@article {pmid40668918,
year = {2025},
author = {Valcárcel, G and Lazarenkov, A and López-Rubio, AV and Berenguer, C and Calafell-Segura, J and Rodríguez-Ubreva, J and Ballestar, E and Sardina, JL},
title = {Modulating immune cell fate and inflammation through CRISPR-mediated DNA methylation editing.},
journal = {Science advances},
volume = {11},
number = {29},
pages = {eadt1644},
doi = {10.1126/sciadv.adt1644},
pmid = {40668918},
issn = {2375-2548},
mesh = {*DNA Methylation/genetics ; Humans ; *Gene Editing/methods ; *Inflammation/genetics/immunology ; *CRISPR-Cas Systems ; Promoter Regions, Genetic ; DNA Methyltransferase 3A ; Cell Differentiation/genetics ; Epigenesis, Genetic ; DNA (Cytosine-5-)-Methyltransferases/genetics ; Gene Expression Regulation ; Myeloid Cells/metabolism/immunology ; Proto-Oncogene Proteins/genetics ; Mixed Function Oxygenases ; Interleukin 1 Receptor Antagonist Protein ; },
abstract = {Immune cell differentiation and activation are associated with widespread DNA methylation changes; however, the causal relationship between these changes and their impact in shaping cell fate decisions still needs to be fully elucidated. Here, we conducted a genome-wide analysis to investigate the relationship between DNA methylation and gene expression at gene regulatory regions in human immune cells. By using CRISPR-dCas9-TET1 and -DNMT3A epigenome editing tools, we successfully established a cause-and-effect relationship between the DNA methylation levels of the promoter of the interleukin-1 receptor antagonist (IL1RN) gene and its expression. We observed that modifying the DNA methylation status of the IL1RN promoter is sufficient to alter human myeloid cell fate and change the cellular response to inflammatory and pathogenic stimuli. Collectively, our findings demonstrate the potential of targeting specific DNA methylation events to directly modulate immune and inflammatory responses, providing a proof of principle for intervening in a broad range of inflammation-related diseases.},
}

*DNA Methylation/genetics
Humans
*Gene Editing/methods
*Inflammation/genetics/immunology
*CRISPR-Cas Systems
Promoter Regions, Genetic
DNA Methyltransferase 3A
Cell Differentiation/genetics
Epigenesis, Genetic
DNA (Cytosine-5-)-Methyltransferases/genetics
Gene Expression Regulation
Myeloid Cells/metabolism/immunology
Proto-Oncogene Proteins/genetics
Mixed Function Oxygenases
Interleukin 1 Receptor Antagonist Protein

@article {pmid40668108,
year = {2025},
author = {Yang, X and Fan, Y and Xu, X and Shen, T and An, X and Zhang, Y and Zhang, Z and Pan, H and Chang, D},
title = {Direct Testing of Blood Samples to Diagnose Bloodstream Infections.},
journal = {ACS infectious diseases},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsinfecdis.5c00109},
pmid = {40668108},
issn = {2373-8227},
abstract = {Bloodstream infection (BSI) is a critical condition with extremely high mortality. Rapid and accurate diagnosis is crucial for effective treatment. The traditional blood culture (BC) method has issues, such as long testing times and limited sensitivity, making it challenging to meet the need for timely diagnosis. To address this problem, various molecular biology methods for directly detecting blood samples (whole blood, plasma, serum, and positive BC samples) have emerged. These include Raman spectroscopy, mass spectrometry, nucleic acid amplification, and hybridization techniques (such as the CRISPR/Cas system, digital droplet PCR (ddPCR), and T2 magnetic resonance (T2MR)), biosensors, and next-generation sequencing (NGS). These methods can quickly identify pathogens and their drug-resistant markers, significantly reducing diagnostic delays and helping to provide earlier targeted treatment. This article systematically analyzes the principles, advantages, and disadvantages of these advanced techniques, explores their value in revolutionizing the BSI diagnostic model, and looks ahead to future development directions, providing a reference for research and clinical applications in this field.},
}

@article {pmid40667754,
year = {2025},
author = {Wu, X and Luteijn, RD and Lozano-Andrés, E and Marougka, K and Li, W and Narimatsu, Y and van Kuppeveld, FJM and Bosch, BJ and Lebbink, RJ and Vries, E and de Haan, CAM},
title = {Identification of β4GALNT2 as an anti-hPIV3 factor through genome-wide CRISPR/Cas9 library screening.},
journal = {Emerging microbes & infections},
volume = {14},
number = {1},
pages = {2529895},
doi = {10.1080/22221751.2025.2529895},
pmid = {40667754},
issn = {2222-1751},
mesh = {Humans ; *CRISPR-Cas Systems ; *N-Acetylgalactosaminyltransferases/genetics/metabolism ; *Parainfluenza Virus 3, Human/physiology/genetics ; *Respirovirus Infections/virology/genetics ; Polypeptide N-acetylgalactosaminyltransferase ; *Host-Pathogen Interactions ; Glycosylation ; Polysaccharides/metabolism ; Animals ; HEK293 Cells ; },
abstract = {Human respirovirus 3 (also known as human parainfluenza virus 3; hPIV3) is a major cause of severe acute respiratory infections in vulnerable populations. Here we conducted a genome-wide CRISPR/Cas9 library screen to identify key host factors for hPIV3 infection. In addition to identifying several host proteins involved in glycosylation as proviral factors, we identified β-1,4-N-Acetyl-Galactosaminyltransferase 2 (β4GALNT2) as a potent restriction factor. Further investigation demonstrated that the addition of a GalNAc residue to α2-3-sialylated glycans by β4GALNT2, resulting in the Sd[a] glycotope, disrupted the interaction between the viral hemagglutinin-neuraminidase (HN) attachment protein and sialoglycan receptors. Specifically, the additional GalNAc residue interfered with the interaction of residue W371 in HN with sub-terminal glycan moieties. β4GALNT2-mediated Sd[a] epitope expression also negatively affected infection by other respiroviruses, with the strongest effect being observed for hPIV3.},
}

Humans
*CRISPR-Cas Systems
*N-Acetylgalactosaminyltransferases/genetics/metabolism
*Parainfluenza Virus 3, Human/physiology/genetics
*Respirovirus Infections/virology/genetics
Polypeptide N-acetylgalactosaminyltransferase
*Host-Pathogen Interactions
Glycosylation
Polysaccharides/metabolism
Animals
HEK293 Cells

@article {pmid40665763,
year = {2025},
author = {Shao, F and Zhou, Y and Shi, J and Zhang, M and Yang, H and Fei, J},
title = {Establishment and Application of Mice Models for Tracing Gene Expression and Protein Product of TNF.},
journal = {Biotechnology journal},
volume = {20},
number = {7},
pages = {e70080},
doi = {10.1002/biot.70080},
pmid = {40665763},
issn = {1860-7314},
support = {2019YFA0905900//National Key Research and Development Program of China/ ; 19DZ2280500//Science and Technology Commission of Shanghai Municipality/ ; 18DZ2293500//Science and Technology Commission of Shanghai Municipality/ ; 18DZ2290700//Science and Technology Commission of Shanghai Municipality/ ; },
mesh = {Animals ; Mice ; *Tumor Necrosis Factor-alpha/genetics/metabolism ; Mice, Transgenic ; Disease Models, Animal ; Lipopolysaccharides ; Genes, Reporter ; CRISPR-Cas Systems ; Promoter Regions, Genetic ; Luciferases/genetics/metabolism ; Sepsis/genetics/chemically induced/metabolism ; Dexamethasone/pharmacology ; Gene Expression ; Inflammation/genetics ; },
abstract = {Tumor necrosis factor α (TNF-α) is a pleiotropic cytokine crucial for immune function, cellular homeostasis, and disease progression, yet its complex roles in vivo remain unclear. Challenges in studying TNF-α include its widespread gene expression, variability in expression levels, diverse protein forms, and low baseline expression, which complicate traditional detection and tracking methods. To address these challenges, we constructed three distinct transgenic luciferase reporter mouse models (TNF-IRES-Luc, TNF-Nanoluc, and TNF-HiBiT) driven by the endogenous TNF-α gene, using CRISPR/Cas9 technology through homology-directed repair. The firefly luciferase gene, secreted NanoLuc gene, and HiBiT gene were individually integrated into the mouse genome under the control of the endogenous TNF-α promoter. Our results demonstrate that endogenous TNF-α expression can be effectively monitored by measuring luciferase levels in vivo and in vitro, using an in vivo imaging system and a luminometer. This was validated during inflammatory processes such as lipopolysaccharide (LPS)-induced sepsis and phorbol ester (TPA)-induced mouse ear edema. Furthermore, the anti-inflammatory drug dexamethasone (DXM) significantly inhibited TNF-α and luciferase expression in both inflammatory models. Our study demonstrates these mouse models are valuable tools for studying TNF-α expression in inflammatory responses and related diseases, as well as evaluating anti-inflammatory drug efficacy.},
}

Animals
Mice
*Tumor Necrosis Factor-alpha/genetics/metabolism
Mice, Transgenic
Disease Models, Animal
Lipopolysaccharides
Genes, Reporter
CRISPR-Cas Systems
Promoter Regions, Genetic
Luciferases/genetics/metabolism
Sepsis/genetics/chemically induced/metabolism
Dexamethasone/pharmacology
Gene Expression
Inflammation/genetics

@article {pmid40665149,
year = {2025},
author = {Wongborisuth, C and Innachai, P and Saisawang, C and Tubsuwan, A and Jearawiriyapaisarn, N and Kaewprommal, P and Piriyapongsa, J and Chiangjong, W and Anurathapan, U and Songdej, D and Tangprasittipap, A and Hongeng, S},
title = {Disrupting ZBTB7A or BCL11A binding sites reactivates fetal hemoglobin in erythroblasts from healthy and β[0]-thalassemia/HbE individuals.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {25580},
pmid = {40665149},
issn = {2045-2322},
support = {Grant ID: BRF1-016/2565//Basic Research Fund: the fiscal year 2022 , Mahidol University/ ; Grant ID: RF_64041//Faculty of Medicine Ramathibodi Hospital, Mahidol University/ ; Grant ID: MRC-IM 02/2565//Mahidol University/ ; },
mesh = {Humans ; *Fetal Hemoglobin/metabolism/genetics ; *beta-Thalassemia/genetics/metabolism ; *Erythroblasts/metabolism ; Binding Sites ; *Transcription Factors/metabolism/genetics ; *DNA-Binding Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Gene Editing ; Repressor Proteins/metabolism/genetics ; Promoter Regions, Genetic ; gamma-Globins/genetics ; *Hemoglobin E/metabolism/genetics ; *Carrier Proteins/metabolism/genetics ; Hematopoietic Stem Cells/metabolism ; *Nuclear Proteins/metabolism/genetics ; },
abstract = {CRISPR/Cas9 genome editing has emerged as a promising treatment for genetic diseases like β-thalassemia. Editing γ-globin promoters to disrupt ZBTB7A/LRF or BCL11A binding sites has shown potential for reactivating fetal hemoglobin and treating sickle cell disease. However, its application to β[0]-thalassemia/HbE disease remains unclear. This study utilized CRISPR/Cas9 to disrupt these sites in mobilized CD34 + hematopoietic stem /progenitor cells from healthy donors and β[0]-thalassemia/HbE patients. The editing efficiency for the BCL11A site (75-92%) was higher than for the ZBTB7A/LRF site (57-60%). Both disruptions similarly increased fetal hemoglobin production in healthy donors (BCL11A 26.2 ± 1.4%, ZBTB7A/LRF 27.9 ± 1.5%) and β[0]-thalassemia/HbE cells (BCL11A 62.7 ± 0.9%, ZBTB7A/LRF 64.0 ± 1.6%). Off-target effects were absent in BCL11A-edited cells but observed at low frequencies in ZBTB7A/LRF-edited cells. Neither disruption significantly affected erythroid differentiation. These findings highlight the comparable contributions of ZBTB7A/LRF and BCL11A binding sites to γ-globin reactivation. CRISPR/Cas9 editing of either site may offer a potential therapeutic strategy for β[0]-thalassemia/HbE disease.},
}

Humans
*Fetal Hemoglobin/metabolism/genetics
*beta-Thalassemia/genetics/metabolism
*Erythroblasts/metabolism
Binding Sites
*Transcription Factors/metabolism/genetics
*DNA-Binding Proteins/metabolism/genetics
CRISPR-Cas Systems
Gene Editing
Repressor Proteins/metabolism/genetics
Promoter Regions, Genetic
gamma-Globins/genetics
*Hemoglobin E/metabolism/genetics
*Carrier Proteins/metabolism/genetics
Hematopoietic Stem Cells/metabolism
*Nuclear Proteins/metabolism/genetics

@article {pmid40242936,
year = {2025},
author = {Guler Kara, H and Dogan, E and Bozok, V and Aktan, C and Cakiroglu, E and Eroglu, Z and Aygunes Jafari, D and Korkmaz, KS and Senturk, S and Kosova, B},
title = {The G protein-coupled receptor GPR89A is a novel potential therapeutic target to overcome cisplatin resistance in NSCLC Calu1 cells.},
journal = {The FEBS journal},
volume = {292},
number = {14},
pages = {3755-3770},
pmid = {40242936},
issn = {1742-4658},
support = {TUB1//Türkiye Bilimsel ve Teknolojik Araştırma Kurumu/ ; TOA-2021-22673//Ege University Research Foundation/ ; },
mesh = {Humans ; *Cisplatin/pharmacology ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Carcinoma, Non-Small-Cell Lung/drug therapy/genetics/pathology/metabolism ; *Receptors, G-Protein-Coupled/genetics/metabolism/antagonists & inhibitors ; *Lung Neoplasms/drug therapy/genetics/pathology/metabolism ; Cell Line, Tumor ; CRISPR-Cas Systems ; Apoptosis/drug effects ; Antineoplastic Agents/pharmacology ; Gene Expression Regulation, Neoplastic/drug effects ; Cell Proliferation/drug effects ; },
abstract = {Lung cancer is the most frequently diagnosed cancer type worldwide and is characterised by its high metastatic potential. Standard therapy for nonsmall cell lung cancer (NSCLC) cases includes chemotherapy with the platinum-based chemotherapeutic agent cisplatin. Although lung cancer cases respond well to cisplatin at the beginning of treatment, ~ 60% develop chemotherapy resistance during this process. In this study, a genome-wide CRISPR-Cas9-based genetic screening approach was employed to identify genes that cisplatin-resistant NSCLC Calu1 cells are more addicted to than sensitive cells. Cisplatin-resistant Calu1 cells were generated by the dose escalation method, and genome-wide CRISPR-Cas9-based genetic screening was performed with the Brunello CRISPR knockout library. Bioinformatics analyses of the obtained next-generation sequencing data revealed 63 potential candidate genes responsible for cisplatin resistance, including G protein-coupled receptor 89A (GPR89A), Poly(U) binding splicing factor 60 (PUF60), NBAS subunit of NRZ tethering complex (NBAS) and GrpE like 1, mitochondrial (GRPEL1). The GPR89A protein is located in the Golgi cisterna and Golgi-associated vesicle membrane, enables voltage-gated anion channel activity, and is involved in intracellular pH reduction. Functional studies carried out with GPR89A-knockout cisplatin-resistant Calu1 cells resulted in cell cycle arrest in the G2/M phase and increased polyploidy, and also prevented colony formation and cell migration. Cisplatin treatment, on the other hand, resulted in increased cell death by apoptosis upon cell cycle arrest in the S phase. In conclusion, this is the first study that identified GPR89A as a potential therapeutic target to overcome cisplatin resistance in NSCLC Calu1 cells.},
}

Humans
*Cisplatin/pharmacology
*Drug Resistance, Neoplasm/genetics/drug effects
*Carcinoma, Non-Small-Cell Lung/drug therapy/genetics/pathology/metabolism
*Receptors, G-Protein-Coupled/genetics/metabolism/antagonists & inhibitors
*Lung Neoplasms/drug therapy/genetics/pathology/metabolism
Cell Line, Tumor
CRISPR-Cas Systems
Apoptosis/drug effects
Antineoplastic Agents/pharmacology
Gene Expression Regulation, Neoplastic/drug effects
Cell Proliferation/drug effects

@article {pmid40664931,
year = {2025},
author = {Besati, M and Safarnejad, MR and Aliahmadi, A and Farzaneh, M and Ruiz, R and Montagud-Martínez, R and Rodrigo, G and Rafati, H},
title = {Detection of tomato brown rugose fruit virus through CRISPR-Cas12a and CRISPR-Cas9 systems.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {25638},
pmid = {40664931},
issn = {2045-2322},
mesh = {*Solanum lycopersicum/virology ; *CRISPR-Cas Systems ; *Plant Diseases/virology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Tomato brown rugose fruit virus (ToBRFV) is a single-stranded positive-sense RNA virus that targets tomato and pepper plants and is causing significant damage to crops in some regions of the world. ToBRFV is a highly contagious virus that is stable and rapidly spreads by mechanical methods and seeds. As a result, it may spread both locally and over long distances, and it is now recognized as a pandemic in plants. This study investigates the effectiveness of the systems CRISPR-Cas12a and CRISPR-Cas9, in conjugation with recombinase polymerase amplification (RPA), to detect ToBRFV in tomato plant samples collected from the field. The trans-cleavage activity of both nucleases, Cas12a and Cas9, was exploited to process a probe labelled with fluorescein and biotin to be resolved on a lateral flow device, thereby enabling a visual readout. We were able to detect the RNA genome of the virus in about 1 h at a low constant temperature. These results could pave way to offer a rapid, sensitive, and specific method for on-site detection of ToBRFV.},
}

