Resistance/genetics ; Zea mays/parasitology/genetics ; Multidrug Resistance-Associated Proteins/genetics/metabolism ; Cadherins/genetics/metabolism ; Larva/drug effects/genetics ; Bacillus thuringiensis/genetics/metabolism ; Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Pest Control, Biological ; Mutation ; },
abstract = {Crops genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have been used extensively to control some major crop pests, but their benefits decrease when pests evolve resistance. Better understanding of the genetic basis of resistance is needed to effectively monitor, manage, and counter pest resistance to Bt crops. Resistance to Bt proteins in at least 11 species of Lepidoptera, including many important crop pests, is associated with naturally occurring mutations that disrupt one or more of three larval midgut proteins: cadherin and ATP-binding cassette proteins ABCC2 and ABCC3. Here, we determined how CRISPR/Cas9-mediated mutations disrupting cadherin, ABCC2, and ABCC3 singly and in pairs affect resistance to Bt proteins Cry1Ab and Cry1Fa in the Asian corn borer (Ostrinia furnacalis), which is the most damaging pest of corn in Asia and is closely related to the European corn borer (Ostrinia nubilalis), a major pest in Europe and North America. The results from bioassays of six knockout strains and their parent susceptible strain support a model in which Cry1Ab can kill larvae via one path requiring ABCC2 or another path requiring cadherin and ABCC3, whereas Cry1Fa uses only the first path. The model's predictions are generally supported by results from genetic linkage analyses and responses to Cry1Ab and Cry1Fa of Sf9 cells and Xenopus oocytes modified to produce cadherin, ABCC2, and ABCC3 singly or in pairs. The functional redundancy identified here for Cry1Ab could sustain its efficacy against O. furnacalis and may exemplify a widespread natural strategy for delaying resistance.},
}

Animals
*Endotoxins/genetics/toxicity/metabolism
Bacillus thuringiensis Toxins
*Bacterial Proteins/toxicity/genetics/metabolism/pharmacology
*Hemolysin Proteins/genetics/toxicity/metabolism/pharmacology
*Moths/genetics/drug effects
Multidrug Resistance-Associated Protein 2
Insecticide Resistance/genetics
Zea mays/parasitology/genetics
Multidrug Resistance-Associated Proteins/genetics/metabolism
Cadherins/genetics/metabolism
Larva/drug effects/genetics
Bacillus thuringiensis/genetics/metabolism
Insect Proteins/genetics/metabolism
CRISPR-Cas Systems
Pest Control, Biological
Mutation

@article {pmid40215828,
year = {2025},
author = {Miskel, D and Kurzella, J and Rings, F and Tholen, E and Tesfaye, D and Schellander, K and Salilew-Wondim, D and Held-Hoelker, E and Große-Brinkhaus, C and Hoelker, M},
title = {Functional COPA is indispensable for early embryo development beyond major genome activation in bovines.},
journal = {Theriogenology},
volume = {241},
number = {},
pages = {117415},
doi = {10.1016/j.theriogenology.2025.117415},
pmid = {40215828},
issn = {1879-3231},
mesh = {Animals ; *Embryonic Development/genetics/physiology ; Cattle/embryology/genetics ; CRISPR-Cas Systems ; Gene Expression Regulation, Developmental/physiology ; *Vesicular Transport Proteins/genetics/metabolism ; Blastocyst ; Female ; },
abstract = {Embryonic genome activation is divided into a minor and a major wave of transition to endogenous transcription. In bovines, minor genome activation begins early in the 2-cell stage and is completed by the 8-cell stage when major genome activation becomes dominant. While the activation of genes known to regulate early development have been studied extensively, genes involved in more central cellular functions have not been examined. Taking advantage of the CRISPR Cas9 system, the present study investigated the effect of knocking out the Golgi retrograde protein transporter COPA on early bovine development. After the electroporation of presumptive zygotes with Cas9 ribonucleoproteins targeting COPA exon 6, sequences of 2 (11 %) and 4-cell (16 %) embryos showed knockouts of COPA whereas 8-cell embryos and blastocysts did not, demonstrating that COPA is necessary for development to the 8-cell stage and beyond. Using a repair template containing silent mutations along the target site, COPA loss of wildtype was observed in 5 blastocysts, with successful knock-in of the template on at least one allele. This shows that an edited yet functional copy of COPA can save the developmental capacity of the embryo and demonstrates that Cas9 activity at the target region itself is not responsible for the loss of function. Together, the present study revealed that COPA is necessary for embryonic development, and that the timing of this necessity is before major genome activation onset. More generally, this study further demonstrates the utility of genome editing within reproductive biotechnology for the interrogation of gene function and early embryonic development.},
}

Animals
*Embryonic Development/genetics/physiology
Cattle/embryology/genetics
CRISPR-Cas Systems
Gene Expression Regulation, Developmental/physiology
*Vesicular Transport Proteins/genetics/metabolism
Blastocyst
Female

@article {pmid38379101,
year = {2025},
author = {Zhang, X and Van Treeck, B and Horton, CA and McIntyre, JJR and Palm, SM and Shumate, JL and Collins, K},
title = {Harnessing eukaryotic retroelement proteins for transgene insertion into human safe-harbor loci.},
journal = {Nature biotechnology},
volume = {43},
number = {1},
pages = {42-51},
pmid = {38379101},
issn = {1546-1696},
support = {DP1 HL156819/HL/NHLBI NIH HHS/United States ; F32 GM139306/GM/NIGMS NIH HHS/United States ; T32 GM007232/GM/NIGMS NIH HHS/United States ; GM139306//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {Humans ; *Transgenes/genetics ; *Retroelements/genetics ; CRISPR-Cas Systems/genetics ; *Mutagenesis, Insertional/methods ; Animals ; Gene Editing/methods ; },
abstract = {Current approaches for inserting autonomous transgenes into the genome, such as CRISPR-Cas9 or virus-based strategies, have limitations including low efficiency and high risk of untargeted genome mutagenesis. Here, we describe precise RNA-mediated insertion of transgenes (PRINT), an approach for site-specifically primed reverse transcription that directs transgene synthesis directly into the genome at a multicopy safe-harbor locus. PRINT uses delivery of two in vitro transcribed RNAs: messenger RNA encoding avian R2 retroelement-protein and template RNA encoding a transgene of length validated up to 4 kb. The R2 protein coordinately recognizes the target site, nicks one strand at a precise location and primes complementary DNA synthesis for stable transgene insertion. With a cultured human primary cell line, over 50% of cells can gain several 2 kb transgenes, of which more than 50% are full-length. PRINT advantages include no extragenomic DNA, limiting risk of deleterious mutagenesis and innate immune responses, and the relatively low cost, rapid production and scalability of RNA-only delivery.},
}

Humans
*Transgenes/genetics
*Retroelements/genetics
CRISPR-Cas Systems/genetics
*Mutagenesis, Insertional/methods
Animals
Gene Editing/methods

@article {pmid40244417,
year = {2025},
author = {Bono, H},
title = {Recent Advances in Genome Editing and Bioinformatics: Addressing Challenges in Genome Editing Implementation and Genome Sequencing.},
journal = {International journal of molecular sciences},
volume = {26},
number = {7},
pages = {},
pmid = {40244417},
issn = {1422-0067},
support = {JPMJPF2010//Japan Science and Technology Agency/ ; },
mesh = {*Gene Editing/methods ; *Computational Biology/methods ; Humans ; CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing/methods ; Animals ; Genome ; },
abstract = {Genome-editing technology has advanced significantly since the 2020 Nobel Prize in Chemistry was awarded for the development of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9). While CRISPR-Cas9 has become widely used in academic research, its social implementation has lagged due to unresolved patent disputes and slower progress in gene function analysis. To address this, new approaches bypassing direct gene function analysis are needed, with bioinformatics and next-generation sequencing (NGS) playing crucial roles. NGS is essential for sequencing the genome of target species, but challenges such as data quality, genome heterogeneity, ploidy, and small individual sizes persist. Despite these issues, advancements in sequencing technologies, like PacBio high-fidelity (HiFi) long reads and high-throughput chromosome conformation capture (Hi-C), have improved genome sequencing. Bioinformatics contributes to genome editing through off-target prediction and target gene selection, both of which require accurate genome sequence information. In this review, I will give updates on the development of genome editing and bioinformatics technologies with a focus on the rapid progress in genome sequencing.},
}

*Gene Editing/methods
*Computational Biology/methods
Humans
CRISPR-Cas Systems
High-Throughput Nucleotide Sequencing/methods
Animals
Genome