*Solanum lycopersicum/virology
*CRISPR-Cas Systems
*Plant Diseases/virology
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40664653,
year = {2025},
author = {Chen, H and Tan, Q and Li, L and Li, L and Fu, J and Zhu, W and Li, J and Wang, Y and Li, S and Li, H and Sun, Y and Sun, Q and Lu, Z and Liu, Z},
title = {Refined DNA repair manipulation enables a universal knock-in strategy in mouse embryos.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6502},
pmid = {40664653},
issn = {2041-1723},
mesh = {Animals ; *Gene Knock-In Techniques/methods ; Mice ; *Embryo, Mammalian/metabolism ; CRISPR-Cas Systems/genetics ; *DNA Repair/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA End-Joining Repair/genetics ; Female ; DNA-Directed DNA Polymerase/genetics/metabolism ; Gene Editing/methods ; DNA Breaks, Double-Stranded ; },
abstract = {The design and screening of sgRNA in CRISPR-dependent gene knock-in is always laborious. Therefore, a universal and highly efficient knock-in strategy suitable for different sgRNA target sites is necessary. In our mouse embryo study, we find that the knock-in efficiency guided by adjacent sgRNAs varies greatly, although similar indel frequency. MMEJ-biased sgRNAs usually lead to high knock-in efficiency, whereas NHEJ-biased sgRNAs result in low knock-in efficiency. Blocking MMEJ repair by knocking down Polq can enhance knock-in efficiency, but inhibiting NHEJ repair shows variable effects. We identify a compound, AZD7648, that can shift DSBs repair towards MMEJ. Finally, by combining AZD7648 treatment with Polq knockdown, we develop a universal and highly efficient knock-in strategy in mouse embryos. This approach is validated at more than ten genomic loci, achieving up to 90% knock-in efficiency, marking a significant advancement toward predictable and highly efficient CRISPR-mediated gene integration.},
}

Animals
*Gene Knock-In Techniques/methods
Mice
*Embryo, Mammalian/metabolism
CRISPR-Cas Systems/genetics
*DNA Repair/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
DNA End-Joining Repair/genetics
Female
DNA-Directed DNA Polymerase/genetics/metabolism
Gene Editing/methods
DNA Breaks, Double-Stranded

@article {pmid40664139,
year = {2025},
author = {Lau, CH and Liang, QL and Chen, X and Li, J and Shi, Y and Huang, Y and Xia, Q and Zhu, H and Chen, G},
title = {CRISPR/Dx-based colorimetric and electrochemical detection systems for POCT applications.},
journal = {Biosensors & bioelectronics},
volume = {288},
number = {},
pages = {117778},
doi = {10.1016/j.bios.2025.117778},
pmid = {40664139},
issn = {1873-4235},
abstract = {To realize point-of-care testing (POCT) for human diseases, CRISPR/Cas technology has been integrated with commercial analytical devices and chromogenic substrates such as personal glucose meter (PGM), pregnancy test strips (PTS), and horseradish peroxidase (HRP). These integrated detection platforms enable portable diagnostics, economical, universal, rapid, and high throughput detection. The change in color and electrochemical signal enables visible signal readout and intuitive results without specialized equipment and skillful operators. Herein, we comprehensively and critically discuss these advancements and describe the molecular principles of each detection system. The current challenges and technical shortcomings associated with these CRISPR-based diagnostics for POCT applications are discussed. We also provide future perspectives and directions in this field that will improve the performance of CRISPR-PGM, CRISPR-PTS, and CRISPR-HRP for pathogens detection and molecular diagnostics. Their applications in early tumor detection and pan-cancer screening are highlighted. The potential ethical and societal impact of home self-tests for cancer are discussed.},
}

@article {pmid40663463,
year = {2025},
author = {Carmichael, JC and Stevens, CS and Atanasoff, KE and Kowdle, S and Reis, RA and Tortorella, D and Lee, B},
title = {Precision engineering of human cytomegalovirus without BAC constraints: a Sendai virus-delivered CRISPR/Cas9 approach.},
journal = {The Journal of general virology},
volume = {106},
number = {7},
pages = {},
doi = {10.1099/jgv.0.002126},
pmid = {40663463},
issn = {1465-2099},
mesh = {Humans ; *Cytomegalovirus/genetics ; *CRISPR-Cas Systems ; *Sendai virus/genetics ; *Chromosomes, Artificial, Bacterial/genetics ; *Gene Editing/methods ; Genome, Viral ; Fibroblasts/virology ; Genetic Vectors/genetics ; Epithelial Cells/virology ; Cell Line ; },
abstract = {Human cytomegalovirus (HCMV) genetic manipulation traditionally relies on bacterial artificial chromosome (BAC) recombineering, necessitated by its large ~236 kb genome. This approach is limited by the scarcity of HCMV strains engineered into BACs and often requires the deletion of 'non-essential' genes to accommodate the BAC cassette. We developed a novel approach using temperature-sensitive Sendai virus (SeV) vectors to deliver CRISPR/Cas9 for targeted HCMV genome editing without these constraints. This system achieves high editing efficiency (80-90%) in fibroblasts, epithelial cells and endothelial cells without BAC intermediates. As proof of principle, we targeted the HCMV (TB40/E strain) pentamer complex (PC) genes UL128 and UL130, crucial for viral entry into non-fibroblast cells. Edited viruses showed significantly reduced infectivity in epithelial cells, confirming functional disruption of the PC. Plaque purification yielded isogenic clones with phenotypes comparable to AD169, a naturally PC-deficient strain. Furthermore, multiplexed editing created precise 663 bp deletions in over 60% of viral genomes. Importantly, this method enables HCMV editing in physiologically relevant cell types without fibroblast passaging, which typically introduces mutations. This SeV-Cas9 system represents a significant advancement for studying HCMV biology in diverse cell types.},
}

Humans
*Cytomegalovirus/genetics
*CRISPR-Cas Systems
*Sendai virus/genetics
*Chromosomes, Artificial, Bacterial/genetics
*Gene Editing/methods
Genome, Viral
Fibroblasts/virology
Genetic Vectors/genetics
Epithelial Cells/virology
Cell Line

@article {pmid40663383,
year = {2025},
author = {Tao, S and Fang, Y and Zheng, L and Zhang, H and Xu, Y and Liang, W},
title = {Mechanistic study of the immune defense function of the CRISPR1-Cas system in Enterococcus faecalis.},
journal = {Virulence},
volume = {16},
number = {1},
pages = {2530665},
doi = {10.1080/21505594.2025.2530665},
pmid = {40663383},
issn = {2150-5608},
mesh = {*Enterococcus faecalis/genetics/immunology/drug effects ; *CRISPR-Cas Systems ; Plasmids/genetics ; Gene Transfer, Horizontal ; Mutation ; Drug Resistance, Bacterial/genetics ; Transformation, Bacterial ; },
abstract = {Enterococci are Gram-positive cocci that are considered to be one of the causative agents of hospital-acquired infections. CRISPR-Cas is an adaptive immune system with targeted defense functions against foreign invading nucleic acids and plays an important role in antibiotic resistance. In this study, we aimed to investigate II-A CRISPR-Cas-mediated immunity and the molecular mechanism underlying the horizontal transfer of drug resistance genes in Enterococcus faecalis. The mutant strains were constructed by the homologous recombination strategy. The interference of plasmid transformation by the Enterococcus faecalis CRISPR1/Cas system was confirmed through plasmid transformation efficiency. The different mutation positions in the protospacer sequence S1 and PAM region recombinant plasmids were constructed through enzyme digestion and sequencing verification to assess the impact of the CRISPR-encoded immunity. In the wild-type strain, the transformation efficiency of plasmids pAT28-S1-S9 containing protospacers and PAM sites decreased (p < 0.05). Single-base mutations at positions 25 and 28 of the protospacer region eliminated the ability of the wild-type strain to prevent plasmid transformation containing the protospacer and PAM sites (p > 0.05), whereas a single mismatch at protospacer positions 2,10,18,23 did not affect the ability of CRISPR-Cas system-positive strains to interfere with plasmid transformation (p < 0.05). There was no significant difference between the wild-type strain and the mutant strain in the transformation efficiency of the pS1-pΔPAM plasmid without PAM and plasmids containing single mutations (p > 0.05). In conclusion, the CRISPR-Cas system can block the transformation of matching protospacer sequences, and mutations near or within the protospacer adjacent motif (PAM) allow the plasmid to escape CRISPR-encoded immunity.},
}

*Enterococcus faecalis/genetics/immunology/drug effects
*CRISPR-Cas Systems
Plasmids/genetics
Gene Transfer, Horizontal
Mutation
Drug Resistance, Bacterial/genetics
Transformation, Bacterial

@article {pmid40663257,
year = {2025},
author = {Yerlikaya, BA and Yerlikaya, S and Gül, B and Yoldaş, H and Kavas, M and Mohamed, HI},
title = {Harnessing CRISPR/Cas9 in engineering biotic stress immunity in crops.},
journal = {Planta},
volume = {262},
number = {3},
pages = {54},
pmid = {40663257},
issn = {1432-2048},
mesh = {*CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics/immunology ; Gene Editing/methods ; *Stress, Physiological/genetics ; Disease Resistance/genetics ; Plant Diseases/immunology/genetics ; Plants, Genetically Modified/genetics ; Genetic Engineering/methods ; },
abstract = {There is significant potential for CRISPR/Cas9 to be used in developing crops that can adapt to biotic stresses such as fungal, bacterial, viral, and pest infections and weeds. The increasing global population and climate change present significant threats to food security by putting stress on plants, making them more vulnerable to diseases and productivity losses caused by pathogens, pests, and weeds. Traditional breeding methods are inadequate for the rapid development of new plant traits needed to counteract this decline in productivity. However, modern advances in genome-editing technologies, particularly CRISPR/Cas9, have transformed crop protection through precise and targeted modifications of plant genomes. This enables the creation of resilient crops with improved resistance to pathogens, pests, and weeds. This review examines various methods by which CRISPR/Cas9 can be utilized for crop protection. These methods include knocking out susceptibility genes, introducing resistance genes, and modulating defense genes. Potential applications of CRISPR/Cas9 in crop protection involve introducing genes that confer resistance to pathogens, disrupting insect genes responsible for survival and reproduction, and engineering crops that are resistant to herbicides. In conclusion, CRISPR/Cas9 holds great promise for advancing crop protection and ensuring food security in the face of environmental challenges and increasing population pressures. The most recent advancements in CRISPR technology for creating resistance to bacteria, fungi, viruses, and pests are covered here. We wrap up by outlining the most pressing issues and technological shortcomings, as well as unanswered questions for further study.},
}

*CRISPR-Cas Systems/genetics
*Crops, Agricultural/genetics/immunology
Gene Editing/methods
*Stress, Physiological/genetics
Disease Resistance/genetics
Plant Diseases/immunology/genetics
Plants, Genetically Modified/genetics
Genetic Engineering/methods

@article {pmid40662362,
year = {2025},
author = {Maillard, M and Nishii, R and Vu, HS and Bhattarai, KR and Yang, W and Li, J and Hofmann, U and Savic, D and Bhatia, S and Schwab, M and Ni, M and Yang, JJ},
title = {The NUDIX hydrolase NUDT5 regulates thiopurine metabolism and cytotoxicity.},
journal = {The Journal of clinical investigation},
volume = {135},
number = {14},
pages = {},
doi = {10.1172/JCI190443},
pmid = {40662362},
issn = {1558-8238},
mesh = {Humans ; *Pyrophosphatases/genetics/metabolism ; *Mercaptopurine/pharmacology ; Cell Line, Tumor ; CRISPR-Cas Systems ; *Neoplasm Proteins/genetics/metabolism ; Drug Resistance, Neoplasm ; Nudix Hydrolases ; },
abstract = {Thiopurines are anticancer agents used for the treatment of leukemia and autoimmune diseases. These purine analogs are characterized by a narrow therapeutic index because of the risk of myelosuppression. With the discovery of NUDIX hydrolase 15 (NUDT15) as a major modulator of thiopurine metabolism and toxicity, we sought to comprehensively examine all members of the NUDIX hydrolase family for their effect on the pharmacologic effects of thiopurine. By performing a NUDIX-targeted CRISPR/Cas9 screen in leukemia cells, we identified NUDT5, whose depletion led to drastic thiopurine resistance. NUDT5 deficiency resulted in a nearly complete depletion of active metabolites of thiopurine and the loss of thioguanine incorporation into DNA. Mechanistically, NUDT5 deletion resulted in substantial alteration in purine nucleotide biosynthesis, as determined by steady-state metabolomics profiling. Stable isotope tracing demonstrated that the loss of NUDT5 was linked to a marked suppression of the purine salvage pathway but with minimal effects on purine de novo synthesis. Finally, we comprehensively identified germline genetic variants in NUDT5 associated with thiopurine-induced myelosuppression in 582 children with acute lymphoblastic leukemia. Collectively, these results pointed to NUDT5 as a key regulator of the thiopurine response primarily through its effects on purine homeostasis, highlighting its potential to inform individualized thiopurine therapy.},
}

Humans
*Pyrophosphatases/genetics/metabolism
*Mercaptopurine/pharmacology
Cell Line, Tumor
CRISPR-Cas Systems
*Neoplasm Proteins/genetics/metabolism
Drug Resistance, Neoplasm
Nudix Hydrolases

@article {pmid40661867,
year = {2025},
author = {Mestanza, M and Hernández-Amasifuen, AD and Pineda-Lázaro, AJ and Eriksson, D and Guerrero-Abad, JC},
title = {Genome editing for sustainable agriculture in Peru: advances, potential applications and regulation.},
journal = {Frontiers in genome editing},
volume = {7},
number = {},
pages = {1611040},
pmid = {40661867},
issn = {2673-3439},
abstract = {Peruvian agriculture is characterize by crops such as potato, maize, rice, asparagus, mango, banana, avocado, cassava, onion, oil palm, chili, papikra, blueberry, coffee, cacao, grapes, quinoa, olive, citrus and others. All of them have challenges in production in their specific agroecosystems under stress due to pests, diseases, salinity, drought, cold among others. Gene editing through CRISPR/Cas is a key tool for addressing critical challenges in agriculture by improving resilience to biotic and abiotic stress, increasing yield and enhancing the nutritional value of the crops. This approach allows precise mutation on site-specific gene at the DNA level, obtaining desirable traits when its function is altered. The CRISPR/Cas system could be used as a transgene-free genome editing tool when the ribonucleoprotein (RNP) acts as a carrier to delivered the CRISPR/Cas components into the plant cell protoplasts, or when the tRNA-like sequence (TLS) motifs are fused to single-guide RNA (sgRNA) and Cas mRNA sequence and expressed in transgenic plants rootstock to produce "mobile" CRISPR/Cas components to upper tissue (scion). Those innovations could be a potential approach to strengthen the Peruvian agriculture, food security and gricultural economy, especially in the tropical, Andean and coastal regions. This review article examines the advances and strategies of gene editing, focusing on transgene-free methodologies that could be adopted for research, development and use, and also identifies potential applications in key crops for Peru and analyzes their impact in the productivity and reduction of agrochemicals dependence. Finally, this review highlights the need to establish regulatory policies that strengthen the use of biotechnological precise innovations, ensuring the conservation and valorization of agrobiodiversity for the benefit of Peruvian farmers.},
}

@article {pmid40661507,
year = {2025},
author = {Shentu, TP and Wu, T and Zhou, Z and Yeh, CF and Zhu, J and Li, J and Miao, BA and Lee, TH and Zhang, L and Huang, RT and Harrison, D and Hodonsky, CJ and Auguste, G and Husain, A and Tirrell, MV and Miller, CL and Dickinson, B and Yang, KC and Fang, Y},
title = {Mechanosensitive Endothelial METTL7A Regulates Internal m [7] G mRNA Methylation and Protects Against Atherosclerosis.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.05.22.655328},
pmid = {40661507},
issn = {2692-8205},
abstract = {BACKGROUND: Internal N [7] -methylguanosine (m [7] G) is a recently identified chemical modification of mammalian mRNA and a component of the epitranscriptome. While the epitranscriptome plays a key role in regulating RNA metabolism and cellular function, the specific contribution of internal m [7] G to cardiovascular health and disease remains unknown. Atherosclerosis preferentially develops at sites where disturbed blood flow activates endothelial cells, but whether internal m [7] G and its regulatory machinery influence endothelial mechanotransduction and atherogenesis is unclear.