@article {pmid40244221,
year = {2025},
author = {Fan, S and Jia, L and Wu, J and Zhao, Y},
title = {Harnessing the Potential of CRISPR/Cas in Targeted Alfalfa Improvement for Stress Resilience.},
journal = {International journal of molecular sciences},
volume = {26},
number = {7},
pages = {},
pmid = {40244221},
issn = {1422-0067},
support = {42320104006//National Natural Science Foundation of China/ ; },
mesh = {*Medicago sativa/genetics/physiology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Stress, Physiological/genetics ; Plant Breeding/methods ; Climate Change ; Genome, Plant ; },
abstract = {Alfalfa (Medicago sativa), recognized as the most valuable legume feed crop, faces significant challenges in enhancing both qualitative and quantitative production amidst the pressures of climate change. This review highlights these challenges, including the underutilization of genomic and genetic resources, while proposing potential solutions through genome editing. Our focus is on leveraging CRISPR/Cas technology in conjunction with decades of advancements in conventional breeding to expedite the improvement of alfalfa. By adopting this approach, we aim to overcome the limitations of traditional alfalfa improvement approaches and accelerate the development of improved cultivars capable of thriving in changing climates. Key candidate traits for CRISPR/Cas genome editing, as reviewed in the latest literature, include nutrient use efficiency, freezing tolerance, and resistance to pests and diseases. We dissect literature on potential gene pathways associated with these traits, providing molecular breeders with valuable insights for utilizing CRISPR/Cas genome editing. Furthermore, we propose editing modalities to expedite the development of stress-resilient, genome-edited alfalfa that can effectively cope with climate change.},
}

*Medicago sativa/genetics/physiology
*CRISPR-Cas Systems
*Gene Editing/methods
*Stress, Physiological/genetics
Plant Breeding/methods
Climate Change
Genome, Plant

@article {pmid40244157,
year = {2025},
author = {Zanatta, CB and Narendja, F and El Jawhary, H and Abou-Sleymane, G and Subburaj, S and Nodari, RO and Agapito-Tenfen, SZ},
title = {Suitability of Real-Time PCR Methods for New Genomic Technique Detection in the Context of the European Regulations: A Case Study in Arabidopsis.},
journal = {International journal of molecular sciences},
volume = {26},
number = {7},
pages = {},
pmid = {40244157},
issn = {1422-0067},
support = {301911//The Research Council of Norway/ ; },
mesh = {*Arabidopsis/genetics ; *Real-Time Polymerase Chain Reaction/methods ; *Plants, Genetically Modified/genetics ; Europe ; Polymorphism, Single Nucleotide ; *Genomics/methods ; CRISPR-Cas Systems ; Gene Editing/methods ; DNA, Plant/genetics ; Genome, Plant ; },
abstract = {PCR methods are widely applied for the detection of genetically modified organisms (GMOs) in Europe, facilitating compliance with stringent regulatory requirements and enabling the accurate identification and quantification of genetically modified traits in various crops and foodstuffs. This manuscript investigates the suitability of real-time PCR methods for detecting organisms generated through new genomic techniques (NGTs), specifically focusing on a case study using Arabidopsis thaliana as a model gene-edited plant. Given the complexities of European regulations regarding genetically modified organisms (GMOs) and the classification of gene-edited plants, there is a pressing need for robust detection methods. Our study highlights the development and validation of a novel single-plex real-time PCR method targeting a specific single nucleotide polymorphism (SNP) in the grf1-3 gene modified using CRISPR-Cas9 technology. We emphasize the effectiveness of locked nucleic acid (LNA)-modified primers in improving specificity. The results demonstrate that while the grf1-3 LNA method successfully detected and quantified gene-edited Arabidopsis DNA, achieving absolute specificity remains a challenge. This study also addresses the significance of the cross-laboratory method for validation, demonstrating that the method developed for an SNP-modified allele can be performed in accordance with the precision and trueness criteria established by the European Network of GMO Laboratories (ENGL). Furthermore, we call for continued collaboration among regulatory agencies, academia, and industry stakeholders to refine detection strategies. This proactive approach is essential not only for regulatory compliance but also for maintaining public trust in the safe integration of gene-edited organisms into food products.},
}

*Arabidopsis/genetics
*Real-Time Polymerase Chain Reaction/methods
*Plants, Genetically Modified/genetics
Europe
Polymorphism, Single Nucleotide
*Genomics/methods
CRISPR-Cas Systems
Gene Editing/methods
DNA, Plant/genetics
Genome, Plant

@article {pmid40215680,
year = {2025},
author = {Ito, R and Nakano, T and Sugawara, A and Yokoyama, A},
title = {A CRISPR-based high-throughput screening system identifies bromodomain inhibitors as transcriptional suppressors of CYP11B1.},
journal = {Biochemical and biophysical research communications},
volume = {762},
number = {},
pages = {151779},
doi = {10.1016/j.bbrc.2025.151779},
pmid = {40215680},
issn = {1090-2104},
mesh = {*Steroid 11-beta-Hydroxylase/genetics/antagonists & inhibitors/metabolism ; *High-Throughput Screening Assays/methods ; Humans ; *Triazoles/pharmacology ; *Azepines/pharmacology ; *CRISPR-Cas Systems ; *Transcription, Genetic/drug effects ; Cell Line ; Promoter Regions, Genetic/drug effects ; *Enzyme Inhibitors/pharmacology ; Epigenesis, Genetic/drug effects ; },
abstract = {CYP11B1 encodes steroid 11β-hydroxylase, the final rate-limiting enzyme for cortisol biosynthesis in the adrenal cortex. Excessive cortisol production is a hallmark of Cushing's disease (CD). While direct enzymatic inhibitors have been explored, achieving specificity remains a challenge due to the high homology between CYP11B1 and CYP11B2, highlighting transcriptional suppression of CYP11B1 as an alternative therapeutic strategy. To identify transcriptional regulators of CYP11B1, we generated genome-edited H295R adrenal cells carrying a luciferase reporter knocked into the endogenous CYP11B1 locus. Using this reporter cell line, we established a high-throughput screening (HTS) platform and screened a focused chemical library targeting epigenetic-related factors, given the importance of epigenetic mechanisms in gene regulation. Among eight candidate compounds identified, we focused on JQ1, a bromodomain inhibitor. JQ1 significantly suppressed Forskolin-induced CYP11B1 promoter activity and mRNA expression without causing cytotoxicity, suggesting the involvement of epigenetic readers in the transcriptional regulation of steroidogenic genes. Furthermore, the reporter-based HTS platform developed here, when combined with our previously established CYP11B2-luciferase system, may facilitate the identification of compounds that selectively modulate adrenal steroidogenic pathways. These findings provide a foundation for the development of novel transcription-targeted therapies for CD.},
}

*Steroid 11-beta-Hydroxylase/genetics/antagonists & inhibitors/metabolism
*High-Throughput Screening Assays/methods
Humans
*Triazoles/pharmacology
*Azepines/pharmacology
*CRISPR-Cas Systems
*Transcription, Genetic/drug effects
Cell Line
Promoter Regions, Genetic/drug effects
*Enzyme Inhibitors/pharmacology
Epigenesis, Genetic/drug effects

@article {pmid40196691,
year = {2025},
author = {Tang, S and Žedaveinytė, R and Burman, N and Pandey, S and Ramirez, JL and Kulber, LM and Wiegand, T and Wilkinson, RA and Ma, Y and Zhang, DJ and Lampe, GD and Berisa, M and Jovanovic, M and Wiedenheft, B and Sternberg, SH},
title = {Protein-primed DNA homopolymer synthesis by an antiviral reverse transcriptase.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {40196691},
issn = {2692-8205},
abstract = {Bacteria defend themselves from viral predation using diverse immune systems, many of which sense and target foreign DNA for degradation. Defense-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this strategy by leveraging DNA synthesis instead. We and others recently showed that DRT2 systems use an RNA template to assemble a de novo gene, leading to expression of an antiviral effector protein, Neo. It remains unknown whether similar mechanisms of defense are employed by other DRT families. Focusing on DRT9, here we uncover an unprecedented mechanism of DNA homopolymer synthesis, in which viral infection triggers polydeoxyadenylate (poly-dA) accumulation in the cell to drive abortive infection and population-level immunity. Cryo-EM structures reveal how a conserved noncoding RNA serves as both a structural scaffold and reverse transcription template to direct hexameric complex assembly and RNA-templated poly-dA synthesis. Remarkably, biochemical and functional experiments identify conserved tyrosine residues within the reverse transcriptase itself that prime DNA synthesis, leading to the formation of high-molecular weight protein-DNA covalent adducts. Synthesis of poly-dA in vivo is regulated by the competing activities of phage-encoded triggers and host-encoded silencers of DRT9. Collectively, our work unveils a novel nucleic acid-driven defense system that expands the paradigm of bacterial immunity and broadens the known functions of reverse transcriptases.},
}

@article {pmid40054045,
year = {2025},
author = {Li, X and Wang, Z and Man, X and Dai, X and Zhou, Q and Zhang, S},
title = {Research advances CRISPR gene editing technology generated models in the study of epithelial ovarian carcinoma.},
journal = {Gynecologic oncology},
volume = {195},
number = {},
pages = {34-44},
doi = {10.1016/j.ygyno.2025.02.022},
pmid = {40054045},
issn = {1095-6859},
mesh = {Female ; *Carcinoma, Ovarian Epithelial/genetics/therapy ; *Gene Editing/methods ; Animals ; Humans ; *Ovarian Neoplasms/genetics/therapy ; *CRISPR-Cas Systems ; Mice ; *Disease Models, Animal ; Genetic Therapy/methods ; },
abstract = {Epithelial ovarian carcinoma (EOC), the most lethal gynecologic cancer, is often diagnosed at advanced stages, which urge us to explore the novel therapeutic strategies. Mouse models have played a crucial role in elucidating the molecular mechanisms for the development ovarian cancer and its therapeutic strategies. However, there are still various challenges in modeling the genetic drivers of ovarian cancer in animal models. Here, we provided an overview of the research advances for the molecular mechanisms underlying EOC development, therapeutic strategies, the CRISPR genome editing technology and its generated EOC models. The review also comprehensively discussed the advantages and obstacles of CRISPR in generating EOC mouse models and the promising therapeutic approach by correcting the oncogenes of EOC through in vivo delivery of gene-edited components. The development of more precise animal models, along with a deeper understanding of EOC molecular mechanisms, will dramatically benefit the investigation and treatment of EOC.},
}

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

@article {pmid40244092,
year = {2025},
author = {Ciapaite, J and Albersen, M and Savelberg, SMC and Bosma, M and Meijer, NWF and Tessadori, F and Bakkers, JPW and van Haaften, G and Jans, JJ and Verhoeven-Duif, NM},
title = {Broad Vitamin B6-Related Metabolic Disturbances in a Zebrafish Model of Hypophosphatasia (TNSALP-Deficiency).},
journal = {International journal of molecular sciences},
volume = {26},
number = {7},
pages = {},
pmid = {40244092},
issn = {1422-0067},
support = {OZF 2014-2015//Wilhelmina Children's Hospital Research Fund/ ; },
mesh = {Animals ; *Zebrafish/metabolism/genetics ; *Vitamin B 6/metabolism ; *Hypophosphatasia/metabolism/genetics ; Disease Models, Animal ; *Alkaline Phosphatase/genetics/metabolism/deficiency ; Pyridoxal Phosphate/metabolism ; Pyridoxine ; Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Hypophosphatasia (HPP) is a rare inborn error of metabolism caused by pathogenic variants in ALPL, coding for tissue non-specific alkaline phosphatase. HPP patients suffer from impaired bone mineralization, and in severe cases from vitamin B6-responsive seizures. To study HPP, we generated alpl[-/-] zebrafish using CRISPR/Cas9 gene-editing technology. At 5 days post fertilization (dpf), no alpl mRNA and 89% lower total alkaline phosphatase activity was detected in alpl[-/-] compared to alpl[+/+] embryos. The survival of alpl[-/-] zebrafish was strongly decreased. Alizarin red staining showed decreased bone mineralization in alpl[-/-] embryos. B6 vitamer analysis revealed depletion of pyridoxal and its degradation product 4-pyridoxic acid in alpl[-/-] embryos. Accumulation of d3-pyridoxal 5'-phosphate (d3-PLP) and reduced formation of d3-pyridoxal in alpl[-/-] embryos incubated with d3-PLP confirmed Alpl involvement in vitamin B6 metabolism. Locomotion analysis showed pyridoxine treatment-responsive spontaneous seizures in alpl[-/-] embryos. Metabolic profiling of alpl[-/-] larvae using direct-infusion high-resolution mass spectrometry showed abnormalities in polyamine and neurotransmitter metabolism, suggesting dysfunction of vitamin B6-dependent enzymes. Accumulation of N-methylethanolaminium phosphate indicated abnormalities in phosphoethanolamine metabolism. Taken together, we generated the first zebrafish model of HPP that shows multiple features of human disease and which is suitable for the study of the pathophysiology of HPP and for the testing of novel treatments.},
}

Animals
*Zebrafish/metabolism/genetics
*Vitamin B 6/metabolism
*Hypophosphatasia/metabolism/genetics
Disease Models, Animal
*Alkaline Phosphatase/genetics/metabolism/deficiency
Pyridoxal Phosphate/metabolism
Pyridoxine
Zebrafish Proteins/genetics/metabolism
CRISPR-Cas Systems

@article {pmid40243700,
year = {2025},
author = {He, J and Liu, J and Yue, Y and Wang, L and Liu, Z and Xi, G and An, L and Tian, J and Wang, Y},
title = {Genome Editing in Mouse Embryo Using the CRISPR/Cas12i3 System.},
journal = {International journal of molecular sciences},
volume = {26},
number = {7},
pages = {},
pmid = {40243700},
issn = {1422-0067},
support = {2022YFD1302202//National Key R&D Program of China/ ; No.2023TC019//Chinese Universities Scientific Fund/ ; 2022TC114//Chinese Universities Scientific Fund/ ; 2022TC123//Chinese Universities Scientific Fund/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Mice ; *Embryo, Mammalian/metabolism ; Nanog Homeobox Protein/genetics ; Female ; Embryonic Development/genetics ; Blastocyst/metabolism ; },
abstract = {The CRISPR/Cas system is a sizable family that is currently a popular and efficient gene editing tool. Cas12i3, as a member of the Type V-I family, has the characteristics of recognizing T-rich PAM sequences and being guided by shorter crRNA and has higher gene editing efficiency than Cas9 in rice. However, as a potential tool in accelerating the breeding process, the application of Cas12i3 in mammalian embryos has not yet been reported. Our study systematically evaluated the feasibility of applying CRISPR/Cas12i3 to gene editing in mouse embryos, with the core pluripotency regulator gene Nanog as the target. We successfully constructed a Nanog loss-of-function mouse embryo model using CRISPR/Cas12i3. At the targeted Nanog locus, its editing efficiency exceeded that of the Cas9 system under matched experimental conditions; no off-target phenomenon was detected. Moreover, the Cas12i3 system exhibited no side effect on mouse embryo development and proliferation of blastocyst cells. Finally, we obtained healthy chimeric gene-edited offspring by optimizing the concentration of the Cas12i3 mixture. These results confirm the feasibility and safety of CRISPR/Cas12i3 for gene editing in mammals, which provides a reliable tool for one-step generation of gene-edited animals for applications in biology, medical research, and large livestock breeding.},
}

Animals
*CRISPR-Cas Systems
*Gene Editing/methods
Mice
*Embryo, Mammalian/metabolism
Nanog Homeobox Protein/genetics
Female
Embryonic Development/genetics
Blastocyst/metabolism

@article {pmid40243657,
year = {2025},
author = {Daliri, K and Hescheler, J and Newby, GA and Clement, K and Liu, DR and Pfannkuche, K},
title = {Modulating Collagen I Expression in Fibroblasts by CRISPR-Cas9 Base Editing of the Collagen 1A1 Promoter.},
journal = {International journal of molecular sciences},
volume = {26},
number = {7},
pages = {},
pmid = {40243657},
issn = {1422-0067},
support = {4346564//Imhoff foundation/ ; },
mesh = {Humans ; *Fibroblasts/metabolism ; *Promoter Regions, Genetic ; *Gene Editing/methods ; *Collagen Type I/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Collagen Type I, alpha 1 Chain ; MCF-7 Cells ; Fibrosis/genetics ; },
abstract = {Fibrotic diseases, contributing to a significant portion of global mortality, highlight the need for innovative therapies. This study explores a novel approach to disrupt the expression of collagen by using adenine base editing to target Col1a1, a key gene driving both fibrosis and cancer metastasis. Editing Col1a1 in fibroblasts demonstrated 18% editing efficiency. An analysis of a specific clone harboring a CCAAT-to-CCGGA mutation in the Col1a1 promoter revealed reduced collagen production. Notably, when wild-type fibroblasts were cultured on the Col1a1-edited matrix, no compensatory collagen upregulation was detected, suggesting a lack of feedback mechanism in fibroblasts. Furthermore, the matrix derived from edited fibroblasts did not support the growth of MCF-7 cancer cells. These findings suggest that Col1a1 gene editing holds promise as a potential therapeutic strategy for fibrotic diseases. Further investigation is warranted to fully elucidate the implications of these findings for fibrosis and cancer.},
}

Humans
*Fibroblasts/metabolism
*Promoter Regions, Genetic
*Gene Editing/methods
*Collagen Type I/genetics/metabolism
*CRISPR-Cas Systems/genetics
Collagen Type I, alpha 1 Chain
MCF-7 Cells
Fibrosis/genetics