METHODS: We integrated epitranscriptomic profiling, human tissue analysis, genetically modified mouse models, and targeted nanomedicine approaches to investigate the role of Methyltransferase-like protein 7A (METTL7A), a putative internal m [7] G methyltransferase, in regulating the flow-sensitive endothelial transcriptome and atherosclerosis. Vascular endothelial cells were subjected to well-defined athero-protective and athero-prone flow waveforms in vitro and in vivo . METTL7A function was assessed using RNA sequencing (RNA-seq), liquid chromatography-tandem mass spectrometry (LC-MS/MS), crosslinking immunoprecipitation sequencing (CLIP-seq), RNA stability assays, and a CRISPR-Cas-inspired RNA targeting system (CIRTS). METTL7A expression in human coronary arteries with and without atherosclerosis was evaluated by RNA-seq and immunostaining. In vivo atherosclerosis studies were conducted in both global and endothelial-specific Mettl7a1 knockout mice. Endothelial METTL7A expression was restored using cationic polymer-based nanoparticles delivering CDH5 promoter-driven METTL7A plasmids or VCAM1-targeted lipid nanoparticles delivering N1-methylpseudouridine (m¹Ψ)-modified METTL7A mRNA.

RESULTS: Athero-protective unidirectional flow significantly induced METTL7A expression, which promoted internal m [7] G methylation of endothelial transcripts, while other major epitranscriptomic marks and cap-associated m [7] G were not affected by METTL7A. METTL7A preferentially binds to AG-enriched motifs in protein-coding mRNAs and plays a key role in regulating KLF4 and NFKBIA transcripts, enhancing their internal m [7] G and stability and supporting vascular homeostasis. In contrast, endothelial METTL7A expression was significantly reduced by disturbed blood flow and in human atherosclerotic lesions. Global or endothelial-specific loss of METTL7A exacerbated disturbed flow-induced atherosclerosis in mice, independent of serum lipid levels. Restoration of endothelial METTL7A, via nanoparticle-mediated plasmid or m [1] Ψ mRNA delivery, markedly reduced lesion formation in Mettl7a1 [⁻/⁻] and ApoE [⁻/⁻] mice.

CONCLUSIONS: These findings establish METTL7A as a previously unrecognized mechanosensitive methyltransferase that maintains endothelial homeostasis by stabilizing key anti-inflammatory transcripts, KLF4 and NFKBIA, through internal m [7] G methylation. Loss of METTL7A disrupts endothelial function and accelerates atherogenesis in response to disturbed flow. Therapeutic restoration of endothelial METTL7A, via targeted nanoparticle-mediated gene or m [1] Ψ mRNA delivery, significantly lessens atherosclerosis. Collectively, these results uncover a novel epitranscriptomic mechanism governing vascular health and position METTL7A as a promising target for precision nanomedicine in atherosclerotic cardiovascular disease.},
}

@article {pmid40661421,
year = {2025},
author = {Xiao, R and Hoffmann, FT and Xie, D and Wiegand, T and Palmieri, AI and Sternberg, SH and Chang, L},
title = {Structural basis of RNA-guided transcription by a dCas12f-σ [E] -RNAP complex.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.06.10.658880},
pmid = {40661421},
issn = {2692-8205},
abstract = {RNA-guided proteins have emerged as critical transcriptional regulators in both natural and engineered biological systems by modulating RNA polymerase (RNAP) and its associated factors [1-5] . In bacteria, diverse clades of repurposed TnpB and CRISPR-associated proteins repress gene expression by blocking transcription initiation or elongation, enabling non-canonical modes of regulatory control and adaptive immunity [1,6,7] . Intriguingly, a distinct class of nuclease-dead Cas12f homologs (dCas12f) instead activates gene expression through its association with unique extracytoplasmic function sigma factors (σ [E]) [8] , though the molecular basis has remained elusive. Here we reveal a novel mode of RNA-guided transcription initiation by determining cryo-electron microscopy structures of the dCas12f-σ [E] system from Flagellimonas taeanensis . We captured multiple conformational and compositional states, including the DNA-bound dCas12f-σ [E] -RNAP holoenzyme complex, revealing how RNA-guided DNA binding leads to σ [E] -RNAP recruitment and nascent mRNA synthesis at a precisely defined distance downstream of the R-loop. Rather than following the classical paradigm of σ [E] -dependent promoter recognition, these studies show that recognition of the -35 element is largely supplanted by CRISPR-Cas targeting, while the melted -10 element is stabilized through unusual stacking interactions rather than insertion into the typical recognition pocket. Collectively, this work provides high-resolution insights into an unexpected mechanism of RNA-guided transcription, expanding our understanding of bacterial gene regulation and opening new avenues for programmable transcriptional control.},
}

@article {pmid40660068,
year = {2025},
author = {Wang, D and Guo, Y and Liu, M and Liu, H},
title = {The function of TaWOX14 in wheat genetic transformation.},
journal = {Plant cell reports},
volume = {44},
number = {8},
pages = {176},
pmid = {40660068},
issn = {1432-203X},
support = {2021YFF1000203//Ministry of Science and Technology of the People's Republic of China/ ; 232300420189//Natural Science Foundation of Henan Province/ ; 242102111117//Henan Provincial Science and Technology Research Project/ ; },
mesh = {*Triticum/genetics ; *Transformation, Genetic ; *Plant Proteins/genetics/metabolism ; Gene Editing ; Plants, Genetically Modified ; CRISPR-Cas Systems/genetics ; Haploidy ; Gene Expression Regulation, Plant ; *Homeodomain Proteins/genetics/metabolism ; },
abstract = {Overexpression of TaWOX14 can significantly improve the genetic transformation and genome editing efficiencies of some wheat immature embryos, and TaPLD serves as a promising candidate gene for haploid induction in wheat. Genetic transformation and genome editing systems have the potential for accelerating the breeding process in crops. However, their effectiveness is often limited by the regeneration efficiency of explants. The WUSCHEL-related homeobox (WOX) family, a group of plant-specific transcription factors, plays a crucial role in somatic embryogenesis. In wheat, WOX genes are categorized into three clades: ancient, intermediate, and WUS. In this study, we systematically analyzed the function of the WUS clade genes in wheat transformation. Our results demonstrated that overexpression of TaWOX14 significantly improved genetic transformation efficiencies in several wheat genotypes, including Fielder, Kenong199, Zhengmai7698, and Yangmai13. Furthermore, by combining the CRISPR-Cas9 system with TaWOX14, we observed enhanced genome editing efficiency in the wheat variety Fielder. Additionally, we explored the potential of TaPLD as a candidate gene for haploid induction in wheat. Based on the amino acid sequence of Zea mays PHOSPHOLIPASE D3 (ZmPLD3), we edited its homologs in wheat and identified a TaPLD-edited plant with a ploidy level intermediate between haploid and hexaploid. Overall, these findings are expected to accelerate the wheat breeding process by improving genetic transformation efficiency and identifying a potential haploid induction gene. Future research will focus on further characterizing the mechanisms of TaWOX14 and TaPLD, and exploring their applications in breeding programs.},
}

*Triticum/genetics
*Transformation, Genetic
*Plant Proteins/genetics/metabolism
Gene Editing
Plants, Genetically Modified
CRISPR-Cas Systems/genetics
Haploidy
Gene Expression Regulation, Plant
*Homeodomain Proteins/genetics/metabolism

@article {pmid40619644,
year = {2025},
author = {Zhong, J and Chen, Y and Dong, Y and Guo, C and Liu, Y},
title = {SPARC: An Orthogonal Cas12a/Cas13a Dual-Channel CRISPR Platform for Reliable SNV Identification and Mutation Confirmation.},
journal = {Analytical chemistry},
volume = {97},
number = {27},
pages = {14629-14636},
doi = {10.1021/acs.analchem.5c02141},
pmid = {40619644},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; Mutation ; *Polymorphism, Single Nucleotide ; *Endodeoxyribonucleases/genetics/metabolism ; Humans ; *CRISPR-Associated Proteins/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {Rapid and reliable detection of single nucleotide variants (SNVs) is essential for accurate pathogen diagnostics, genetic mutation screening, and personalized medicine. However, existing CRISPR-based nucleic acid detection platforms frequently suffer from ambiguous signal interpretation, specificity limitations, and complex assay workflows. Herein, we introduce SPARC (specific and precise mutation recognition with Cas12a/Cas13a), a novel orthogonal dual-channel CRISPR assay that significantly enhances the SNV detection reliability. SPARC integrates Acidaminococcus sp. Cas12a (AsCas12a), which specifically detects a conserved region as an internal reference, with our recently identified DNA-activated Leptotrichia buccalis Cas13a (LbuCas13a), which exhibits exceptionally high intrinsic SNV specificity without requiring engineered crRNA mismatches. The orthogonal design uniquely resolves the common diagnostic ambiguity between genuine SNVs and target absence. Combined with recombinase polymerase amplification (RPA) and T7 exonuclease digestion, the SPARC platform achieved a sensitivity as low as 1 aM. We demonstrated the platform's robust clinical applicability through successful detection and accurate differentiation of hepatitis B virus (HBV) and clinically significant YMDD resistance mutations. This work presents an innovative and versatile CRISPR-based solution, highlighting substantial potential for advancing clinical diagnostics and precision medicine.},
}

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

@article {pmid40590162,
year = {2025},
author = {Xia, L and Yin, J and Wang, Y and Gui, Y and Tong, J and Yin, W and Xiao, D and Mu, Y},
title = {Wide Dynamic Range Multiplex Digital Clustered Regularly Interspaced Short Palindromic Repeat Chip for Rapid Detection and Absolute Quantification of Nucleic Acids.},
journal = {ACS nano},
volume = {19},
number = {27},
pages = {24874-24883},
doi = {10.1021/acsnano.5c03382},
pmid = {40590162},
issn = {1936-086X},
mesh = {CRISPR-Cas Systems ; Limit of Detection ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Nucleic Acids/analysis/genetics ; *Lab-On-A-Chip Devices ; Animals ; Nucleic Acid Amplification Techniques ; },
abstract = {Rapid detection and accurate quantification of multiplex nucleic acids are crucial for infectious diagnosis and pathogen identification, yet they still represent a significant challenge that deserves further attention and investigation. Herein, this research developed a wide dynamic range multiplex digital clustered regularly interspaced short palindromic repeat (CRISPR) (WDRM-dCRISPR) chip and established a rapid, sensitive, multiplexed nucleic acid detection (RSMND) platform that can achieve absolute quantification of multiplex nucleic acids with a limit of detection of 90 copies/mL within 30 min. The chip integrates the rapid amplification capability of RPA, the specific cleavage activity of CRISPR/Cas12a, and the portability of microfluidics, enabling precise and reliable digital absolute quantification of multiplex nucleic acids. As a proof of concept, this chip allows for multiplex absolute quantification of foodborne pathogens with a linear dynamic range of 9 × 10[1] to 1.8 × 10[6] copies/mL (R[2] > 0.9999), high sensitivity, high specificity, and strong tolerance to background interference. The platform was successfully used for the analysis of foodborne pathogens in milk. Compared to qPCR, the RSMND platform exhibited superior accuracy and sensitivity in nucleic acid quantification. Overall, this study demonstrates that the platform provides a powerful tool for multiplex nucleic acid detection and holds significant potential for applications in point-of-care testing.},
}

CRISPR-Cas Systems
Limit of Detection
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Nucleic Acids/analysis/genetics
*Lab-On-A-Chip Devices
Animals
Nucleic Acid Amplification Techniques

@article {pmid40586733,
year = {2025},
author = {Liu, F and Chen, L and Zhu, L and Liu, J and Li, M and Chen, Y and Yang, G and Qu, L},
title = {Aptamer-Amplified and CRISPR-Cas12a-Assisted Dual-Mode PEC-SERS Biosensor for Ultrasensitive Detection of Inflammatory Bowel Disease Biomarkers.},
journal = {Analytical chemistry},
volume = {97},
number = {27},
pages = {14377-14387},
doi = {10.1021/acs.analchem.5c01481},
pmid = {40586733},
issn = {1520-6882},
mesh = {*Aptamers, Nucleotide/chemistry ; *Biosensing Techniques/methods ; Animals ; *Inflammatory Bowel Diseases/diagnosis/metabolism ; Biomarkers/analysis ; *Electrochemical Techniques/methods ; *Matrix Metalloproteinase 9/analysis ; Humans ; *CRISPR-Cas Systems ; Mice ; *Alkaline Phosphatase/analysis ; Spectrum Analysis, Raman/methods ; Limit of Detection ; *CRISPR-Associated Proteins/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Inflammatory bowel disease (IBD) has proven to be a critical global health problem characterized by severe life-threatening complications; thus, the development of noninvasive, reliable, and cost-effective diagnostic methods remains an urgent clinical need. Herein, a novel photoelectrochemical (PEC) and surface-enhanced Raman scattering (SERS) dual-mode platform was successfully developed for ultrasensitive detection of IBD-associated biomarkers, matrix metalloproteinases-9 (MMP-9), and intestinal alkaline phosphatase (IAP). A bifunctional covalent organic frameworks/MXene-Au substrate was synthesized with excellent PEC and SERS properties. An aptamer-based amplification strategy was first employed for MMP-9 detection, which was also the basis for the detection of IAP. The magnetic bead-conjugated double-stranded DNA was then designed to generate activator DNA in the presence of IAP, which activated the trans-cleavage activity of the CRISPR-Cas12a system. The resultant Cas12a specifically cleaved the electrode-immobilized single-stranded DNA (ssDNA), triggering the release of methylene blue as a dual-signal reporter, thereby enabling synchronized PEC-SERS detection for MMP-9 and IAP. The biosensor exhibited a wide linear range with detection limits of 0.074 pg/mL (PEC) and 0.016 pg/mL (SERS) for MMP-9, and 0.38 pg/mL (PEC) and 0.16 pg/mL (SERS) for IAP, respectively. Significantly, clinical validation was performed using a murine IBD model and human intestinal inflammation specimens, confirming the practical utility of the PEC-SERS platform. This study establishes a robust dual-mode biosensing strategy with multicomponent detection, enabling advanced biological analysis and precision health monitoring.},
}

*Aptamers, Nucleotide/chemistry
*Biosensing Techniques/methods
Animals
*Inflammatory Bowel Diseases/diagnosis/metabolism
Biomarkers/analysis
*Electrochemical Techniques/methods
*Matrix Metalloproteinase 9/analysis
Humans
*CRISPR-Cas Systems
Mice
*Alkaline Phosphatase/analysis
Spectrum Analysis, Raman/methods
Limit of Detection
*CRISPR-Associated Proteins/metabolism
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid39375448,
year = {2025},
author = {Gu, J and Iyer, A and Wesley, B and Taglialatela, A and Leuzzi, G and Hangai, S and Decker, A and Gu, R and Klickstein, N and Shuai, Y and Jankovic, K and Parker-Burns, L and Jin, Y and Zhang, JY and Hong, J and Niu, X and Costa, JA and Pezet, MG and Chou, J and Chen, C' and Paiva, M and Snoeck, HW and Landau, DA and Azizi, E and Chan, EM and Ciccia, A and Gaublomme, JT},
title = {Mapping multimodal phenotypes to perturbations in cells and tissue with CRISPRmap.},
journal = {Nature biotechnology},
volume = {43},
number = {7},
pages = {1101-1115},
pmid = {39375448},
issn = {1546-1696},
support = {1DP2CA281605//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; 5R01CA197774-04//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; 5R01CA227450-04//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; 1R21HG012639-01A1//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; 1R01HG012875-01//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; Lloyd J. Old STAR award//Cancer Research Institute (CRI)/ ; },
mesh = {Humans ; Phenotype ; *CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Breast Neoplasms/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Induced Pluripotent Stem Cells ; Mice ; },
abstract = {Unlike sequencing-based methods, which require cell lysis, optical pooled genetic screens enable investigation of spatial phenotypes, including cell morphology, protein subcellular localization, cell-cell interactions and tissue organization, in response to targeted CRISPR perturbations. Here we report a multimodal optical pooled CRISPR screening method, which we call CRISPRmap. CRISPRmap combines in situ CRISPR guide-identifying barcode readout with multiplexed immunofluorescence and RNA detection. Barcodes are detected and read out through combinatorial hybridization of DNA oligos, enhancing barcode detection efficiency. CRISPRmap enables in situ barcode readout in cell types and contexts that were elusive to conventional optical pooled screening, including cultured primary cells, embryonic stem cells, induced pluripotent stem cells, derived neurons and in vivo cells in a tissue context. We conducted a screen in a breast cancer cell line of the effects of DNA damage repair gene variants on cellular responses to commonly used cancer therapies, and we show that optical phenotyping pinpoints likely pathogenic patient-derived mutations that were previously classified as variants of unknown clinical significance.},
}

Humans
Phenotype
*CRISPR-Cas Systems/genetics
Cell Line, Tumor
Breast Neoplasms/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Induced Pluripotent Stem Cells
Mice

@article {pmid39349835,
year = {2025},
author = {Lei, X and Huang, A and Chen, D and Wang, X and Ji, R and Wang, J and Zhang, Y and Zhang, Y and Lu, S and Zhang, K and Chen, Q and Zhang, Y and Yin, H},
title = {Rapid generation of long, chemically modified pegRNAs for prime editing.},
journal = {Nature biotechnology},
volume = {43},
number = {7},
pages = {1156-1167},
pmid = {39349835},
issn = {1546-1696},
mesh = {Humans ; *Gene Editing/methods ; Ribonucleoproteins/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {The editing efficiencies of prime editing (PE) using ribonucleoprotein (RNP) and RNA delivery are not optimal due to the challenges in solid-phase synthesis of long PE guide RNA (pegRNA) (>125 nt). Here, we develop an efficient, rapid and cost-effective method for generating chemically modified pegRNA (125-145 nt) and engineered pegRNA (epegRNA) (170-190 nt). We use an optimized splint ligation approach and achieve approximately 90% production efficiency for these RNAs, referred to as L-pegRNA and L-epegRNA. L-epegRNA demonstrates enhanced editing efficiencies across various cell lines and human primary cells with improvements of up to more than tenfold when using RNP delivery and several hundredfold with RNA delivery of PE, compared to epegRNA produced by in vitro transcription. L-epegRNA-mediated RNP delivery also outperforms plasmid-encoded PE in most comparisons. Our study provides a solution to obtaining high-quality pegRNA and epegRNA with desired chemical modifications, paving the way for the use of PE in therapeutics and various other fields.},
}

Humans
*Gene Editing/methods
Ribonucleoproteins/genetics/metabolism
*RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems/genetics
HEK293 Cells

@article {pmid39134754,
year = {2025},
author = {Karasu, ME and Toufektchan, E and Chen, Y and Albertelli, A and Cullot, G and Maciejowski, J and Corn, JE},
title = {Removal of TREX1 activity enhances CRISPR-Cas9-mediated homologous recombination.},
journal = {Nature biotechnology},
volume = {43},
number = {7},
pages = {1168-1176},
pmid = {39134754},
issn = {1546-1696},
mesh = {Humans ; *Exodeoxyribonucleases/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Phosphoproteins/genetics/metabolism ; *Homologous Recombination/genetics ; Gene Editing/methods ; },
abstract = {CRISPR-Cas9-mediated homology-directed repair (HDR) can introduce desired mutations at targeted genomic sites, but achieving high efficiencies is a major hurdle in many cell types, including cells deficient in DNA repair activity. In this study, we used genome-wide screening in Fanconi anemia patient lymphoblastic cell lines to uncover suppressors of CRISPR-Cas9-mediated HDR. We found that a single exonuclease, TREX1, reduces HDR efficiency when the repair template is a single-stranded or linearized double-stranded DNA. TREX1 expression serves as a biomarker for CRISPR-Cas9-mediated HDR in that the high TREX1 expression present in many different cell types (such as U2OS, Jurkat, MDA-MB-231 and primary T cells as well as hematopoietic stem and progenitor cells) predicts poor HDR. Here we demonstrate rescue of HDR efficiency (ranging from two-fold to eight-fold improvement) either by TREX1 knockout or by the use of single-stranded DNA templates chemically protected from TREX1 activity. Our data explain why some cell types are easier to edit than others and indicate routes for increasing CRISPR-Cas9-mediated HDR in TREX1-expressing contexts.},
}

Humans
*Exodeoxyribonucleases/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Phosphoproteins/genetics/metabolism
*Homologous Recombination/genetics
Gene Editing/methods

@article {pmid40659848,
year = {2025},
author = {Karimi, M and Ghorbani, A and Niazi, A and Rostami, M and Tahmasebi, A},
title = {Integrating AI and CRISPR Cas13a for rapid detection of tomato brown rugose fruit virus.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {25422},
pmid = {40659848},
issn = {2045-2322},
mesh = {*Solanum lycopersicum/virology ; *Plant Diseases/virology ; *CRISPR-Cas Systems ; *Plant Viruses/genetics/isolation & purification ; Computational Biology/methods ; RNA, Viral/genetics ; },
abstract = {The Tomato Brown Rugose Fruit Virus (ToBRFV) has recently emerged as a serious threat to global tomato production, underscoring the need for rapid and sensitive diagnostic tools. Here, we present an AI-driven CRISPR-Cas13a pipeline for designing crRNAs with high specificity to enable the detection of ToBRFV. A computational pipeline that retrieves viral sequences, aligns them in multiple sequence alignments, analyzes their conservation, and screens for off-targets-all coupled with machine learning to optimize crRNA sequences. Experimentally validated crRNAs were evaluated with a fluorescence-based Cas13a assay and showed better sensitivity than RT-PCR, RT-qPCR, and RT-LAMP. By the CRISPR-Cas13a system, ToBRFV was detected at 1:200 (1 ng/µL) dilutions, which performed superior to conventional methods. Integrating bioinformatics with experimental workflows, this pipeline provides a powerful framework for rapid diagnostics that can be deployed in the field, addressing significant challenges in plant virus surveillance and management.},
}

*Solanum lycopersicum/virology
*Plant Diseases/virology
*CRISPR-Cas Systems
*Plant Viruses/genetics/isolation & purification
Computational Biology/methods
RNA, Viral/genetics

@article {pmid40659683,
year = {2025},
author = {Li, P and Wu, Z and Liu, T and Deng, C and Liu, Q and Ni, J},
title = {The defensome of prokaryotes in aquifers.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6482},
pmid = {40659683},
issn = {2041-1723},
support = {U2240205//National Natural Science Foundation of China (National Science Foundation of China)/ ; 51721006//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Groundwater/microbiology ; *Bacteria/genetics/immunology/virology ; China ; *Prokaryotic Cells ; Metagenomics ; Bacteriophages ; CRISPR-Cas Systems/genetics ; Ecosystem ; Metagenome ; Phylogeny ; },
abstract = {Groundwater harbors a pristine biosphere where microbes co-evolve with less human interference, yet the ancient and ongoing arms race between prokaryotes and viruses remains largely unknown in such ecosystems. Based on our recent nationwide groundwater monitoring campaign across China, we construct a metagenomic groundwater prokaryotic defensome catalogue (GPDC), encompassing 190,810 defense genes, 90,824 defense systems, 139 defense families, and 669 defense islands from 141 prokaryotic phyla. Over 94% of the defense genes in GPDC are novel and contribute vast microbial immune resources in groundwater. We find that candidate phyla radiation (CPR) bacteria possess higher defense system density and diversity against intense phage infection, while microbes as a whole exhibit an inverse relationship between defense systems and adaptive traits like resistance genes in groundwater. We further identify five first-line defense families covering 69.2% of the total defense systems, and high-turnover accessory immune genes are mostly conveyed to defense islands by mobile genetic elements. Our study also reveals viral resistance to microbial defense through co-localized anti-defense genes and interactions between CRISPR-Cas9 and anti-CRISPR protein. These findings expand our understanding of microbial immunity in pristine ecosystems and offer valuable immune resources for potential biotechnological applications.},
}

*Groundwater/microbiology
*Bacteria/genetics/immunology/virology
China
*Prokaryotic Cells
Metagenomics
Bacteriophages
CRISPR-Cas Systems/genetics
Ecosystem
Metagenome
Phylogeny

@article {pmid40659672,
year = {2025},
author = {Viswanatha, R and Entwisle, S and Hu, Y and Kim, AR and Reap, K and Butnaru, M and Qadiri, M and Mohr, SE and Perrimon, N},
title = {Higher resolution pooled genome-wide CRISPR knockout screening in Drosophila cells using integration and anti-CRISPR (IntAC).},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6498},
pmid = {40659672},
issn = {2041-1723},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Drosophila melanogaster/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; Genome, Insect ; Humans ; Drosophila Proteins/genetics ; Plasmids/genetics ; },
abstract = {CRISPR screens enable systematic, scalable genotype-to-phenotype mapping. We previously developed a CRISPR screening method for Drosophila melanogaster and mosquito cell lines using plasmid transfection and site-specific integration to introduce single guide (sgRNA) libraries. The method relies on weak sgRNA promoters to avoid early CRISPR-Cas9 activity causing discrepancies between genome edits and integrated sgRNAs. To address this issue and utilize higher strength sgRNA expression, we introduce a method to co-transfect a plasmid expressing anti-CRISPR protein to suppress early CRISPR-Cas9 activity which we term "IntAC" (integrase with anti-CRISPR). IntAC dramatically improves precision-recall of fitness genes across the genome, allowing us to generate the most comprehensive list of cell fitness genes yet assembled for Drosophila. Drosophila fitness genes show strong correlation with human fitness genes and underscore the effects of paralogs on gene essentiality. We also perform a resistance screen to proaerolysin, a glycosylphosphatidylinositol-(GPI)-binding pore-forming toxin, retrieving 18/23 expected and one previously uncharacterized GPI synthesis gene. We also demonstrate that an IntAC sublibrary enables precise positive selection of a transporter under solute overload. IntAC represents a straightforward enhancement to existing Drosophila CRISPR screening methods, dramatically increasing accuracy, and might also be broadly applicable to virus-free CRISPR screens in other cell and species types.},
}

Animals
*CRISPR-Cas Systems/genetics
*Drosophila melanogaster/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Knockout Techniques/methods
Cell Line
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Gene Editing/methods
Genome, Insect
Humans
Drosophila Proteins/genetics
Plasmids/genetics

@article {pmid40659638,
year = {2025},
author = {Sewgoolam, B and Jim, KK and de Bakker, V and Bock, FP and Gibson, PS and Veening, JW},
title = {Genome-wide antibiotic-CRISPRi profiling identifies LiaR activation as a strategy to resensitize fluoroquinolone-resistant Streptococcus pneumoniae.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6491},
pmid = {40659638},
issn = {2041-1723},
support = {310030_192517//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 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)/ ; },
mesh = {*Streptococcus pneumoniae/genetics/drug effects/metabolism ; *Fluoroquinolones/pharmacology ; *Anti-Bacterial Agents/pharmacology ; Animals ; *Bacterial Proteins/genetics/metabolism ; *Drug Resistance, Bacterial/genetics/drug effects ; Zebrafish ; Regulon/genetics ; Gene Expression Regulation, Bacterial/drug effects ; Humans ; Bacitracin/pharmacology ; Ciprofloxacin/pharmacology ; Microbial Sensitivity Tests ; Pneumococcal Infections/drug therapy/microbiology ; Genome, Bacterial ; CRISPR-Cas Systems ; },
abstract = {Streptococcus pneumoniae is a human pathogen that has become increasingly resistant to synthetic fluoroquinolone antibiotics that target bacterial topoisomerases. To identify pathways essential under fluoroquinolone stress and potential novel targets to revitalize use of this antibiotic class, we perform genome-wide CRISPRi-seq screens and generate antibiotic-gene essentiality signatures. Expectedly, genes involved in DNA recombination and repair become more important under fluoroquinolone-induced DNA damage, including recA, recJ, recF, recO, rexAB, and ruvAB. Surprisingly, specific downregulation of the gene encoding the histidine kinase LiaS caused fluoroquinolone hypersensitivity. LiaS is part of the LiaFSR (VraTSR) three-component regulatory system involved in cell envelope homeostasis. We show that LiaS keeps the response regulator LiaR inactive, and that liaS deletion causes LiaR hyperphosphorylation and upregulation of the LiaR regulon. We use RNA-seq to refine the LiaR regulon, highlighting the role of heat-shock response and pleiotropic regulator SpxA2 in fluoroquinolone sensitivity. Activating the LiaR-regulon by the cell envelope-targeting antibiotic bacitracin synergized with ciprofloxacin and levofloxacin, restoring sensitivity in fluoroquinolone-resistant strains in vitro. Furthermore, bacitracin/levofloxacin combination therapy is effective in vivo and improved treatment of fluoroquinolone-resistant S. pneumoniae infection in a zebrafish meningitis model. These findings offer a starting point for identification and validation of potent combination therapies to treat antibiotic-resistant pneumococcal infections.},
}

*Streptococcus pneumoniae/genetics/drug effects/metabolism
*Fluoroquinolones/pharmacology
*Anti-Bacterial Agents/pharmacology
Animals
*Bacterial Proteins/genetics/metabolism
*Drug Resistance, Bacterial/genetics/drug effects
Zebrafish
Regulon/genetics
Gene Expression Regulation, Bacterial/drug effects
Humans
Bacitracin/pharmacology
Ciprofloxacin/pharmacology
Microbial Sensitivity Tests
Pneumococcal Infections/drug therapy/microbiology
Genome, Bacterial
CRISPR-Cas Systems

@article {pmid40659249,
year = {2025},
author = {Storz, U},
title = {The CRISPR Cas patent files, part 3: Prime Editing and integrase-based variants.},
journal = {Journal of biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jbiotec.2025.07.006},
pmid = {40659249},
issn = {1873-4863},
abstract = {The epic patent disputes regarding CRISPR Cas9 and, to a lesser extent, CRISPR Cas12a, have somehow overcast a new patent debate that is materializing at the horizon, namely the one that relates to Prime Editing and its integrase-based siblings. While these techniques offer great promise for the precise integration of large DNA stretches into a host genome, it appears that a new chain of dependencies is about to unfold that makes the establishment of Freedom to Operate complicated for interested parties.[[1]].},
}

@article {pmid40658808,
year = {2025},
author = {Sekiba, K and Miyake, N and Miyakawa, Y and Shibata, C and Seimiya, T and Kishikawa, T and Fujishiro, M and Otsuka, M},
title = {CRISPR-mediated proximity labeling unveils ABHD14B as a host factor to regulate HBV cccDNA transcriptional activity.},
journal = {Hepatology communications},
volume = {9},
number = {8},
pages = {},
doi = {10.1097/HC9.0000000000000757},
pmid = {40658808},
issn = {2471-254X},
mesh = {Humans ; *Hepatitis B virus/genetics ; *DNA, Circular/genetics/metabolism ; *DNA, Viral/genetics/metabolism ; Virus Replication ; CRISPR-Cas Systems ; RNA, Viral/genetics ; Hep G2 Cells ; Transcription, Genetic ; *Viral Transcription ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {BACKGROUND: The long-term goal of chronic hepatitis B research is a functional cure (HBsAg seroclearance). Although currently used nucleos(t)ide analogs can efficiently inhibit viral replication, they do not reduce viral RNAs or proteins produced from covalently closed circular DNA (cccDNA), and rarely achieve a functional cure. To overcome this situation, revealing the mode of the existence of cccDNA is required, including identifying the interreacting proteins with cccDNA. Here, we aimed to identify novel proteins that interact with cccDNA.

METHODS: Using an in vitro HBV infection model and a sequence-specific proximity labelling method consisting of dead Cas9 and biotin identification (BioID2), we comprehensively determined proteins that possibly interact with cccDNA. After identifying the candidate proteins, the HBV RNA transcription levels were examined by knocking out the associated genes.

RESULTS: We identified ABHD14B as a protein that interacts with cccDNA and inhibits HBV RNA transcription from cccDNA. ABHD14B decreases the acetylation levels of histone proteins that control the transcription levels of HBV RNA in cccDNA. Moreover, ABHD14B interacts with TFII-I, which binds directly to cccDNA in a sequence-dependent manner. These results suggest that the host protein, ABHD14B, is recruited to cccDNA via the TFII-I protein, inhibiting HBV RNA transcription from cccDNA by deacetylating cccDNA histones.