@article {pmid40243589,
year = {2025},
author = {Kim, T and Park, BS and Heo, S and Jeon, H and Kim, J and Kim, D and Kook Lee, S and Jung, SY and Kong, SY and Lu, T},
title = {Combinatorial CRISPR screen reveals FYN and KDM4 as targets for synergistic drug combination for treating triple negative breast cancer.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {40243589},
issn = {2050-084X},
support = {2021R1A2C1093499//National Research Foundation of Korea/ ; 2E32331//Korea Institute of Science and Technology/ ; 2020M3A9A5036362//National Research Foundation of Korea/ ; },
mesh = {*Triple Negative Breast Neoplasms/drug therapy/genetics ; Humans ; Animals ; *Proto-Oncogene Proteins c-fyn/genetics/metabolism/antagonists & inhibitors ; Female ; Cell Line, Tumor ; Mice ; *Jumonji Domain-Containing Histone Demethylases/genetics/metabolism/antagonists & inhibitors ; Drug Synergism ; *Protein Kinase Inhibitors/pharmacology ; CRISPR-Cas Systems ; *Antineoplastic Agents/pharmacology ; Drug Resistance, Neoplasm ; },
abstract = {Tyrosine kinases play a crucial role in cell proliferation and survival and are extensively investigated as targets for cancer treatment. However, the efficacy of most tyrosine kinase inhibitors (TKIs) in cancer therapy is limited due to resistance. In this study, we identify a synergistic combination therapy involving TKIs for the treatment of triple negative breast cancer. By employing pairwise tyrosine kinase knockout CRISPR screens, we identify FYN and KDM4 as critical targets whose inhibition enhances the effectiveness of TKIs, such as NVP-ADW742 (IGF-1R inhibitor), gefitinib (EGFR inhibitor), and imatinib (ABL inhibitor) both in vitro and in vivo. Mechanistically, treatment with TKIs upregulates the transcription of KDM4, which in turn demethylates H3K9me3 at FYN enhancer for FYN transcription. This compensatory activation of FYN and KDM4 contributes to the resistance against TKIs. FYN expression is associated with therapy resistance and persistence by demonstrating its upregulation in various experimental models of drug-tolerant persisters and residual disease following targeted therapy, chemotherapy, and radiotherapy. Collectively, our study provides novel targets and mechanistic insights that can guide the development of effective combinatorial targeted therapies, thus maximizing the therapeutic benefits of TKIs.},
}

*Triple Negative Breast Neoplasms/drug therapy/genetics
Humans
Animals
*Proto-Oncogene Proteins c-fyn/genetics/metabolism/antagonists & inhibitors
Female
Cell Line, Tumor
Mice
*Jumonji Domain-Containing Histone Demethylases/genetics/metabolism/antagonists & inhibitors
Drug Synergism
*Protein Kinase Inhibitors/pharmacology
CRISPR-Cas Systems
*Antineoplastic Agents/pharmacology
Drug Resistance, Neoplasm

@article {pmid40243584,
year = {2025},
author = {Zhao, X and Mai, C and Xia, L and Jia, G and Li, X and Lu, Y and Li, Z and Yang, H and Wang, L},
title = {Molecular Insights into the Positive Role of Soybean Nodulation by GmWRKY17.},
journal = {International journal of molecular sciences},
volume = {26},
number = {7},
pages = {},
pmid = {40243584},
issn = {1422-0067},
support = {32241046, 32472158//National Natural Science Foundation of China/ ; 2025xczx03//Shanxi Breeding Innovation Joint research and development projects/ ; 2023ZD040350103//Scientific and Technological Innovation 2030-Major Projects/ ; 202204051001020//Science and Technology Innovation Young Talent Team of Shanxi Province/ ; 2021xG003, 2022xG0014//Scientific research fund for talents of Shanxi Agricultural University/ ; },
mesh = {*Glycine max/genetics/microbiology/metabolism ; *Plant Root Nodulation/genetics ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Root Nodules, Plant/genetics/metabolism/microbiology ; CRISPR-Cas Systems ; Symbiosis ; *Transcription Factors/genetics/metabolism ; Cytokinins/metabolism ; Plants, Genetically Modified ; Abscisic Acid/metabolism ; RNA Interference ; Nitrogen Fixation ; },
abstract = {Soybean is an important economic oilseed crop, being rich in protein and plant oil, it is widely cultivated around the world. Soybeans have been shown to establish a symbiotic nitrogen fixation (SNF) with their compatible rhizobia, resulting in the formation of nodules. Previous studies have demonstrated the critical roles of phytohormones, such as abscisic acid and cytokinin, in the process of legume nodulation. The present study investigated the role of GmWRKY17, a homolog of Rosa hybrida (Rh)WRKY13 in regulating plant immunity through cytokinin content and abscisic acid signaling in soybean nodulation. Utilizing real-time PCR and histochemical staining, we demonstrated that GmWRKY17 is predominantly expressed in soybean root nodules. Subsequently, we analyzed the function of GmWRKY17-overexpression, RNA interference (RNAi), and the CRISPR/Cas9 system. Overexpression of GmWRKY17 significantly increases soybean nodule number, while RNAi or CRISPR/Cas9-mediated knockout of GmWRKY17 resulted in a dramatic repression of nodule formation in soybeans. These results highlight that GmWRKY17 functions as a positive regulator involved in soybean nodulation. Furthermore, manipulation of GmWRKY17 expression impacts the expression of genes associated with the nod factor (NF) signaling pathway, thereby influencing soybean nodulation. This study demonstrated that WRKY-type transcription factors are involved in the regulation of legume nodulation, offering new light on the molecular basis of the symbiotic interaction between legumes and rhizobia.},
}

*Glycine max/genetics/microbiology/metabolism
*Plant Root Nodulation/genetics
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
Root Nodules, Plant/genetics/metabolism/microbiology
CRISPR-Cas Systems
Symbiosis
*Transcription Factors/genetics/metabolism
Cytokinins/metabolism
Plants, Genetically Modified
Abscisic Acid/metabolism
RNA Interference
Nitrogen Fixation

@article {pmid40199056,
year = {2025},
author = {Li, M and Pan, L and Ma, C and Wu, H and Xiang, G and Li, LF and Wang, T and Luo, R and Li, Y and Liu, D and Zhai, H and Assad, M and Song, X and Wang, Y and Gallardo, F and Qiu, HJ and Sun, Y},
title = {Tracking of single virus: Dual fluorescent labeling of pseudorabies virus for observing entry and replication in the N2a cells.},
journal = {Veterinary microbiology},
volume = {304},
number = {},
pages = {110503},
doi = {10.1016/j.vetmic.2025.110503},
pmid = {40199056},
issn = {1873-2542},
mesh = {*Herpesvirus 1, Suid/physiology/genetics ; Animals ; *Virus Replication ; Mice ; *Virus Internalization ; Genome, Viral ; Staining and Labeling/methods ; Cell Line ; Pseudorabies/virology ; Cell Line, Tumor ; Luminescent Proteins/genetics ; CRISPR-Cas Systems ; Red Fluorescent Protein ; },
abstract = {Pseudorabies virus (PRV) is a neurotropic herpesvirus. It is not easy to be track the whole replication progress of PRV, especially the nascent viral genome in the host cells. In this study, we developed a dual-fluorescence-labeled PRV (rPRV-Anchor3-mCherry) with the viral genome and the envelope protein gM labeled by ANCHOR DNA labeling system and mCherry, respectively. Through single-virus tracking of rPRV-Anchor3-mCherry, we observed that PRV invaded mouse neuroblastoma Neuro-2a cells via both endocytosis and plasma membrane fusion pathway. During the replication stage, parental and progeny viral genome of rPRV-Anchor3-mCherry in the cell nuclei could be visible, and viral nucleocapsid appeared more specifically than traditional capsid protein labeled PRV particles (rPRV-VP26-EGFP). We found that numerous progeny viral particles were produced in the nuclear, causing the nucleus membrane to break using three-dimensional (3D) live-cell imaging and electron microscopy. Moreover, our findings confirmed that simultaneously targeting of the UL9 and UL54 genes using a CRISPR-Cas9 system led to the complete inhibition PRV replication. rPRV-Anchor3-mCherry can be used to research multiple steps of the viral cycle.},
}

*Herpesvirus 1, Suid/physiology/genetics
Animals
*Virus Replication
Mice
*Virus Internalization
Genome, Viral
Staining and Labeling/methods
Cell Line
Pseudorabies/virology
Cell Line, Tumor
Luminescent Proteins/genetics
CRISPR-Cas Systems
Red Fluorescent Protein