CONCLUSIONS: ABHD14B was newly identified as a suppressor of HBV RNA transcription from cccDNA, which may improve our understanding of the mode of existence of cccDNA, providing a basis for development of a functional cure.},
}

Humans
*Hepatitis B virus/genetics
*DNA, Circular/genetics/metabolism
*DNA, Viral/genetics/metabolism
Virus Replication
CRISPR-Cas Systems
RNA, Viral/genetics
Hep G2 Cells
Transcription, Genetic
*Viral Transcription
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40657403,
year = {2025},
author = {Akram, F and Safdar, M and Shabbir, I and Fatima, T and Ikram-Ul-Haq, },
title = {Insight into the eminent biotechnological applications of xylanolytic enzymes for sustainable bioprocessing.},
journal = {3 Biotech},
volume = {15},
number = {8},
pages = {249},
pmid = {40657403},
issn = {2190-572X},
abstract = {Xylan is one of the most abundant polysaccharides in nature and presents a structural complexity characterized by a heterogeneous polymer composition. Comprising various sugar subunits and associated acids linked through a diverse array of bonds, xylan poses challenges for complete degradation. This review article provides a comprehensive overview of xylan's structure, the role of xylanolytic enzymes in its degradation, and the industrial applications of xylanases in sectors, such as paper and pulp, food, textiles, and pharmaceuticals. Furthermore, it also discusses the use of advanced biotechnology tools, such as nano-biotechnology and genetic engineering, particularly through CRISPR/CAS technology, for enhancing the thermostability of xylanases. This article also provides insights into emerging trends in xylanase research, including bioprospecting novel thermostable xylanases from metagenomes, protein engineering, synthetic biology, and the integration of biorefinery. Finally, it highlights the importance of regulatory frameworks and standardization initiatives for ensuring the quality and the sustainability of xylanase-based technologies. Overall, this review offers valuable insights into the multifaceted role of xylanases in biotechnology and industrial bioprocessing while outlining future directions for research and innovation in this field.},
}

@article {pmid40655875,
year = {2025},
author = {Okesanya, OJ and Ayeni, RA and Amadin, P and Ngwoke, I and Amisu, BO and Ukoaka, BM and Ahmed, MM and Oso, TA and Musa, SS and Lucero-Prisno, DE},
title = {Advances in HIV Treatment and Vaccine Development: Emerging Therapies and Breakthrough Strategies for Long-Term Control.},
journal = {AIDS research and treatment},
volume = {2025},
number = {},
pages = {6829446},
pmid = {40655875},
issn = {2090-1240},
abstract = {Since its identification in 1981, HIV has posed a global public health challenge, witnessing transformative advancements in treatment and prevention. This review summarizes recent novel therapeutic and preventive approaches for long-term HIV control, management, and elimination, and how global collaboration and technological innovations may advance HIV control efforts. This study highlights the progress and challenges in HIV treatment, emphasizing the effectiveness of current antiretroviral therapy (ART) in suppressing viral replication, reducing transmission, and preventing end-organ damage. However, adherence remains a significant barrier due to pill burden, side effects, and psychosocial factors affecting patients. ART-related toxicities include neuropathy, hepatotoxicity, metabolic disorders, and neuropsychiatric effects. Long-acting ART (LA-ART) offers a promising alternative to daily dosing; however, challenges such as injection site reactions persist. Broadly neutralizing antibodies (bNAbs) have shown enhanced efficacy in viral suppression and immune response activation, offering potential for treatment and vaccine design. Innovative gene-editing tools, such as CRISPR-Cas systems, are being explored for their ability to excise or silence proviral DNA; however, their clinical application is limited by off-target effects and delivery challenges. Latency-targeting strategies like "shock and kill" and "block and lock" remain experimental with limited clinical success, and nanotechnology-based drug delivery systems offer targeted, sustained, and less toxic treatment options. Despite the challenges posed by the virus's rapid mutation rate and immune evasion mechanisms, novel vaccine approaches, such as mRNA technology, vector-based platforms, and epitope-targeting strategies, are being explored. In addition, artificial intelligence and machine learning are enhancing the design of vaccines, predictive modeling, and fast-tracking progress in this area. Socio-economic bottlenecks in HIV control, such as stigma, gender disparities, and inequitable healthcare access, exacerbate the epidemic, particularly in sub-Saharan Africa. Enhancing global collaboration, providing sustainable funding, and integrating emerging and innovative technologies are critical for advancing HIV prevention and management. Achieving an AIDS-free generation and ultimately eliminating the epidemic will depend on effectively addressing the social, structural, and scientific barriers that hinder progress in this regard. Trial Registration: ClinicalTrials.gov identifier: NCT02120352, NCT02938520, NCT03639311, NCT03497676, NCT03635788.},
}

@article {pmid40655388,
year = {2025},
author = {Jhalora, V and Bist, R},
title = {A Comprehensive Review of Molecular Mechanisms Leading to the Emergence of Multidrug Resistance in Bacteria.},
journal = {Indian journal of microbiology},
volume = {65},
number = {2},
pages = {844-865},
pmid = {40655388},
issn = {0046-8991},
abstract = {UNLABELLED: Multidrug resistance (MDR) in bacteria poses a serious global health threat, compromising the effectiveness of antibiotics. MDR causes approximately 700,000 deaths annually, with MDR tuberculosis alone claiming 230,000 lives. While bacteria inherently possess intrinsic resistance, acquired resistance stands out as the primary culprit in MDR development. Acquired resistance mechanisms mediated by the bacterial cell wall, nucleic acids, and proteins play a pivotal role in the genesis of MDR. Bacteria can modify their cell wall structure, produce resistant enzymes, exhibit mutations in antibiotic-targeted genes, and acquire resistant genes through horizontal gene transfer. Bacteria can produce proteins that act as enzymes, chemically modifying or directly degrading the antibiotic molecules, leading to the loss of their functionality. Apart from these mechanisms, biofilms also play a pivotal role in MDR expansion. Despite the development of several antibiotics since the discovery of penicillin, continuous structural and molecular modifications in bacteria render these antibiotics ineffective against MDR. The most recent approaches such as clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR-Cas), nanotechnology, a combination of CRISPR-Cas, and nanoparticles, show promise in treating MDR. Thus, this review delves deep into the molecular mechanisms of MDR, emphasizing the limitations of current antibiotics due to bacterial evolution and highlighting current strategies in the fight against MDR bacteria. This will drive comprehensive research to uncover additional resistance mechanisms and develop innovative strategies to combat resistant bacteria effectively.

SUPPLEMENTARY INFORMATION: The online version supplementary material available at 10.1007/s12088-024-01384-6.},
}

@article {pmid40654865,
year = {2025},
author = {Taranenko, D and Kotovskaya, O and Kuznedelov, K and Yanovskaya, D and Demkina, A and Fardeeva, S and Mamontov, V and Vierra, K and Burman, N and Li, D and Wang, M and Wiedenheft, B and Severinov, K and Semenova, E and Isaev, A},
title = {A census of anti-CRISPR proteins reveals AcrIE9 as an inhibitor of Escherichia coli K12 Type IE CRISPR-Cas system.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.05.07.652737},
pmid = {40654865},
issn = {2692-8205},
abstract = {CRISPR-Cas adaptive immunity systems provide defense against mobile genetic elements and are often countered by diverse anti-CRISPR (Acr) proteins. The Type IE CRISPR-Cas of Escherichia coli K12 has been a model for structural and functional studies and is a part of the species' core genome. However, this system is transcriptionally silent, which has fueled questions about its true biological function. To clarify the role of this system in defense, we carried out a census of Acr proteins found in Enterobacterales and identified AcrIE9 as a potent inhibitor of the E. coli K12 Type IE CRISPR-Cas system. While sharing little sequence identity, AcrIE9 proteins from Pseudomonas and Escherichia both interact with the Cas7 subunit of the Cascade complex, thus preventing its binding to DNA. We further show that AcrIE9 is genetically linked to AcrIE10, forming the most widespread anti-CRISPR cluster in Enterobacterales , and this module often co-occurs with a novel HTH-like protein with unusual architecture.},
}

@article {pmid40654372,
year = {2025},
author = {Xu, Y and Le, H and Wu, Q and Wang, N and Gong, C},
title = {Advancements in CRISPR/Cas systems for disease treatment.},
journal = {Acta pharmaceutica Sinica. B},
volume = {15},
number = {6},
pages = {2818-2844},
pmid = {40654372},
issn = {2211-3835},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) is an adaptive immune system present in most bacteria and archaea, protecting them from infection by exogenous genetic elements. Due to its simplicity, cost-effectiveness, and precise gene editing capabilities, CRISPR/Cas technology has emerged as a promising tool for treating diseases. The continuous refinement of derivative systems has further broadened its scope in disease treatment. Nevertheless, the heterogeneous physiopathological nature of diseases and variations in disease onset sites pose significant challenges for in vivo applications of CRISPR systems. The efficiency of CRISPR systems in disease treatment is directly influenced by the performance of the delivery system. Additionally, concerns such as off-target effects present crucial hurdles in the clinical implementation of CRISPR systems. This review provides a comprehensive overview of the development of CRISPR systems, vector technologies, and their applications in disease treatment, while also addressing the challenges encountered in clinical settings. Furthermore, future research directions are outlined to pave the way for advancements in CRISPR-based therapies.},
}

@article {pmid40204636,
year = {2025},
author = {Steiner, KK and Young, AC and Patterson, AR and Sugiura, A and Watson, MJ and Preston, SEJ and Zhelonkin, A and Jennings, EQ and Chi, C and Heintzman, DR and Pahnke, AP and Toudji, YT and Hatem, Z and Madden, MZ and Arner, EN and Sewell, AE and Blount, AK and Okparaugo, R and Fallman, E and Krystofiak, ES and Sheldon, RD and Gibson-Corley, KN and Voss, K and Nowinski, SM and Jones, RG and Mogilenko, DA and Rathmell, JC},
title = {Mitochondrial fatty acid synthesis and MECR regulate CD4+ T cell function and oxidative metabolism.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {214},
number = {5},
pages = {958-976},
pmid = {40204636},
issn = {1550-6606},
support = {S10 OD034315/OD/NIH HHS/United States ; P30EY008126/NH/NIH HHS/United States ; T32 CA009582/CA/NCI NIH HHS/United States ; R01 AI165722/AI/NIAID NIH HHS/United States ; R01 AI153167/AI/NIAID NIH HHS/United States ; U2C DK059637/DK/NIDDK NIH HHS/United States ; R38HL143619//Breakthrough T1D Advanced Postdoctoral Fellowship/ ; P30CA068485//National Institutes of Health Cancer Center/ ; P30DK058404/NH/NIH HHS/United States ; P30CA068485/NH/NIH HHS/United States ; //Distinguished Innovator/ ; T32GM007347//Breakthrough T1D Advanced Postdoctoral Fellowship/ ; F31 DK139660/NH/NIH HHS/United States ; R01 HL136664/HL/NHLBI NIH HHS/United States ; //Lupus Research Alliance/ ; P30DK020593/NH/NIH HHS/United States ; U2CDK059637/NH/NIH HHS/United States ; F31 DK139660/DK/NIDDK NIH HHS/United States ; //The Vanderbilt Cell Imaging Shared Resource/ ; K00CA253718//Breakthrough T1D Advanced Postdoctoral Fellowship/ ; T32CA009582//Breakthrough T1D Advanced Postdoctoral Fellowship/ ; R21AR080233/NH/NIH HHS/United States ; P30 DK058404/DK/NIDDK NIH HHS/United States ; K00 CA253718/CA/NCI NIH HHS/United States ; R01AI165722//Paul G. Allen Frontiers Group Distinguished Investigator Program/ ; R01 DK105550/DK/NIDDK NIH HHS/United States ; //The Translational Pathology Shared Resource/ ; R21 AR080233/AR/NIAMS NIH HHS/United States ; 1S10OD034315/NH/NIH HHS/United States ; R01 AI153167/NH/NIH HHS/United States ; R38 HL143619/HL/NHLBI NIH HHS/United States ; 2T32CA009592-32/NH/NIH HHS/United States ; R01DK105550/NH/NIH HHS/United States ; F30CA239367//Breakthrough T1D Advanced Postdoctoral Fellowship/ ; DRG-2495-23//Breakthrough T1D Advanced Postdoctoral Fellowship/ ; P30 EY008126/EY/NEI NIH HHS/United States ; R01HL136664/NH/NIH HHS/United States ; F30 CA239367/CA/NCI NIH HHS/United States ; T32 GM007347/GM/NIGMS NIH HHS/United States ; P30 DK020593/DK/NIDDK NIH HHS/United States ; P30 CA068485/CA/NCI NIH HHS/United States ; P30CA068485//Breakthrough T1D Advanced Postdoctoral Fellowship/ ; T32 CA009592/CA/NCI NIH HHS/United States ; //Mark Foundation Endeavor/ ; R01 CA217987/CA/NCI NIH HHS/United States ; R01CA217987/NH/NIH HHS/United States ; },
mesh = {*CD4-Positive T-Lymphocytes/immunology/metabolism ; *Mitochondria/metabolism ; *Fatty Acids/biosynthesis ; Animals ; Mice ; CRISPR-Cas Systems ; *Oxidoreductases Acting on CH-CH Group Donors/genetics/metabolism ; Mice, Knockout ; Ferroptosis ; Male ; Female ; *Oxygen/metabolism ; Colitis/physiopathology ; },
abstract = {Imbalanced effector and regulatory CD4+ T cell subsets drive many inflammatory diseases. These T cell subsets rely on distinct metabolic programs, modulation of which differentially affects T cell fate and function. Lipid metabolism is fundamental yet remains poorly understood across CD4+ T cell subsets. Therefore, we performed targeted in vivo CRISPR/Cas9 screens to identify lipid metabolism genes and pathways essential for T cell functions. These screens established mitochondrial fatty acid synthesis genes Mecr, Mcat, and Oxsm as key metabolic regulators. Of these, the inborn error of metabolism gene Mecr was most dynamically regulated. Mecrfl/fl; Cd4cre mice had normal naïve CD4+ and CD8+ T cell numbers, demonstrating that MECR is not essential in homeostatic conditions. However, effector and memory T cells were reduced in Mecr knockout and MECR-deficient CD4+ T cells and proliferated, differentiated, and survived less well than control T cells. Interestingly, T cells ultimately showed signs of mitochondrial stress and dysfunction in the absence of MECR. Mecr-deficient T cells also had decreased mitochondrial respiration, reduced tricarboxylic acid intermediates, and accumulated intracellular iron, which appeared to contribute to increased cell death and sensitivity to ferroptosis. Importantly, MECR-deficient T cells exhibited fitness disadvantages and were less effective at driving disease in an in vivo model of inflammatory bowel disease. Thus, MECR-mediated metabolism broadly supports CD4+ T cell proliferation and survival in vivo. These findings may also provide insight to the immunological state of MECR- and other mitochondrial fatty acid synthesis-deficient patients.},
}

*CD4-Positive T-Lymphocytes/immunology/metabolism
*Mitochondria/metabolism
*Fatty Acids/biosynthesis
Animals
Mice
CRISPR-Cas Systems
*Oxidoreductases Acting on CH-CH Group Donors/genetics/metabolism
Mice, Knockout
Ferroptosis
Male
Female
*Oxygen/metabolism
Colitis/physiopathology

@article {pmid40653316,
year = {2025},
author = {Kouvela, A and Jaramillo Ponce, JR and Giarimoglou, N and Chicher, J and Marzi, S and Stathopoulos, C and Stamatopoulou, V},
title = {Coupling tRNAGly gene redundancy with staphylococcal cell wall integrity, antibiotic susceptibility, and virulence potential.},
journal = {Nucleic acids research},
volume = {53},
number = {13},
pages = {},
doi = {10.1093/nar/gkaf599},
pmid = {40653316},
issn = {1362-4962},
support = {//University of Patras/ ; },
mesh = {*Cell Wall/genetics/drug effects/metabolism ; *Staphylococcus aureus/genetics/pathogenicity/drug effects ; Humans ; Anti-Bacterial Agents/pharmacology ; Virulence/genetics ; CRISPR-Cas Systems ; Biofilms/growth & development/drug effects ; Gene Expression Regulation, Bacterial ; Riboswitch ; Staphylococcal Infections/microbiology ; Protein Biosynthesis ; Gene Editing ; },
abstract = {Redundancy in transfer RNA (tRNA) gene copies across species remains poorly understood and, in many cases, largely unexplored. In Staphylococcus aureus, multiple tRNAGly genes encode isoacceptors involved in protein synthesis and cell wall formation, aminoacylated by a sole glycyl-tRNA synthetase (GlyRS) which is under the transcription regulation of a species-specific glyS T-box riboswitch. The T-box can interact with all tRNAGly isoacceptors to adopt species-specific conformations and affect both pathways. Using CRISPR/Cas9 editing, we ablated a gene copy corresponding to the proteinogenic P1 tRNAGlyGCC. Surprisingly, the growth and the overall translational activity of the edited strain were found unaffected, suggesting functional compensation by the remaining tRNAGly genes. On the other hand, transcriptomics and proteomics analyses combined with functional assays revealed nutrient-dependent stress responses with surprisingly impaired cell wall integrity and increased susceptibility to cell wall-targeting antibiotics. Additionally, the edited strain displayed reduced biofilm formation but retained the ability to invade human cells in vitro. Overall, the present study underscores the critical role of tRNA gene redundancy in the physiology of S. aureus and highlights tRNAs as regulators of metabolic homeostasis in pathogenic bacteria.},
}

*Cell Wall/genetics/drug effects/metabolism
*Staphylococcus aureus/genetics/pathogenicity/drug effects
Humans
Anti-Bacterial Agents/pharmacology
Virulence/genetics
CRISPR-Cas Systems
Biofilms/growth & development/drug effects
Gene Expression Regulation, Bacterial
Riboswitch
Staphylococcal Infections/microbiology
Protein Biosynthesis
Gene Editing

@article {pmid40652883,
year = {2025},
author = {Zhang, S and Kim, JC and Ahn, J},
title = {Phage engineering strategies to expand host range for controlling antibiotic-resistant pathogens.},
journal = {Microbiological research},
volume = {300},
number = {},
pages = {128278},
doi = {10.1016/j.micres.2025.128278},
pmid = {40652883},
issn = {1618-0623},
abstract = {Bacteriophages are known as a promising alternative to control rising bacterial resistance. The adsorption phase is critical for the successful infection of phages, as it determines their ability to recognize and attach to specific bacterial host cells. However, their limited host ranges due to narrow host specificity significantly limit their potential applications and overall effectiveness. Receptor-binding proteins (RBPs) are crucial in the recognition process, and modifying these proteins provides a valuable opportunity to broaden host ranges and enhance adsorption rates. Therefore, gaining a more comprehensive understanding of the interactions between phages and their bacterial hosts is essential. To overcome this challenge, various in vivo and in vitro engineering platforms have been developed, including recombineering, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) systems, yeast-based technologies, and cell-free systems. These methods provide diverse strategies and flexibility for constructing customized phage genomes with desired characteristics, ultimately enhancing phage application efficiency. This review discusses different types of RBPs in phages and their mechanisms of adsorption, highlighting their relevance for adaptable engineering strategies. We also summarize various phage engineering platforms and explore the design of synthetic phages with expanded host ranges. Finally, we highlight the advantages and limitations of current engineering methods, providing insights to guide future research efforts.},
}

@article {pmid40652308,
year = {2025},
author = {Oliver-Caldes, A and Mañe Pujol, J and Battram, AM and Perez-Amill, L and Bachiller, M and Calderon, H and Castella, M and Carpio, J and Salsench, SV and Tovar, N and Cardus, O and Urbano-Ispizua, A and Moreno, DF and Rodríguez-Lobato, LG and Lozano, E and Rosiñol, L and Juan, M and Martín-Antonio, B and Fernández de Larrea, C},
title = {TIGIT blockade in the context of BCMA-CART cell therapy does not augment efficacy in a multiple myeloma mouse model.},
journal = {Oncoimmunology},
volume = {14},
number = {1},
pages = {2529632},
doi = {10.1080/2162402X.2025.2529632},
pmid = {40652308},
issn = {2162-402X},
mesh = {Animals ; *Multiple Myeloma/therapy/immunology/pathology ; *Receptors, Immunologic/antagonists & inhibitors/immunology/genetics ; Mice ; *Immunotherapy, Adoptive/methods ; Humans ; Disease Models, Animal ; *Receptors, Chimeric Antigen/immunology ; *B-Cell Maturation Antigen/immunology ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; Female ; },
abstract = {BCMA-directed CAR-T therapies have shown promising results in multiple myeloma (MM). However, patients continue to relapse. T cell exhaustion with increased TIGIT expression is a resistance mechanism which was confirmed in CAR-T cells from ARI0002h trial, an academic CAR-T developed in our institution. We aimed to analyze the impact of blocking TIGIT on the efficacy of ARI0002h. We used three different strategies to block TIGIT: (1) Addition of an external blocking anti-TIGIT-antibody (Ab), (2) Modify ARI0002h into a 4[th] generation CAR-T, named ARITIGIT, capable of secreting a soluble TIGIT-blocking scFv and (3) TIGIT knock-out in ARI0002h using CRISPR/Cas9. Each strategy was evaluated in vitro and in vivo. Adding a TIGIT-blocking Ab to ARI0002h improved in vitro cytotoxicity, but failed to enhance mice survival. The new 4[th] generation CAR-T, ARITIGIT, was also unable to achieve better survival outcomes despite favoring the in vivo model by using a myeloma cell line with high expression of the TIGIT ligand PVR. Interestingly, when mice were challenged with a second infusion of tumor cells, mimicking a relapse model, a trend for improved survival with ARITIGIT was observed (p = 0.11). Finally, TIGIT-knock-out on ARI0002h (KO-ARI0002h) using CRISPR/Cas9 showed similar in vitro activity to ARI0002h. In an in vivo stress model, TIGIT KO-ARI0002h prolonged survival (p = 0.02). However, this improvement was not significant compared to ARI0002h (p = 0.07). This study failed to demonstrate a significant benefit of TIGIT-blockade on ARI0002h cells despite using three different approaches, suggesting that targeting a single immune checkpoint may be insufficient.},
}

Animals
*Multiple Myeloma/therapy/immunology/pathology
*Receptors, Immunologic/antagonists & inhibitors/immunology/genetics
Mice
*Immunotherapy, Adoptive/methods
Humans
Disease Models, Animal
*Receptors, Chimeric Antigen/immunology
*B-Cell Maturation Antigen/immunology
Cell Line, Tumor
Xenograft Model Antitumor Assays
CRISPR-Cas Systems
Female

@article {pmid40490045,
year = {2025},
author = {Ye, X and Jia, H and Zu, Y},
title = {lmod2a mutations affect F-actin and SRF pathway leading to cardiac dysfunction in zebrafish.},
journal = {Developmental biology},
volume = {525},
number = {},
pages = {306-316},
doi = {10.1016/j.ydbio.2025.06.002},
pmid = {40490045},
issn = {1095-564X},
mesh = {Animals ; *Zebrafish/genetics/embryology/metabolism ; *Actins/metabolism/genetics ; *Serum Response Factor/metabolism/genetics ; *Zebrafish Proteins/genetics/metabolism ; Mutation/genetics ; Signal Transduction/genetics ; Cardiomyopathy, Dilated/genetics ; *Heart/embryology/physiopathology ; *Muscle Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Microfilament Proteins/genetics/metabolism ; Disease Models, Animal ; },
abstract = {Leiomodin 2 (LMOD2), a critical pathogenic gene associated with human dilated cardiomyopathy (DCM), is essential in regulating thin filament length during cardiac development. This study generated a homozygous knockout zebrafish line (lmod2a[-/-]) using CRISPR/Cas9 genome editing. lmod2a[-/-] embryos exhibited impaired locomotor activity alongside irregular heart rhythms, reduced cardiac output, compromised contractility, and delayed calcium transients, as revealed by high-speed imaging and calcium optical mapping. Immunofluorescence staining demonstrated a marked reduction in filamentous actin (F-actin), corroborated by QPCR data showing downregulation of the F-actin marker gene acta1b. Moreover, expression levels of key downstream targets of the serum response factor (SRF) signaling pathway were markedly reduced in mutants. These findings indicate that lmod2a deficiency disrupts F-actin homeostasis and SRF-mediated gene regulation, ultimately leading to defective cardiac performance. This study establishes a novel zebrafish model for investigating LMOD-associated cardiomyopathies and provides valuable insights for future therapeutic interventions targeting actin-related cardiac disorders.},
}

Animals
*Zebrafish/genetics/embryology/metabolism
*Actins/metabolism/genetics
*Serum Response Factor/metabolism/genetics
*Zebrafish Proteins/genetics/metabolism
Mutation/genetics
Signal Transduction/genetics
Cardiomyopathy, Dilated/genetics
*Heart/embryology/physiopathology
*Muscle Proteins/genetics/metabolism
CRISPR-Cas Systems
*Microfilament Proteins/genetics/metabolism
Disease Models, Animal

@article {pmid40404079,
year = {2025},
author = {Ferrari, K and Gurung, S and Loges, LN and Batta, SPR and Hammond, MA and Griciunaite, M and DeMoya, R and Restrepo, NK and Sumanas, S},
title = {Zebrafish Kelch-like family member 4 is required for vasculogenesis and hematopoiesis.},
journal = {Developmental biology},
volume = {525},
number = {},
pages = {1-12},
pmid = {40404079},
issn = {1095-564X},
mesh = {Animals ; *Zebrafish/embryology/genetics/metabolism ; *Hematopoiesis/physiology/genetics ; *Zebrafish Proteins/genetics/metabolism ; *Neovascularization, Physiologic/genetics/physiology ; Gene Expression Regulation, Developmental ; Hematopoietic Stem Cells/metabolism/cytology ; Endothelial Cells/metabolism ; Apoptosis ; Embryo, Nonmammalian/metabolism ; CRISPR-Cas Systems ; Cell Proliferation ; Transcription Factors/metabolism/genetics ; Mutation ; },
abstract = {Molecular mechanisms regulating vascular development and hematopoiesis are still incompletely understood. The KLHL (Kelch-like) family of proteins function as adapters to target proteins for ubiquitination. However, their role in vascular development has not been previously analyzed. Here we have characterized a novel regulator of vascular development, kelch-like family member 4 (klhl4) in zebrafish. We show that zebrafish klhl4 is expressed in early vascular endothelial and hematopoietic progenitors, while its expression is restricted to vascular endothelial cells during later developmental stages. To determine the functional role of klhl4, we generated loss-of-function zebrafish mutants using CRISPR/Cas9 genome editing. klhl4 mutant embryos were viable, yet they exhibited delayed sprouting of intersegmental vessels (ISVs), which correlated with reduced expression of vascular endothelial and erythroid specific molecular markers. Time-lapse imaging showed that vascular endothelial and hematopoietic progenitor cells exhibit delayed migration towards the midline and undergo increased apoptosis and reduced proliferation in klhl4 mutants. Expression of npas4l and etv2/etsrp, two master regulators of endothelial and hematopoietic development, was reduced in klhl4 mutants, suggesting that some vascular defects could be caused by the reduction of npas4l and etv2 expression. However, npas4l or etv2 overexpression failed to rescue ISV sprouting defects in klhl4 mutants, suggesting that klhl4 may promote vasculogenesis by additional mechanisms. In summary, our findings demonstrate a novel role for zebrafish klhl4 in regulating vascular endothelial and hematopoietic development during embryogenesis. Because the Klhl4 protein sequence is highly conserved between different vertebrates, it is likely that it may play a similar role in other organisms.},
}

Animals
*Zebrafish/embryology/genetics/metabolism
*Hematopoiesis/physiology/genetics
*Zebrafish Proteins/genetics/metabolism
*Neovascularization, Physiologic/genetics/physiology
Gene Expression Regulation, Developmental
Hematopoietic Stem Cells/metabolism/cytology
Endothelial Cells/metabolism
Apoptosis
Embryo, Nonmammalian/metabolism
CRISPR-Cas Systems
Cell Proliferation
Transcription Factors/metabolism/genetics
Mutation

@article {pmid40651052,
year = {2025},
author = {Arutselvan, R and Kumar, S and Akash, AU and Greeshma, K and Sinha, SS and Khan, AS and Pati, K and Chauhan, VBS and Gowda, KH and Pradhan, S and Jeeva, ML and Veena, SS and Makeshkumar, T and Meena, M and Sangeetha, BG and Laxminarayana, K and Nedunchezhiyan, M},
title = {Deciphering the complex signaling networks in phytophthora infected plants: Insights into microbiome interactions and plant defense mechanisms.},
journal = {Plant physiology and biochemistry : PPB},
volume = {228},
number = {},
pages = {110222},
doi = {10.1016/j.plaphy.2025.110222},
pmid = {40651052},
issn = {1873-2690},
abstract = {Phytophthora species are destructive plant pathogens that cause severe economic losses in agriculture and natural ecosystems, known for their rapid spread through soil and water and resistance to conventional control methods. Understanding the complex signaling networks activated in plants during Phytophthora infection is crucial for developing effective management strategies. This review summarizes research findings on Phytophthora-plant interactions, with special emphasis on Phytophthora-plant microbiome interactions. Initially, molecular mechanisms involved in the plant response to Phytophthora infection are discussed, further emphasizing key signaling pathways activated by Phytophthora in host plants. The role of phytohormones in imparting resistance to Phytophthora infections is explored in depth. Additionally, the interaction and effects of Phytophthora and the plant immune system with the plant microbiome are examined, highlighting how these interactions facilitate disease and/or aid in plant defense. Various biotechnological approaches for enhancing plant resistance to Phytophthora, including recent technologies like CRISPR-Cas systems, are also reviewed. The conclusion addresses the need for further research into signaling networks within Phytophthora-plant-microbiome interactions and their future implications for crop protection.},
}

@article {pmid40650977,
year = {2025},
author = {Faber, A and Politan, RJ and Stukenberg, D and Morris, KM and Kim, R and Jeon, E and Inckemann, R and Becker, A and Thuronyi, B and Fritz, G},
title = {Expanding genetic engineering capabilities in Vibrio natriegens with the Vnat Collection.},
journal = {Nucleic acids research},
volume = {53},
number = {13},
pages = {},
doi = {10.1093/nar/gkaf580},
pmid = {40650977},
issn = {1362-4962},
support = {FT230100283//Australian Research Council/ ; //Forrest Research Foundation/ ; //Forrest Research Foundation Scholarship/ ; GRK 2937//Deutsche Forschungsgemeinschaft/ ; //Williams College/ ; //Australian Research Council/ ; },
mesh = {*Vibrio/genetics/metabolism ; *Genetic Engineering/methods ; Promoter Regions, Genetic ; Synthetic Biology/methods ; Metabolic Engineering/methods ; Gene Editing/methods ; CRISPR-Cas Systems ; Operon ; Cloning, Molecular ; },
abstract = {Vibrio natriegens, with its exceptionally fast growth rate, has great promise as a revolutionary chassis for synthetic biology, yet the realization of its full potential has been limited by the lack of robust, standardized genetic tools. Here, we present the Vnat Collection, a comprehensive, modular toolkit specifically engineered to overcome these limitations. Leveraging optimized Golden Gate cloning strategies, we introduce improved junction sequences and a highly efficient dropout part system, achieving up to a 300-fold increase in assembly efficiency. Our toolkit significantly expands the synthetic biology toolbox by providing a wide array of characterized inducible promoters, enabling precise, orthogonal gene regulation, and novel operon connectors to streamline the construction of multi-gene pathways critical for metabolic engineering. Furthermore, we enhance genome editing workflows through refined NT-CRISPR methods, incorporating homology-flanked targeting constructs and demonstrating a simplified protocol that eliminates intermediate purification steps. With over 220 rigorously validated modular components, the Vnat Collection establishes an advanced standard for genetic engineering of V. natriegens, empowering researchers to efficiently harness this organism's unparalleled potential for diverse biotechnology applications.},
}

*Vibrio/genetics/metabolism
*Genetic Engineering/methods
Promoter Regions, Genetic
Synthetic Biology/methods
Metabolic Engineering/methods
Gene Editing/methods
CRISPR-Cas Systems
Operon
Cloning, Molecular

@article {pmid40650973,
year = {2025},
author = {Chi, H and McMahon, S and Daniel-Pedersen, L and Graham, S and Gloster, TM and White, MF},
title = {SAM-AMP lyases in type III CRISPR defence.},
journal = {Nucleic acids research},
volume = {53},
number = {13},
pages = {},
doi = {10.1093/nar/gkaf655},
pmid = {40650973},
issn = {1362-4962},
support = {BB/T004789/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; 101018608//European Research Council Advanced Grant/ ; //University of St Andrews/ ; },
mesh = {S-Adenosylmethionine/metabolism ; *CRISPR-Cas Systems ; Escherichia coli/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; Thionucleosides/metabolism/chemistry ; Models, Molecular ; *Lyases/chemistry/metabolism/genetics ; Deoxyadenosines/metabolism/chemistry ; Crystallography, X-Ray ; },
abstract = {Type III CRISPR systems detect non-self RNA and activate the enzymatic Cas10 subunit, which generates nucleotide second messengers for activation of ancillary effectors. Although most signal via cyclic oligoadenylate, an alternative class of signalling molecule SAM-AMP, formed by conjugating ATP and S-adenosylmethionine, was described recently. SAM-AMP activates a trans-membrane effector of the CorA magnesium transporter family to provide anti-phage defence. Intriguingly, immunity also requires SAM-AMP degradation by means of a specialized CRISPR-encoded NrN family phosphodiesterase in Bacteroides fragilis. In Clostridium botulinum, the nrn gene is replaced by a gene encoding a SAM-AMP lyase. Here, we investigate the structure and activity of C. botulinum SAM-AMP lyase, which can substitute for the nrn gene to provide CorA-mediated immunity in Escherichia coli. The structure of SAM-AMP lyase bound to its reaction product 5'-methylthioadenosine-AMP reveals key details of substrate binding and turnover by this PII superfamily protein. Bioinformatic analysis revealed a phage-encoded SAM-AMP lyase that degrades SAM-AMP efficiently in vitro, consistent with an anti-CRISPR function.},
}

S-Adenosylmethionine/metabolism
*CRISPR-Cas Systems
Escherichia coli/genetics
*Bacterial Proteins/chemistry/metabolism/genetics
Thionucleosides/metabolism/chemistry
Models, Molecular
*Lyases/chemistry/metabolism/genetics
Deoxyadenosines/metabolism/chemistry
Crystallography, X-Ray