@article {pmid40185099,
year = {2025},
author = {Lee, JS and Dan, T and Zhang, H and Cheng, Y and Rehfeld, F and Brugarolas, J and Mendell, JT},
title = {An ultraconserved snoRNA-like element in long noncoding RNA CRNDE promotes ribosome biogenesis and cell proliferation.},
journal = {Molecular cell},
volume = {85},
number = {8},
pages = {1543-1560.e10},
doi = {10.1016/j.molcel.2025.03.006},
pmid = {40185099},
issn = {1097-4164},
mesh = {*RNA, Long Noncoding/genetics/metabolism ; Humans ; *Cell Proliferation/genetics ; *RNA, Small Nucleolar/genetics/metabolism ; *Ribosomes/metabolism/genetics ; *Carcinoma, Renal Cell/genetics/pathology/metabolism ; *Kidney Neoplasms/genetics/pathology/metabolism ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Cell Nucleolus/metabolism/genetics ; Alternative Splicing ; HEK293 Cells ; RNA, Ribosomal/genetics/metabolism ; Conserved Sequence ; CRISPR-Cas Systems ; },
abstract = {Cancer cells frequently upregulate ribosome production to support tumorigenesis. While small nucleolar RNAs (snoRNAs) are critical for ribosome biogenesis, the roles of other classes of noncoding RNAs in this process remain largely unknown. Here, we performed CRISPR interference (CRISPRi) screens to identify essential long noncoding RNAs (lncRNAs) in renal cell carcinoma (RCC) cells. This revealed that an alternatively spliced isoform of lncRNA colorectal neoplasia differentially expressed (CRNDE) containing an ultraconserved element (UCE), referred to as CRNDE[UCE], is required for RCC cell proliferation. CRNDE[UCE] localizes to the nucleolus and promotes 60S ribosomal subunit biogenesis. The UCE of CRNDE functions as an unprocessed C/D box snoRNA that directly interacts with ribosomal RNA precursors. This facilitates delivery of eukaryotic initiation factor 6 (eIF6), a key 60S biogenesis factor, which binds to CRNDE[UCE] through a sequence element adjacent to the UCE. These findings highlight the functional versatility of snoRNA sequences and expand the known mechanisms through which noncoding RNAs orchestrate ribosome biogenesis.},
}

*RNA, Long Noncoding/genetics/metabolism
Humans
*Cell Proliferation/genetics
*RNA, Small Nucleolar/genetics/metabolism
*Ribosomes/metabolism/genetics
*Carcinoma, Renal Cell/genetics/pathology/metabolism
*Kidney Neoplasms/genetics/pathology/metabolism
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
Cell Nucleolus/metabolism/genetics
Alternative Splicing
HEK293 Cells
RNA, Ribosomal/genetics/metabolism
Conserved Sequence
CRISPR-Cas Systems

@article {pmid40174266,
year = {2025},
author = {Khan, TG and Bragazzi Cunha, J and Raut, C and Burroughs, M and Vyas, HS and Leix, K and Goonewardena, SN and Smrcka, AV and Speliotes, EK and Emmer, BT},
title = {Functional interrogation of cellular Lp(a) uptake by genome-scale CRISPR screening.},
journal = {Atherosclerosis},
volume = {403},
number = {},
pages = {119174},
doi = {10.1016/j.atherosclerosis.2025.119174},
pmid = {40174266},
issn = {1879-1484},
mesh = {Humans ; *Lipoprotein(a)/metabolism/blood/genetics ; *Receptors, LDL/genetics/metabolism ; Ubiquitin-Protein Ligases/genetics/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; United Kingdom ; *Hepatocytes/metabolism ; },
abstract = {BACKGROUND AND AIMS: An elevated level of lipoprotein(a), or Lp(a), in the bloodstream has been causally linked to the development of atherosclerotic cardiovascular disease and calcific aortic valve stenosis. Steady state levels of circulating lipoproteins are modulated by their rate of clearance, but the identity of the Lp(a) uptake receptor(s) has been controversial.

METHODS: We performed a genome-scale CRISPR screen to functionally interrogate all potential Lp(a) uptake regulators in HuH7 cells. Screen validation was performed by single gene disruption and overexpression. Direct binding between purified lipoproteins and recombinant protein was tested using biolayer interferometry. An association between human genetic variants and circulating Lp(a) levels was analyzed in the UK Biobank cohort.

RESULTS: The top positive and negative regulators of Lp(a) uptake in our screen were LDLR and MYLIP, encoding the LDL receptor and its ubiquitin ligase IDOL, respectively. We also found a significant correlation for other genes with established roles in LDLR regulation. No other gene products, including those previously proposed as Lp(a) receptors, exhibited a significant effect on Lp(a) uptake in our screen. We validated the functional influence of LDLR expression on HuH7 Lp(a) uptake, confirmed in vitro binding between the LDLR extracellular domain and purified Lp(a), and detected an association between loss-of-function LDLR variants and increased circulating Lp(a) levels in the UK Biobank cohort.

CONCLUSIONS: Our findings support a central role for the LDL receptor in mediating Lp(a) uptake by hepatocytes.},
}

Humans
*Lipoprotein(a)/metabolism/blood/genetics
*Receptors, LDL/genetics/metabolism
Ubiquitin-Protein Ligases/genetics/metabolism
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
United Kingdom
*Hepatocytes/metabolism

@article {pmid40173337,
year = {2025},
author = {Kandlbinder, A and Peter-Spiess, MH and Leeners, B and Mollaysa, A and Cavazza, T and Meier, A and Braunschweig, M and Ioannidi, E and Schwank, G and Krauthammer, M},
title = {Strategies for Interdisciplinary Human Gene Editing Research: Insights from a Swiss Project.},
journal = {The CRISPR journal},
volume = {8},
number = {2},
pages = {79-88},
doi = {10.1089/crispr.2024.0041},
pmid = {40173337},
issn = {2573-1602},
mesh = {*Gene Editing/methods/ethics ; Humans ; CRISPR-Cas Systems ; Genetic Therapy/methods ; *Interdisciplinary Research ; Switzerland ; },
abstract = {CRISPR gene editing is a cutting-edge technology that has advanced tremendously in recent years. The first clinical CRISPR applications have been approved, and more gene editing therapies are to be expected in human medicine. Consequently, continuous basic research is needed to assess possibilities and prime future clinical applications. Because this technology not only offers new possibilities for treating diseases but also raises important ethical and societal questions, collaboration between human, life, biomedical, and medical sciences is needed. In this article, we discuss the practical challenges of such interdisciplinary projects and present strategies for addressing them based on our experience of conducting an interdisciplinary project on CRISPR. This work aims to help and encourage interdisciplinary collaborations and discussions on modern scientific endeavors that, such as gene editing, tend to blur the lines between traditional disciplines. The strategies suggested include realistic expectations, shared goals, space setting, and expert and lay dialogue.},
}

*Gene Editing/methods/ethics
Humans
CRISPR-Cas Systems
Genetic Therapy/methods
*Interdisciplinary Research
Switzerland

@article {pmid40163416,
year = {2025},
author = {Tordoff, J and Alfonse, LE and Makarova, KS and Ornstein, A and Garrity, AJ and Yan, WX and Scott, DA and Koonin, EV and Cheng, DR},
title = {Initial Characterization of 12 New Subtypes and Variants of Type V CRISPR Systems.},
journal = {The CRISPR journal},
volume = {8},
number = {2},
pages = {149-154},
doi = {10.1089/crispr.2024.0100},
pmid = {40163416},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Bacteriophages/genetics ; *CRISPR-Associated Proteins/genetics ; Gene Editing ; Prophages/genetics ; Genetic Variation ; },
abstract = {Type V CRISPR systems are highly diverse in sequence, mechanism, and function. Although recent efforts have greatly expanded our understanding of their evolution, the diversity of type V systems remains to be completely explored, and many clades have not been experimentally characterized. In this work, we mined metagenomic databases to identify three new subtypes and nine new variants of Cas12, the effector of Type V systems, and provide experimental and computational characterization of their Protospacer-Adjacent Motif (PAM), interference activity, loci architecture, and tracrRNA dependence. Half of the new Cas12s are found in phages or prophages. New subtypes Cas12o and Cas12p lack the canonical RuvC catalytic residues, suggesting they interfere with the target without cleavage, possibly by blocking transcription or replication. One variant, Cas12f10, displays substantial activity on PAM-less targets. Our work expands the diversity of the functionally characterized Cas12 effectors and provides some promising candidates for genome engineering tools.},
}

*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Bacteriophages/genetics
*CRISPR-Associated Proteins/genetics
Gene Editing
Prophages/genetics
Genetic Variation