@article {pmid40650969,
year = {2025},
author = {Madariaga-Marcos, J and Baltramonaitis, M and Henkel-Heinecke, S and Kauert, DJ and Irmisch, P and Bigelyte-Stankeviciene, G and Silanskas, A and Karvelis, T and Siksnys, V and Sasnauskas, G and Seidel, R},
title = {Structural and mechanistic insights into the sequential dsDNA cleavage by SpCas12f1.},
journal = {Nucleic acids research},
volume = {53},
number = {13},
pages = {},
doi = {10.1093/nar/gkaf588},
pmid = {40650969},
issn = {1362-4962},
support = {S-MIP-19-32//Research Council of Lithuania/ ; 02-002-P-0001//Mission-driven Implementation of Science and Innovation Programmes/ ; //New Generation Lithuania/ ; GA 724863/ERC_/European Research Council/International ; SPP2141//Deutsche Forschungsgemeinschaft/ ; SE 1646/8-1//Deutsche Forschungsgemeinschaft/ ; //Alexander von Humboldt Foundation/ ; //Leipzig University/ ; //German Research Foundation/ ; },
mesh = {*DNA Cleavage ; *DNA/metabolism/chemistry/genetics ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; Models, Molecular ; Kinetics ; Catalytic Domain ; R-Loop Structures ; Protein Binding ; *Bacterial Proteins/chemistry/metabolism/genetics ; },
abstract = {Miniature CRISPR-Cas12f1 effector complexes have recently attracted considerable interest for genome engineering applications due to their compact size. Unlike other Class 2 effectors, Cas12f1 functions as a homodimer bound to a single ∼200 nt RNA. While the basic biochemical properties of Cas12f1, such as its use of a single catalytic center for catalysis, have been characterized, the orchestration of the different events occurring during Cas12f1 reactions remained little explored. To gain insights into the dynamics and mechanisms involved in DNA recognition and cleavage by Cas12f1 from Syntrophomonas palmitatica (SpCas12f1), we solved the structure of SpCas12f1 bound to target DNA and employed single-molecule magnetic tweezers measurements in combination with ensemble kinetic measurements. Our data indicate that SpCas12f1 forms 18 bp R-loops, in which local contacts of the protein to the R-loop stabilize R-loop intermediates. DNA cleavage is catalyzed by a single SpCas12f1 catalytic center, which first rapidly degrades a ∼11 bp region on the nontarget strand by cutting at random sites. Subsequent target strand cleavage is slower and requires at least a nick in the nontarget strand.},
}

*DNA Cleavage
*DNA/metabolism/chemistry/genetics
*CRISPR-Associated Proteins/chemistry/metabolism/genetics
CRISPR-Cas Systems
Models, Molecular
Kinetics
Catalytic Domain
R-Loop Structures
Protein Binding
*Bacterial Proteins/chemistry/metabolism/genetics

@article {pmid40650785,
year = {2025},
author = {Samuels, M and Besta, S and Betrán, AL and Nia, RS and Xie, X and Gu, X and Shu, Q and Giamas, G},
title = {CRISPR screening approaches in breast cancer research.},
journal = {Cancer metastasis reviews},
volume = {44},
number = {3},
pages = {59},
pmid = {40650785},
issn = {1573-7233},
mesh = {Humans ; *Breast Neoplasms/genetics/diagnosis/pathology/therapy ; Female ; *CRISPR-Cas Systems ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The emergence of CRISPR-Cas9 technology has transformed functional genomics, offering unmatched opportunities to dissect and understand biological pathways and identify novel therapeutic targets in cancer. Breast cancer is a complex, heterogeneous disease and remains a major cause of morbidity and mortality in women, particularly when diagnosed at advanced or metastatic stages where effective treatments are limited. High-throughput CRISPR screening is undoubtedly a powerful tool to discover novel drug targets, uncover synthetic lethal interactions, and identify vulnerabilities in cancer. This review focuses on advances in our understanding of breast cancer developed through CRISPR-based screening technology, particularly in identifying drivers of breast cancer progression, growth, and metastasis, as well as in identifying potential new therapeutic targets and combination therapies. We discuss recent discoveries, current challenges, and limitations of this approach and explore how advancements in CRISPR technology could have a profound impact on the future of breast cancer treatment.},
}

Humans
*Breast Neoplasms/genetics/diagnosis/pathology/therapy
Female
*CRISPR-Cas Systems
Animals
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40650287,
year = {2025},
author = {Gao, Z and Long, T and Guo, P and Luo, J and Nie, X and Xie, Q and Chen, G and Hu, Z},
title = {The Nuclear Transcription Factor SlNF-YC9 Regulates the Protrusion of Tomato Fruit Tip.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136511},
pmid = {40650287},
issn = {1422-0067},
support = {31872121//National Natural Science Foundation of China/ ; },
mesh = {*Solanum lycopersicum/genetics/metabolism/growth & development ; *Fruit/genetics/growth & development/metabolism ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; Promoter Regions, Genetic ; *CCAAT-Binding Factor/genetics/metabolism ; Indoleacetic Acids/metabolism ; CRISPR-Cas Systems ; },
abstract = {NF-Y transcriptional regulators play crucial roles in diverse biological processes in plants, primarily through the formation of NF-Y complexes that bind to specific DNA motifs. These complexes modulate the expression of downstream genes, which influence plant development and growth. In our research, the function of the NF-Y family C subunit member SlNF-YC9 gene in tomato was investigated with the CRISPR/Cas9 method. In contrast to the WT (wild type), the mutant CR-SlNF-YC9 exhibited a prominent protrusion at the fruit tip. The quantitative PCR analysis displayed that the transcription levels of genes associated with auxin transport (PIN4, PIN5, and PIN9) as well as auxin response genes (ARF7 and LAX3) were enhanced in the CR-SlNF-YC9 fruits than in the WT. Analysis of dual-luciferase reporter and EMSA assays showed that the SlNF-YC9-YB13b-YA7a trimer specifically binds the FUL2 promoter and represses its expression. In conclusion, our results suggest that SlNF-YC9 is crucial in influencing tomato fruit shape by the formation of NF-Y heterotrimeric complexes.},
}

*Solanum lycopersicum/genetics/metabolism/growth & development
*Fruit/genetics/growth & development/metabolism
Gene Expression Regulation, Plant
*Plant Proteins/genetics/metabolism
*Transcription Factors/genetics/metabolism
Promoter Regions, Genetic
*CCAAT-Binding Factor/genetics/metabolism
Indoleacetic Acids/metabolism
CRISPR-Cas Systems

@article {pmid40650152,
year = {2025},
author = {Kovalev, MA and Mamaeva, NY and Kristovskiy, NV and Feskin, PG and Vinnikov, RS and Oleinikov, PD and Sosnovtseva, AO and Yakovlev, VA and Glukhov, GS and Shaytan, AK},
title = {Epigenome Engineering Using dCas Systems for Biomedical Applications and Biotechnology: Current Achievements, Opportunities and Challenges.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136371},
pmid = {40650152},
issn = {1422-0067},
support = {075-15-2024-539//Russian Ministry of Science and Higher Education/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Epigenome/genetics ; *Biotechnology/methods ; Animals ; *Epigenesis, Genetic ; *Epigenomics/methods ; Epigenome Editing ; },
abstract = {Epigenome engineering, particularly utilizing CRISPR/dCas-based systems, is a powerful strategy to modulate gene expression and genome functioning without altering the DNA sequence. In this review we summarized current achievements and prospects in dCas-mediated epigenome editing, primarily focusing on its applications in biomedicine, but also providing a wider context for its applications in biotechnology. The diversity of CRISPR/dCas architectures is outlined, recent innovations in the design of epigenetic editors and delivery methods are highlighted, and the therapeutic potential across a wide range of diseases, including hereditary, neurodegenerative, and metabolic disorders, is examined. Opportunities for the application of dCas-based tools in animal, agricultural, and industrial biotechnology are also discussed. Despite substantial progress, challenges, such as delivery efficiency, specificity, stability of induced epigenetic modifications, and clinical translation, are emphasized. Future directions aimed at enhancing the efficacy, safety, and practical applicability of epigenome engineering technologies are proposed.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*Epigenome/genetics
*Biotechnology/methods
Animals
*Epigenesis, Genetic
*Epigenomics/methods
Epigenome Editing

@article {pmid40650151,
year = {2025},
author = {Jung, YJ and Kim, JY and Cho, YG and Kang, KK},
title = {CRISPR/Cas9-Mediated Knockout of OsbZIP76 Reveals Its Role in ABA-Associated Immune Signaling in Rice.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136374},
pmid = {40650151},
issn = {1422-0067},
support = {RS-2024-00322378//New Breeding Technologies Development Program/ ; },
mesh = {*Oryza/genetics/immunology/microbiology/metabolism ; *Abscisic Acid/metabolism/pharmacology ; *CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; Signal Transduction ; Plant Diseases/microbiology/genetics/immunology ; Gene Expression Regulation, Plant ; Gene Knockout Techniques ; Xanthomonas/pathogenicity ; Disease Resistance/genetics ; *Basic-Leucine Zipper Transcription Factors/genetics/metabolism ; *Plant Immunity/genetics ; Ascomycota ; },
abstract = {The basic leucine zipper (bZIP) transcription factors are involved in a wide range of physiological processes in plants, including hormone signaling, stress responses, and growth and development regulation. They play a key role in abscisic acid (ABA)-mediated immune regulation. However, the immune-related function of OsbZIP76 in rice remains poorly understood. In this study, we generated OsbZIP76 knockout (KO) lines using CRISPR/Cas9-mediated genome editing and examined their phenotypic responses to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo) and the fungal pathogen Magnaporthe oryzae. The KO lines showed increased susceptibility to both pathogens compared to wild-type (WT) plants. Furthermore, qRT-PCR analysis revealed that, upon pathogen infection, the expression of pathogenesis-related genes such as PR1a, PR5, and NPR1 was significantly suppressed in the KO lines. ABA treatment experiments showed that KO lines were hypersensitive to exogenous ABA, indicating a role for OsbZIP76 in ABA perception and signaling. Notably, the expression of the OsbZIP76 gene itself was strongly induced by both ABA treatment and pathogen infection, supporting its role as a positive regulator in ABA-associated immune signaling. Overall, this study demonstrates that OsbZIP76 functions as an important immune regulator by integrating defense gene expression with ABA signaling, providing new insights into the molecular crosstalk between hormonal signaling and pathogen defense mechanisms.},
}

*Oryza/genetics/immunology/microbiology/metabolism
*Abscisic Acid/metabolism/pharmacology
*CRISPR-Cas Systems
*Plant Proteins/genetics/metabolism
Signal Transduction
Plant Diseases/microbiology/genetics/immunology
Gene Expression Regulation, Plant
Gene Knockout Techniques
Xanthomonas/pathogenicity
Disease Resistance/genetics
*Basic-Leucine Zipper Transcription Factors/genetics/metabolism
*Plant Immunity/genetics
Ascomycota

@article {pmid40650142,
year = {2025},
author = {Becchi, G and Whitehead, M and Harvey, JP and Sladen, PE and Dushti, M and Chapple, JP and Yu-Wai-Man, P and Cheetham, ME},
title = {CRISPRa-Mediated Increase of OPA1 Expression in Dominant Optic Atrophy.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136364},
pmid = {40650142},
issn = {1422-0067},
support = {5167/5168//Fight for Sight/ ; },
mesh = {*GTP Phosphohydrolases/genetics/metabolism ; Humans ; *Optic Atrophy, Autosomal Dominant/genetics/metabolism/therapy/pathology ; HEK293 Cells ; *CRISPR-Cas Systems ; Mitochondria/metabolism/genetics ; Gene Editing/methods ; Gene Expression Regulation ; Retinal Ganglion Cells/metabolism/pathology ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Dominant Optic Atrophy (DOA) is the most common inherited optic neuropathy and presents as gradual visual loss caused by the loss of retinal ganglion cells (RGCs). Over 60% of DOA cases are caused by pathogenic variants in the OPA1 gene, which encodes a mitochondrial GTPase essential in mitochondrial fusion. Currently, there are no treatments for DOA. Here, we tested the therapeutic potential of an approach to DOA using CRISPR activation (CRISPRa). Homology directed repair was used to introduce a common OPA1 pathogenic variant (c.2708_2711TTAGdel) into HEK293T cells as an in vitro model of DOA. Heterozygous c.2708_2711TTAGdel cells had reduced levels of OPA1 mRNA transcript, OPA1 protein, and mitochondrial network alterations. The effect of inactivated Cas9 fused to an activator (dCas9-VPR) was tested with a range of guide RNAs (gRNA) targeted to the promotor region of OPA1. gRNA3 and dCas9-VPR increased OPA1 expression at the RNA and protein level towards control levels. Importantly, the correct ratio of OPA1 isoform transcripts was maintained by CRISPRa. CRISPRa-treated cells showed an improvement in mitochondrial networks compared to untreated cells, indicating partial rescue of a disease-associated phenotype. Collectively, these data support the potential application of CRISPRa as a therapeutic intervention in DOA.},
}

*GTP Phosphohydrolases/genetics/metabolism
Humans
*Optic Atrophy, Autosomal Dominant/genetics/metabolism/therapy/pathology
HEK293 Cells
*CRISPR-Cas Systems
Mitochondria/metabolism/genetics
Gene Editing/methods
Gene Expression Regulation
Retinal Ganglion Cells/metabolism/pathology
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40650127,
year = {2025},
author = {El Hazzouri, S and Al-Rifai, R and Surges, N and Rath, M and Singer, H and Oldenburg, J and El-Maarri, O},
title = {FVIII Trafficking Dynamics Across Subcellular Organelles Using CRISPR/Cas9 Specific Gene Knockouts.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136349},
pmid = {40650127},
issn = {1422-0067},
support = {2019-7-05; 2020-7-05; 2022-7-02//University Hospital Bonn/ ; IISR-2023-DE-200366//Takeda (Germany)/ ; 91724372//German Academic Exchange Service/ ; Internal Funding//Institute of Experimental Hematology and Transfusion Medicine - University Hospital Bonn/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; Protein Transport ; Gene Knockout Techniques ; *Factor VIII/metabolism/genetics ; Calnexin/metabolism/genetics ; Endoplasmic Reticulum/metabolism ; rab GTP-Binding Proteins/metabolism ; *Organelles/metabolism ; rab7 GTP-Binding Proteins ; Calreticulin/metabolism/genetics ; Golgi Apparatus/metabolism ; },
abstract = {Factor VIII (FVIII) interacts with Endoplasmic Reticulum (ER) chaperones Calnexin (CANX) and Calreticulin (CALR) and with ER-Golgi Intermediate Compartment (ERGIC) transporters, Lectin, mannose-binding 1 (LMAN1) and Multiple Coagulation Deficiency 2 (MCFD2). We previously reported that the Gamma-aminobutyric Acid Receptor-associated proteins (GABARAPs) also influence FVIII secretion. Here, we further investigated the intracellular dynamics of FVIII using single and double CRISPR/Cas9 Knockout (KO) models of the abovementioned chaperones as well as the GABARAP proteins in HEK293 cells expressing FVIII. Cellular pathways were manipulated by Brefeldin A (BFA), Chloroquine (CQ), a Rab7 inhibitor, and subjected to glucose starvation. The effect of each KO on FVIII secretion and organelle distribution was assessed by a two-stage chromogenic assay and immunofluorescence (IF) microscopy, prior and upon cell treatments. Using these approaches, we first observed distinct effects of each studied protein on FVIII trafficking. Notably, intracellular localization patterns revealed clustering of FVIII phenotypes in GABARAP[KO], CANX[KO], and CALR[KO] cells together under both basal and treated conditions, an observation that was also reflected in their respective double KO combinations. Besides, a clear involvement of additional components of the endomembrane system was evident, specifically at the trans-Golgi space, as marked by FVIII colocalization with the Ras-like proteins in brain (Rab8 and Rab7) and with the Vesicle-Associated Membrane Protein (VAMP8), along with the observed impact of the selected cell treatments on FVIII phenotypes. These outcomes enhance our understanding of the molecular mechanisms regulating FVIII and pave the way for new perspectives, which could be further projected into FVIII replacement, cell and gene therapies.},
}

Humans
*CRISPR-Cas Systems
HEK293 Cells
Protein Transport
Gene Knockout Techniques
*Factor VIII/metabolism/genetics
Calnexin/metabolism/genetics
Endoplasmic Reticulum/metabolism
rab GTP-Binding Proteins/metabolism
*Organelles/metabolism
rab7 GTP-Binding Proteins
Calreticulin/metabolism/genetics
Golgi Apparatus/metabolism