@article {pmid40163415,
year = {2025},
author = {Noel, EA and Sahu, SU and Wyman, SK and Krishnappa, N and Jeans, C and Wilson, RC},
title = {Hairpin Internal Nuclear Localization Signals in CRISPR-Cas9 Enhance Editing in Primary Human Lymphocytes.},
journal = {The CRISPR journal},
volume = {8},
number = {2},
pages = {105-119},
doi = {10.1089/crispr.2024.0080},
pmid = {40163415},
issn = {2573-1602},
mesh = {Humans ; *Gene Editing/methods ; *Nuclear Localization Signals/genetics ; *CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; *Lymphocytes/metabolism ; T-Lymphocytes/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Nucleus ; },
abstract = {The incorporation of nuclear localization signal (NLS) sequences at one or both termini of CRISPR enzymes is a widely adopted strategy to facilitate genome editing. Engineered variants of CRISPR enzymes with diverse NLS sequences have demonstrated superior performance, promoting nuclear localization and efficient DNA editing. However, limiting NLS fusion to the CRISPR protein's termini can negatively impact protein yield via recombinant expression. Here we present a distinct strategy involving the installation of hairpin internal NLS sequences (hiNLS) at rationally selected sites within the backbone of CRISPR-Cas9. We evaluated the performance of these hiNLS Cas9 variants by editing genes in human primary T cells following the delivery of ribonucleoprotein enzymes via either electroporation or co-incubation with amphiphilic peptides. We show that hiNLS Cas9 variants can improve editing efficiency in T cells compared with constructs with terminally fused NLS sequences. Furthermore, many hiNLS Cas9 constructs can be produced with high purity and yield, even when these constructs contain as many as nine NLS. These hiNLS Cas9 constructs represent a key advance in optimizing CRISPR effector design and may contribute to improved editing outcomes in research and therapeutic applications.},
}

Humans
*Gene Editing/methods
*Nuclear Localization Signals/genetics
*CRISPR-Cas Systems/genetics
CRISPR-Associated Protein 9/genetics/metabolism
*Lymphocytes/metabolism
T-Lymphocytes/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Cell Nucleus

@article {pmid40151952,
year = {2025},
author = {Yuan, H and Song, C and Xu, H and Sun, Y and Anthon, C and Bolund, L and Lin, L and Benabdellah, K and Lee, C and Hou, Y and Gorodkin, J and Luo, Y},
title = {An Overview and Comparative Analysis of CRISPR-SpCas9 gRNA Activity Prediction Tools.},
journal = {The CRISPR journal},
volume = {8},
number = {2},
pages = {89-104},
doi = {10.1089/crispr.2024.0058},
pmid = {40151952},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Machine Learning ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; Deep Learning ; Computational Biology/methods ; Computer Simulation ; Software ; Animals ; },
abstract = {Design of guide RNA (gRNA) with high efficiency and specificity is vital for successful application of the CRISPR gene editing technology. Although many machine learning (ML) and deep learning (DL)-based tools have been developed to predict gRNA activities, a systematic and unbiased evaluation of their predictive performance is still needed. Here, we provide a brief overview of in silico tools for CRISPR design and assess the CRISPR datasets and statistical metrics used for evaluating model performance. We benchmark seven ML and DL-based CRISPR-Cas9 editing efficiency prediction tools across nine CRISPR datasets covering six cell types and three species. The DL models CRISPRon and DeepHF outperform the other models exhibiting greater accuracy and higher Spearman correlation coefficient across multiple datasets. We compile all CRISPR datasets and in silico prediction tools into a GuideNet resource web portal, aiming to facilitate and streamline the sharing of CRISPR datasets. Furthermore, we summarize features affecting CRISPR gene editing activity, providing important insights into model performance and the further development of more accurate CRISPR prediction models.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
Machine Learning
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Associated Protein 9/genetics/metabolism
Deep Learning
Computational Biology/methods
Computer Simulation
Software
Animals

@article {pmid40147800,
year = {2025},
author = {Yu, WB and Ye, ZH and Shi, JJ and Deng, WQ and Chen, J and Lu, JJ},
title = {Dual blockade of GSTK1 and CD47 improves macrophage-mediated phagocytosis on cancer cells.},
journal = {Biochemical pharmacology},
volume = {236},
number = {},
pages = {116898},
doi = {10.1016/j.bcp.2025.116898},
pmid = {40147800},
issn = {1873-2968},
mesh = {*CD47 Antigen/antagonists & inhibitors/metabolism/immunology/genetics ; *Phagocytosis/physiology/drug effects ; Humans ; *Macrophages/metabolism/drug effects/immunology ; *Glutathione Transferase/antagonists & inhibitors/metabolism/genetics ; Cell Line, Tumor ; Animals ; Mice ; RAW 264.7 Cells ; CRISPR-Cas Systems ; *Neoplasms/metabolism ; },
abstract = {CD47 is a crucial anti-phagocytic signal in regulating macrophage responses and its manipulation offers the therapeutic potential in cancer treatment. However, in many cases, blockade of CD47 by itself is insufficient to activate macrophage effectively, indicating other unidentified phagocytosis-regulating factors to resist the macrophage activity. In this study, a genome-wide human CRISPR-Cas9 library was developed for comprehensive screening of phagocytosis-regulating factors in the context of CD47 blockade. The screening results identified GSTK1 as a potential anti-phagocytic signal counteracting the efficacy of CD47-based phagocytosis. The disruption of GSTK1 significantly increased the phagocytosis rate of cancer cells by macrophages in combination with anti-CD47 antibody. Further mechanism investigation unveiled that GSTK1 blockade increased the membrane exposure of calreticulin in different cancer cells, which might be the primary mechanism driving enhanced macrophage-mediated phagocytosis. To this end, siGSTK1-loaded nanoparticles (siGSTK1-LNPs) were designed to suppress the GSTK1 expression efficiently. The comparable phagocytosis efficacy was also observed when combining siGSTK1-LNPs with anti-CD47 antibody. Above all, GSTK1 blockade was identified as a promising and feasible stimulus for enhancing the effectiveness of anti-CD47 antibody, introducing a novel and effective combination approach in cancer immunotherapy.},
}

*CD47 Antigen/antagonists & inhibitors/metabolism/immunology/genetics
*Phagocytosis/physiology/drug effects
Humans
*Macrophages/metabolism/drug effects/immunology
*Glutathione Transferase/antagonists & inhibitors/metabolism/genetics
Cell Line, Tumor
Animals
Mice
RAW 264.7 Cells
CRISPR-Cas Systems
*Neoplasms/metabolism

@article {pmid40118290,
year = {2025},
author = {Cros-Perrial, E and Beaumel, S and Gimbert, M and Camus, N and Vicente, C and Sekiou, I and Figuet, L and Duruisseaux, M and Dumontet, C and Jordheim, LP},
title = {SLX4 and XPF are involved in cell migration and EMT in a cell-specific manner.},
journal = {Biochemical pharmacology},
volume = {236},
number = {},
pages = {116885},
doi = {10.1016/j.bcp.2025.116885},
pmid = {40118290},
issn = {1873-2968},
mesh = {Humans ; *Epithelial-Mesenchymal Transition/physiology/drug effects ; *Cell Movement/physiology/drug effects ; Animals ; *DNA-Binding Proteins/genetics/metabolism/deficiency ; Mice ; HEK293 Cells ; Cell Line, Tumor ; A549 Cells ; HT29 Cells ; CRISPR-Cas Systems ; Cell Proliferation ; },
abstract = {SLX4 and XPF are two proteins involved in DNA repair, but very little is known about their potential roles in other processes of cancer cell biology. We developed original cell models with CRISPR-Cas9-mediated knock-out of SLX4 and/or XPF using five different cell lines (A549, NCI-H1703, COLO-357, HT-29 and HEK-293 T), and performed characterization with cell biology experiments including migration assays, drug sensitivity testing, cell proliferation assessment and Western blots for relevant proteins. Results showed decreased migration of all models in HT-29 cells, of XPF-deficient COLO-357 cells and of SLX4-deficient HEK-293 T cells. Modified cell models had overall increased sensitivity to cisplatin and mitomycine C, and some models showed an increased frequency of double-stranded DNA damages. One NCI-H1703 cell model showed major karyotypic modifications, and epithelial to mesenchymal transition (EMT)-related proteins were modified in several models. Finally, knocking out one or both proteins in A549 cells had not the same impact on in vivo growth in mice. These original cell models allowed us to identify new and DNA repair-unrelated cellular roles of SLX4 and XPF in cancer cell biology. Our results should be considered within work on Nucleotide Excision Repair (NER) inhibition targeting SLX, XPF or other related proteins.},
}

Humans
*Epithelial-Mesenchymal Transition/physiology/drug effects
*Cell Movement/physiology/drug effects
Animals
*DNA-Binding Proteins/genetics/metabolism/deficiency
Mice
HEK293 Cells
Cell Line, Tumor
A549 Cells
HT29 Cells
CRISPR-Cas Systems
Cell Proliferation

@article {pmid40099789,
year = {2025},
author = {Huang, L and Zhou, Y and Feng, Y and Jia, S and Wang, S and Zhong, C},
title = {Tailoring Bacterial Cellulose through the CRISPR/Cas9-Mediated Gene Editing Tool in Komagataeibacter xylinus.},
journal = {ACS synthetic biology},
volume = {14},
number = {4},
pages = {1161-1172},
doi = {10.1021/acssynbio.4c00785},
pmid = {40099789},
issn = {2161-5063},
mesh = {*Gene Editing/methods ; *Cellulose/genetics/biosynthesis/metabolism/chemistry ; *CRISPR-Cas Systems/genetics ; *Gluconacetobacter xylinus/genetics/metabolism ; Metabolic Engineering/methods ; *Acetobacteraceae/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Bacterial cellulose (BC) is a nanocellulose produced by bacteria, formed by glucose units linked through β-1,4 glycosidic bonds. It features a three-dimensional network structure, superior water retention capacity, high porosity, and outstanding biocompatibility, among other notable characteristics. Komagataeibacter xylinus was the predominant strain used for BC production. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associate-protein 9)-mediated gene editing tool has been applied in various species; however, its application in K. xylinus has not been reported. To facilitate metabolic pathway engineering in K. xylinus, a CRISPR/Cas9-mediated gene editing tool specific to this strain was developed, achieving a gene editing efficiency exceeding 73%. Upon application of the CRISPR/Cas9-mediated gene editing tool in K. xylinus, the strain's ability to synthesize BC was enhanced by 23.6% (5.75 g/L), and the impact of BC synthase-correlated genes (bcsH, bcsX, bcsY, and bcsZ) on BC structure was investigated. The advancement of CRISPR/Cas9-mediated gene editing tools in K. xylinus is expected to accelerate genetic modification of this organism. This advancement has the potential to significantly improve our understanding of the genetic regulatory mechanisms that govern the structure and production of BC, thereby facilitating cost-effective synthesis of BC with tailored structural properties.},
}

*Gene Editing/methods
*Cellulose/genetics/biosynthesis/metabolism/chemistry
*CRISPR-Cas Systems/genetics
*Gluconacetobacter xylinus/genetics/metabolism
Metabolic Engineering/methods
*Acetobacteraceae/genetics/metabolism
Bacterial Proteins/genetics/metabolism

@article {pmid40081369,
year = {2025},
author = {Binan, L and Jiang, A and Danquah, SA and Valakh, V and Simonton, B and Bezney, J and Manguso, RT and Yates, KB and Nehme, R and Cleary, B and Farhi, SL},
title = {Simultaneous CRISPR screening and spatial transcriptomics reveal intracellular, intercellular, and functional transcriptional circuits.},
journal = {Cell},
volume = {188},
number = {8},
pages = {2141-2158.e18},
doi = {10.1016/j.cell.2025.02.012},
pmid = {40081369},
issn = {1097-4172},
mesh = {Humans ; Animals ; *Transcriptome/genetics ; Induced Pluripotent Stem Cells/metabolism/cytology ; Single-Cell Analysis/methods ; Astrocytes/metabolism/cytology ; Mice ; *CRISPR-Cas Systems/genetics ; Monocytes/metabolism ; *Gene Regulatory Networks ; Autism Spectrum Disorder/genetics ; Immunity, Innate ; Gene Expression Profiling/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Lipopolysaccharides/pharmacology ; },
abstract = {Pooled optical screens have enabled the study of cellular interactions, morphology, or dynamics at massive scale, but they have not yet leveraged the power of highly plexed single-cell resolved transcriptomic readouts to inform molecular pathways. Here, we present a combination of imaging spatial transcriptomics with parallel optical detection of in situ amplified guide RNAs (Perturb-FISH). Perturb-FISH recovers intracellular effects that are consistent with single-cell RNA-sequencing-based readouts of perturbation effects (Perturb-seq) in a screen of lipopolysaccharide response in cultured monocytes, and it uncovers intercellular and density-dependent regulation of the innate immune response. Similarly, in three-dimensional xenograft models, Perturb-FISH identifies tumor-immune interactions altered by genetic knockout. When paired with a functional readout in a separate screen of autism spectrum disorder risk genes in human-induced pluripotent stem cell (hIPSC) astrocytes, Perturb-FISH shows common calcium activity phenotypes and their associated genetic interactions and dysregulated molecular pathways. Perturb-FISH is thus a general method for studying the genetic and molecular associations of spatial and functional biology at single-cell resolution.},
}

Humans
Animals
*Transcriptome/genetics
Induced Pluripotent Stem Cells/metabolism/cytology
Single-Cell Analysis/methods
Astrocytes/metabolism/cytology
Mice
*CRISPR-Cas Systems/genetics
Monocytes/metabolism
*Gene Regulatory Networks
Autism Spectrum Disorder/genetics
Immunity, Innate
Gene Expression Profiling/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Lipopolysaccharides/pharmacology

@article {pmid40073612,
year = {2025},
author = {Durazo-Martínez, K and Chaudhari, J and Sherry, LM and Webster, DA and Martins, K and Bostrom, JR and Carlson, DF and Sonstegard, TS and Vu, HLX},
title = {Modification of the splice acceptor in CD163 exon 7 of pigs is insufficient to confer resistance to PRRSV.},
journal = {Veterinary microbiology},
volume = {304},
number = {},
pages = {110450},
doi = {10.1016/j.vetmic.2025.110450},
pmid = {40073612},
issn = {1873-2542},
mesh = {Animals ; CD163 Antigen ; Swine ; *Receptors, Cell Surface/genetics ; *Porcine respiratory and reproductive syndrome virus ; *Porcine Reproductive and Respiratory Syndrome/genetics/immunology/virology ; *Antigens, CD/genetics ; *Antigens, Differentiation, Myelomonocytic/genetics ; *Exons/genetics ; *RNA Splice Sites/genetics ; *Disease Resistance/genetics ; CRISPR-Cas Systems ; RNA, Messenger/genetics ; Gene Editing ; },
abstract = {CD163 is the primary receptor for PRRSV, and its SRCR5 domain, encoded by exon 7, is crucial for supporting PRRSV infection. Previous studies have used CRISPR/Cas9 technology to remove exon 7 from the host genome, and the edited pigs were completely resistant to PRRSV infection. In this study, we used CRISPR/Cas9 technology mimicking an adenine base editor (ABE) to edit the splice acceptor site of exon 7, rendering it nonfunctional. This alteration was intended to cause exon 6 to join directly to exon 8 during mRNA processing, resulting in a mature mRNA transcript that lacks exon 7, which encodes the SRCR5 domain. Piglets carrying the exon 7 splice site modification (CD163Ex7-ABE) were successfully generated. However, these pigs remained fully susceptible to infection with a PRRSV-2 isolate. Analysis of CD163 mRNA from the CD163Ex7-ABE pigs revealed that they predominantly expressed a mature CD163 mRNA lacking exon 7. However, due to cryptic splice sites, two additional mRNA isoforms were expressed, including an in-frame variant containing all of exon 7 and an extra 48 base pairs. This likely resulted in the expression of a full-length CD163 with a 16-amino-acid insertion upstream of the SRCR5 domain, which was sufficient to render the animals susceptible to PRRSV. Overall, our results demonstrate that merely modifying the splice acceptor site of CD163 exon 7 is not sufficient to generate PRRSV-resistant pigs.},
}

Animals
CD163 Antigen
Swine
*Receptors, Cell Surface/genetics
*Porcine respiratory and reproductive syndrome virus
*Porcine Reproductive and Respiratory Syndrome/genetics/immunology/virology
*Antigens, CD/genetics
*Antigens, Differentiation, Myelomonocytic/genetics
*Exons/genetics
*RNA Splice Sites/genetics
*Disease Resistance/genetics
CRISPR-Cas Systems
RNA, Messenger/genetics
Gene Editing

@article {pmid40019800,
year = {2025},
author = {Major, RM and Mills, CA and Xing, L and Krantz, JL and Wolter, JM and Zylka, MJ},
title = {Exploring the Cytoplasmic Retention of CRISPR-Cas9 in Eukaryotic Cells: The Role of Nuclear Localization Signals and Ribosomal Interactions.},
journal = {The CRISPR journal},
volume = {8},
number = {2},
pages = {120-136},
doi = {10.1089/crispr.2024.0074},
pmid = {40019800},
issn = {2573-1602},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Ribosomes/metabolism/genetics ; *Cytoplasm/metabolism/genetics ; Cell Nucleus/metabolism/genetics ; *Nuclear Localization Signals/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics ; *Eukaryotic Cells/metabolism ; Kelch-Like ECH-Associated Protein 1/metabolism/genetics ; Gene Editing/methods ; Neurons/metabolism ; },
abstract = {Cas9 must be localized to the nucleus to access the genome of mammalian cells. For most proteins, adding a single nuclear localization signal (NLS) is sufficient to promote nuclear entry. However, Cas9 nuclear entry appears to be inefficient as multiple NLSs are typically added to Cas9. Here, we found that three different Cas9 variants interact with the ribosome in HEK293T cells, and that this interaction is RNA mediated. Following immunoprecipitation-mass spectrometry of cytoplasmic-localized Cas9-0NLS and nuclear-localized Cas9-4NLS constructs, we identified novel Cas9 interactors in postmitotic neurons, including KEAP1 and additional ribosomal subunits, the latter were enriched in Cas9-0NLS samples. Collectively, our results suggest that Cas9 is sequestered in the cytoplasm of mammalian cells, in part, via interaction with the ribosome. Increasing the number of NLSs on Cas9 and/or increasing the amount of cytoplasmic guide RNA has the potential to outcompete ribosomal RNA binding and promote efficient nuclear localization of CRISPR-Cas9 variants.},
}

Humans
*CRISPR-Cas Systems/genetics
HEK293 Cells
*Ribosomes/metabolism/genetics
*Cytoplasm/metabolism/genetics
Cell Nucleus/metabolism/genetics
*Nuclear Localization Signals/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Associated Protein 9/metabolism/genetics
*Eukaryotic Cells/metabolism
Kelch-Like ECH-Associated Protein 1/metabolism/genetics
Gene Editing/methods
Neurons/metabolism

@article {pmid39792480,
year = {2025},
author = {Buyukyoruk, M and Krishna, P and Santiago-Frangos, A and Wiedenheft, B},
title = {Discovery of Diverse CRISPR Leader Motifs, Putative Functions, and Applications for Enhanced CRISPR Detection and Subtype Annotation.},
journal = {The CRISPR journal},
volume = {8},
number = {2},
pages = {137-148},
doi = {10.1089/crispr.2024.0093},
pmid = {39792480},
issn = {2573-1602},
support = {K99 GM147842/GM/NIGMS NIH HHS/United States ; R00 GM147842/GM/NIGMS NIH HHS/United States ; },
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Nucleotide Motifs ; *Archaea/genetics ; CRISPR-Cas Systems ; *Bacteria/genetics ; Genome, Archaeal ; Genome, Bacterial ; Conserved Sequence ; },
abstract = {Bacteria and archaea acquire resistance to genetic parasites by preferentially integrating short fragments of foreign DNA at one end of a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR). "Leader" DNA upstream of CRISPR loci regulates transcription and foreign DNA integration into the CRISPR. Here, we analyze 37,477 CRISPRs from 39,277 bacterial and 556 archaeal genomes to identify conserved sequence motifs in CRISPR leaders. A global analysis of all leader sequences fails to identify universally conserved motifs. However, an analysis of leader sequences that have been grouped by 16S rRNA-based taxonomy and CRISPR subtype reveals 87 specific motifs in type I, II, III, and V CRISPR leaders. Fourteen of these leader motifs have biochemically demonstrated roles in CRISPR biology including integration, transcription, and CRISPR RNA processing. Another 28 motifs are related to DNA binding sites for proteins with functions that are consistent with regulating CRISPR activity. In addition, we show that these leader motifs can be used to improve existing CRISPR detection methods and enhance the accuracy of CRISPR classification.},
}

*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Nucleotide Motifs
*Archaea/genetics
CRISPR-Cas Systems
*Bacteria/genetics
Genome, Archaeal
Genome, Bacterial
Conserved Sequence

@article {pmid40243059,
year = {2025},
author = {Liu, Q and Jiang, Z and Li, S and Li, Y and Wan, Y and Hu, Z and Ma, S and Zou, Z and Yang, R},
title = {Nonequilibrium hybridization-driven CRISPR/Cas adapter with extended energetic penalty for discrimination of single-nucleotide variants.},
journal = {Nucleic acids research},
volume = {53},
number = {7},
pages = {},
doi = {10.1093/nar/gkaf287},
pmid = {40243059},
issn = {1362-4962},
support = {22334005//National Natural Science Foundation of China/ ; 2022JJ20038//Natural Science Foundation of Hunan Province/ ; 23A0059//Scientific Research Fund of Hunan Provincial Education Department/ ; 2022XKQ0205//Hunan Normal University/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Polymorphism, Single Nucleotide ; Humans ; Nucleic Acid Hybridization ; Proto-Oncogene Proteins B-raf/genetics ; ErbB Receptors/genetics ; CRISPR-Associated Proteins/metabolism/genetics ; Mutation ; Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Base Pair Mismatch ; },
abstract = {Accurate identification of single-nucleotide variants (SNVs) is critical in clinical diagnostics but remains challenging due to subtle free energy variations, particularly for hard-to-detect SNVs such as wobble base pairs and those in high guanine-cytosine (GC) regions. Here we report a high-energetic-penalty SNV detection (HEPSD) platform that redesigns the hybridization regions of CRISPR RNA (crRNA) in the CRISPR/Cas12a system. This system employs a binary crRNA architecture design that enables the activation of the cleavage activity of Cas12a while amplifying the energetic penalty for single-nucleotide mismatches through nonequilibrium hybridization-driven regulation. Consequently, the entire targeting region of CRISPR/Cas exhibits a marked preference for mutations in genomic DNA, while preventing false activation induced by sequences containing a single mismatched nucleotide. Moreover, HEPSD exhibits exceptional differentiation performance for hard-to-detect SNVs including wobble mutations at extreme GC contents. As proof of principle, profiling of BRAF V600E and EGFR L858R tumor mutations down to a 0.01% variant allele frequency was achieved, enabling accurate discrimination of 132 clinical sample pairs, which showed high consistency with quantitative polymerase chain reaction-based techniques and next-generation sequencing. The proven effectiveness of this platform showcases its potential for clinical molecular diagnostics and expands the fundamental scope of hybridization-based protocols.},
}

*CRISPR-Cas Systems/genetics
*Polymorphism, Single Nucleotide
Humans
Nucleic Acid Hybridization
Proto-Oncogene Proteins B-raf/genetics
ErbB Receptors/genetics
CRISPR-Associated Proteins/metabolism/genetics
Mutation
Endodeoxyribonucleases/genetics/metabolism
Bacterial Proteins/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Base Pair Mismatch

@article {pmid40242952,
year = {2025},
author = {Aloliqi, AA and Alnuqaydan, AM and Albutti, A and Alharbi, BF and Rahmani, AH and Khan, AA},
title = {Current updates regarding biogenesis, functions and dysregulation of microRNAs in cancer: Innovative approaches for detection using CRISPR/Cas13‑based platforms (Review).},
journal = {International journal of molecular medicine},
volume = {55},
number = {6},
pages = {},
doi = {10.3892/ijmm.2025.5531},
pmid = {40242952},
issn = {1791-244X},
mesh = {Humans ; *MicroRNAs/genetics/metabolism ; *Neoplasms/genetics/diagnosis ; *CRISPR-Cas Systems/genetics ; *Gene Expression Regulation, Neoplastic ; Animals ; Biomarkers, Tumor/genetics ; },
abstract = {MicroRNAs (miRNAs) are short non‑coding RNAs, which perform a key role in cellular differentiation and development. Most human diseases, particularly cancer, are linked to miRNA functional dysregulation implicated in the expression of tumor‑suppressive or oncogenic targets. Cancer hallmarks such as continued proliferative signaling, dodging growth suppressors, invasion and metastasis, triggering angiogenesis, and avoiding cell death have all been demonstrated to be affected by dysregulated miRNAs. Thus, for the treatment of different cancer types, the detection and quantification of this type of RNA is significant. The classical and current methods of RNA detection, including northern blotting, reverse transcription‑quantitative PCR, rolling circle amplification and next‑generation sequencing, may be effective but differ in efficiency and accuracy. Furthermore, these approaches are expensive, and require special instrumentation and expertise. Thus, researchers are constantly looking for more innovative approaches for miRNA detection, which can be advantageous in all aspects. In this regard, an RNA manipulation tool known as the CRISPR and CRISPR‑associated sequence 13 (CRISPR/Cas13) system has been found to be more advantageous in miRNA detection. The Cas13‑based miRNA detection approach is cost effective and requires no special instrumentation or expertise. However, more research and validation are required to confirm the growing body of CRISPR/Cas13‑based research that has identified miRNAs as possible cancer biomarkers for diagnosis and prognosis, and as targets for treatment. In the present review, current updates regarding miRNA biogenesis, structural and functional aspects, and miRNA dysregulation during cancer are described. In addition, novel approaches using the CRISPR/Cas13 system as a next‑generation tool for miRNA detection are discussed. Furthermore, challenges and prospects of CRISPR/Cas13‑based miRNA detection approaches are described.},
}

Humans
*MicroRNAs/genetics/metabolism
*Neoplasms/genetics/diagnosis
*CRISPR-Cas Systems/genetics
*Gene Expression Regulation, Neoplastic
Animals
Biomarkers, Tumor/genetics