@article {pmid40650046,
year = {2025},
author = {Cattaneo, M and Giagnorio, E and Lauria, G and Marcuzzo, S},
title = {Therapeutic Approaches for C9ORF72-Related ALS: Current Strategies and Future Horizons.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136268},
pmid = {40650046},
issn = {1422-0067},
support = {//Italian Ministry of Health (RRC)/ ; T4-AN-09 prog. ZRPOS2//CALabria HUB per Ricerca Innovativa ed Avanzata- CALHUB.RIA "Creazione di Hub delle Sci-enze della Vita"/ ; prog. ZRA124//AriSLA foundation, "Bulb-Omics"/ ; PNRR-MCNT2-2023-12377336//the European Union - Next Generation EU - NRRP M6C2 - Investment 2.1 Enhancement and strengthening of biomedical research in the NHS,/ ; },
mesh = {Humans ; *C9orf72 Protein/genetics/metabolism ; *Amyotrophic Lateral Sclerosis/genetics/therapy/metabolism/pathology ; Animals ; DNA Repeat Expansion ; Gene Editing ; Genetic Therapy/methods ; Mutation ; Oligonucleotides, Antisense/therapeutic use ; },
abstract = {Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of upper and lower motor neurons. One of its major genetic causes is C9ORF72, where mutations lead to hexanucleotide repeat expansions in the C9ORF72 gene. These expansions drive disease progression through mechanisms, including the formation of toxic RNAs and the accumulation of damaged proteins such as dipeptide repeats (DPRs). This review highlights these pathogenic mechanisms, focusing on RNA foci formation and the accumulation of toxic DPRs, which contribute to neuronal damage. It also discusses promising targeted therapies, including small molecules and biological drugs, designed to counteract these specific molecular events. Small molecules such as G-quadruplex stabilizers, proteasome and autophagy modulators, and RNase-targeting chimeras show potential in reducing RNA foci and DPR accumulation. Furthermore, targeting enzymes involved in repeat-associated non-AUG (RAN) translation and nucleocytoplasmic transport, which are crucial for disease pathogenesis, opens new therapeutic avenues. Even some anti-viral drugs show encouraging results in preclinical studies. Biological drugs, such as antisense oligonucleotides and gene-editing technologies like CRISPR-Cas, were explored for their potential to specifically target C9ORF72 mutations and modify the disease's molecular foundations. While preclinical and early clinical data show promise, challenges remain in optimizing delivery methods, ensuring long-term safety, and improving efficacy. This review concludes by emphasizing the importance of continued research and the potential for these therapies to alter the disease trajectory and improve patient outcomes.},
}

Humans
*C9orf72 Protein/genetics/metabolism
*Amyotrophic Lateral Sclerosis/genetics/therapy/metabolism/pathology
Animals
DNA Repeat Expansion
Gene Editing
Genetic Therapy/methods
Mutation
Oligonucleotides, Antisense/therapeutic use

@article {pmid40649992,
year = {2025},
author = {Reddy, KD and Rathnayake, SNH and Idrees, S and Boedijono, F and Xenaki, D and Padula, MP and Berge, MVD and Faiz, A and Oliver, BGG},
title = {A Novel Regulatory Role for RPS4Y1 in Inflammatory and Fibrotic Processes.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136213},
pmid = {40649992},
issn = {1422-0067},
support = {GNT1158186//National Health and Research Council, Australia (NHMRC)/ ; },
mesh = {Humans ; *Asthma/genetics/metabolism/pathology ; *Ribosomal Proteins/genetics/metabolism ; Female ; Male ; *Inflammation/genetics/metabolism/pathology ; Fibrosis ; Interleukin-6/genetics/metabolism ; Tenascin/genetics/metabolism ; Animals ; CRISPR-Cas Systems ; },
abstract = {Asthma is a chronic inflammatory respiratory disease well-known to demonstrate sexual dimorphism in incidence and severity, although the mechanisms causing these differences remain incompletely understood. RPS4X and RPS4Y1 are X and Y-chromosome-linked genes coding ribosomal subunits previously associated with inflammation, airway remodelling and asthma medication efficacy. Particularly, RPS4Y1 has been under-investigated within the context of disease, with little examination of molecular mechanisms and pathways regulated by this gene. The ribosome, a vital cellular machinery, facilitates the translation of mRNA into peptides and then proteins. Imbalance or dysfunction in ribosomal components may lead to malfunctioning proteins. Using CRISPR-Cas9 knockout cellular models for RPS4Y1 and RPS4X, we characterised the function of RPS4Y1 in the context of the asthma-relevant processes, inflammation and fibrosis. No viable RPS4X knockouts could be generated. We highlight novel molecular mechanisms such as specific translation of IL6 and tenascin-C mRNA by RPS4Y1 containing ribosomes. Furthermore, an RPS4Y1-centric gene signature correlates with clinical lung function measurements, specifically in adult male asthma patients. These findings inform the current understanding of sex differences in asthma, as females do not produce the RPS4Y1 protein. Therefore, the pathologically relevant functions of RPS4Y1 may contribute to the complex sexually dimorphic pattern of asthma susceptibility and progression.},
}

Humans
*Asthma/genetics/metabolism/pathology
*Ribosomal Proteins/genetics/metabolism
Female
Male
*Inflammation/genetics/metabolism/pathology
Fibrosis
Interleukin-6/genetics/metabolism
Tenascin/genetics/metabolism
Animals
CRISPR-Cas Systems

@article {pmid40649886,
year = {2025},
author = {Chang, TY and Lin, LC and Kao, CY and Lu, JJ},
title = {Study of lug Operon, SCCmec Elements, Antimicrobial Resistance, MGEs, and STs of Staphylococcus lugdunensis Clinical Isolates Through Whole-Genome Sequencing.},
journal = {International journal of molecular sciences},
volume = {26},
number = {13},
pages = {},
doi = {10.3390/ijms26136106},
pmid = {40649886},
issn = {1422-0067},
support = {TCRD-TPE-NSTC-113-18 and TCRD-TPE-114-04(1/3)//Taipei Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation/ ; NSTC 113-2320-B-303-006//National Science and Technology Council, Taiwan/ ; },
mesh = {*Staphylococcus lugdunensis/genetics/drug effects/isolation & purification ; Whole Genome Sequencing ; *Operon ; Humans ; *Staphylococcal Infections/microbiology ; Multilocus Sequence Typing ; Genome, Bacterial ; Anti-Bacterial Agents/pharmacology ; *Drug Resistance, Bacterial/genetics ; Drug Resistance, Multiple, Bacterial/genetics ; Interspersed Repetitive Sequences ; Phylogeny ; Microbial Sensitivity Tests ; *Bacterial Proteins/genetics ; CRISPR-Cas Systems ; },
abstract = {Staphylococcus lugdunensis is a coagulase-negative staphylococcus known for its significant pathogenic potential, often causing severe infections such as endocarditis and bacteremia, with virulence comparable to S. aureus. Despite general susceptibility to most antibiotics, the emergence of oxacillin-resistant strains is increasingly concerning. This study conducted whole-genome sequencing on 20 S. lugdunensis isolates from Chang Gung Memorial Hospital to explore their genetic diversity, antimicrobial resistance mechanisms, and mobile genetic elements. The lugdunin biosynthetic operon, essential for antimicrobial peptide production, was present in multilocus sequence typing (MLST) types 1, 3, and 6 but absent in STs 4, 27, and 29. Additionally, IS256 insertion elements, ranging from 7 to 17 copies, were identified in four strains and linked to multidrug resistance. CRISPR-Cas systems varied across STs, with type III-A predominant in ST1 and ST6 and type IIC in ST4, ST27, and ST29; notably, ST3 lacked CRISPR systems, correlating with a higher diversity of SCCmec elements and an increased potential for horizontal gene transfer. Phage analysis revealed stable phage-host associations in ST1, ST6, and ST29, whereas ST4 displayed a varied prophage profile. Phenotypic resistance profiles generally aligned with genomic predictions, although discrepancies were observed for aminoglycosides and clindamycin. These findings highlight the complex genetic landscape and evolutionary dynamics of S. lugdunensis, emphasizing the need for genomic surveillance to inform clinical management and prevent the spread of resistant strains.},
}

*Staphylococcus lugdunensis/genetics/drug effects/isolation & purification
Whole Genome Sequencing
*Operon
Humans
*Staphylococcal Infections/microbiology
Multilocus Sequence Typing
Genome, Bacterial
Anti-Bacterial Agents/pharmacology
*Drug Resistance, Bacterial/genetics
Drug Resistance, Multiple, Bacterial/genetics
Interspersed Repetitive Sequences
Phylogeny
Microbial Sensitivity Tests
*Bacterial Proteins/genetics
CRISPR-Cas Systems

@article {pmid40648011,
year = {2025},
author = {Fan, X and Zhang, Y and Gu, P and Naz, M},
title = {Epitranscriptomic Control of Drought Tolerance in Rice: The Role of RNA Methylation.},
journal = {Plants (Basel, Switzerland)},
volume = {14},
number = {13},
pages = {},
doi = {10.3390/plants14132002},
pmid = {40648011},
issn = {2223-7747},
support = {(2023-BS-209 and 2024-BS-281)//Liaoning Provincial Department of Science and Technology Doctoral Research Initiation Fund Program/ ; K20241057//Wuxi's "Taihu Light" Scientific and Technological Innovation Initiative/ ; JC2024019//Natural science foundation of Nantong/ ; },
abstract = {Drought stress is a predominant abiotic constraint adversely affecting global rice (Oryza sativa) production and threatening food security. While the transcriptional and post-transcriptional regulation of drought-responsive pathways has been widely investigated, the emerging field of epitranscriptomics, particularly RNA chemical modifications such as N6-methyladenosine (m[6]A), adds a new dimension to gene regulation under stress. The most prevalent internal modification in eukaryotic messenger RNA influences RNA metabolism by interacting dynamically with enzymes that add, remove, or recognize the modification. Recent studies in rice reveal that m[6]A deposition is not static but dynamically regulated in response to water-deficit conditions, influencing transcript stability, splicing, nuclear export, and translation efficiency of key drought-responsive genes. This review critically synthesizes current findings on the distribution and functional implications of m[6]A and other epitranscriptomic marks (e.g., 5-methylcytosine [m[5]C], pseudouridine [Ψ]) in modulating rice responses to drought. We discuss the regulatory circuitry involving m[6]A effectors such as OsMTA, OsFIP37, and YTH domain proteins and their integration with known drought-signaling pathways including ABA and reactive oxygen species (ROS) cascades. We also highlight emerging high-resolution technologies such as m[6]A-seq, direct RNA sequencing, and nanopore-based detection that facilitate epitranscriptomic profiling in rice. Finally, we propose future directions for translating epitranscriptomic knowledge into crop improvement, including CRISPR/Cas-based modulation of RNA modification machinery to enhance drought tolerance.},
}

@article {pmid40647992,
year = {2025},
author = {Sutula, M and Tussipkan, D and Kali, B and Manabayeva, S},
title = {Molecular Mechanisms Underlying Defense Responses of Potato (Solanum tuberosum L.) to Environmental Stress and CRISPR/Cas-Mediated Engineering of Stress Tolerance.},
journal = {Plants (Basel, Switzerland)},
volume = {14},
number = {13},
pages = {},
doi = {10.3390/plants14131983},
pmid = {40647992},
issn = {2223-7747},
support = {BR21882180//Ministry of Science and Higher Education of the Republic of Kazakhstan/ ; },
abstract = {Environmental stresses, such as drought, salinity, and pathogen attacks, significantly affect potato growth, development, and yield by disrupting key physiological and biochemical processes. Plant responses to these stresses are mediated by changes in gene expression, transcriptional regulation, and the activity of various functional proteins, all of which contribute to the molecular mechanisms of stress tolerance. Genome editing using the CRISPR/Cas9 system has been effectively used to enhance the resistance of potato to environmental stresses and to improve its nutritional value. This article provides a comprehensive review of recent studies retrieved from academic databases focusing on the effects of various environmental stressors on potato growth, yield, and postharvest storage. It also examines the influence of these stresses on the production of secondary metabolites and their associated molecular pathways. Finally, the review highlights advancements in the application of CRISPR/Cas-based genome editing technologies between 2021 and 2025 to improve stress tolerance and nutritional traits in potato plants.},
}

@article {pmid40647524,
year = {2025},
author = {Khan, R and Phely, L and Ehrenfeld, S and Schmitz, T and Veratti, P and Wolfes, J and Shoumariyeh, K and Andrieux, G and Martens, US and Bra, S and Auer, M and Schilling, O and Boerries, M and Speicher, M and Illert, AL and Duyster, J and Miething, C},
title = {Modeling the t(2;5) Translocation of Anaplastic Large Cell Lymphoma Using CRISPR-Mediated Chromosomal Engineering.},
journal = {Cancers},
volume = {17},
number = {13},
pages = {},
doi = {10.3390/cancers17132226},
pmid = {40647524},
issn = {2072-6694},
abstract = {BACKGROUND/OBJECTIVES: ALK+ Anaplastic Large Cell Lymphoma (ALCL) is an aggressive T-cell lymphoma that is characterized by expression of the Anaplastic Lymphoma Kinase (ALK), which is induced by the t(2;5) chromosomal rearrangement, leading to the expression of the NPM-ALK fusion oncogene. Most previous preclinical models of ALK+ ALCL were based on overexpression of the NPM-ALK cDNA from heterologous promoters. Due to the enforced expression, this approach is prone to artifacts arising from synthetic overexpression, promoter competition and insertional variation.

METHODS: To improve the existing ALCL models and more closely recapitulate the oncogenic events in ALK+ ALCL, we employed CRISPR/Cas-based chromosomal engineering to selectively introduce translocations between the Npm1 and Alk gene loci in murine cells.

RESULTS: By inducing precise DNA cleavage at the syntenic loci on chromosome 11 and 17 in a murine IL-3-dependent Ba/F3 reporter cell line, we generated de novo Npm-Alk translocations in vivo, leading to IL-3-independent cell growth. To verify efficient recombination, we analyzed the expression of the NPM-ALK fusion protein in the recombined cells and could also show the t(11;17) in the IL-3 independent Ba/F3 cells. Subsequent functional testing of these cells using an Alk-inhibitor showed exquisite responsiveness towards Crizotinib, demonstrating strong dependence on the newly generated ALK fusion oncoprotein. Furthermore, a comparison of the gene expression pattern between Ba/F3 cells overexpressing the Npm-Alk cDNA with Ba/F3 cells transformed by CRISPR-mediated Npm-Alk translocation indicated that, while broadly overlapping, a set of pathways including the unfolded protein response pathway was increased in the Npm-Alk overexpression model, suggesting increased reactive changes induced by exogenous overexpression of Npm-Alk. Furthermore, we observed clustered expression changes in genes located in chromosomal regions close to the breakpoint in the new CRISPR-based model, indicating positional effects on gene expression mediated by the translocation event, which are not part of the older models.

CONCLUSIONS: Thus, CRISPR-mediated recombination provides a novel and more faithful approach to model oncogenic translocations, which may lead to an improved understanding of the molecular pathogenesis of ALCL and enable more accurate therapeutic models of malignancies driven by oncogenic fusion proteins.},
}

@article {pmid40647454,
year = {2025},
author = {Sidabraite, A and Mosert, PL and Ahmed, U and Jones, SK and Gulla, A},
title = {Advancing Cholangiocarcinoma Diagnosis: The Role of Liquid Biopsy and CRISPR/Cas Systems in Biomarker Detection.},
journal = {Cancers},
volume = {17},
number = {13},
pages = {},
doi = {10.3390/cancers17132155},
pmid = {40647454},
issn = {2072-6694},
support = {101078247/ERC_/European Research Council/International ; IG 5728-2024//European Molecular Biology Organization (EMBO)/ ; P-SV-24-447//Research Council of Lithuania/ ; P-LT-TW-24-21//Research Council of Lithuania/ ; },
abstract = {Background/Objectives: Cholangiocarcinoma (CCA) is a highly heterogeneous malignancy of the biliary tract with limited diagnostic tools for early detection. Current serum markers, such as CA19-9, lack specificity and sensitivity, particularly in early-stage disease, which hinders the effectiveness of curative interventions. This narrative review evaluates the limitations of existing diagnostic approaches and explores the potential of combining liquid biopsy (LB) technologies with CRISPR/Cas-based systems for precise, minimally invasive biomarker detection. Methods: A narrative review was conducted, synthesizing literature from 2018 to 2025 across PubMed, MDPI, Web of Science, Google Scholar, and Embase using MeSH terms such as "cholangiocarcinoma," "liquid biopsy," "miRNA," and "CRISPR/Cas." Results: Circulating microRNAs (e.g., miR-21, miR-16, miR-877) exhibit high diagnostic accuracy. The RACE (Rolling Circle Amplification-assisted CRISPR/Cas9 Cleavage) platform shows promise for detecting extracellular vesicle (EV)-derived miRNAs with high sensitivity and single-nucleotide specificity. When paired with liquid biopsy, CRISPR-based assays enable real-time, cost-effective, and multiplexed detection of tumor-specific biomarkers. Conclusions: The introduction of LB combined with CRISPR/Cas systems could potentially revolutionize the early and accurate diagnosis of CCA, thereby advancing the overall treatment strategy. However, this method is still under development and requires further testing before it can be incorporated into routine diagnostics.},
}

@article {pmid40647071,
year = {2025},
author = {Nascimento, APS and Barros, AN},
title = {Sustainable Innovations in Food Microbiology: Fermentation, Biocontrol, and Functional Foods.},
journal = {Foods (Basel, Switzerland)},
volume = {14},
number = {13},
pages = {},
doi = {10.3390/foods14132320},
pmid = {40647071},
issn = {2304-8158},
abstract = {The growing demand for more sustainable food systems has driven the development of solutions based on food microbiology, capable of integrating safety, functionality, and environmental responsibility. This paper presents a critical and up-to-date review of the most relevant advances at the interface between microbiology, sustainability, and food innovation. The analysis is structured around three main axes: (i) microbial fermentation, with a focus on traditional practices and precision technologies aimed at valorizing agro-industrial waste and producing functional foods; (ii) microbial biocontrol, including the use of bacteriocins, protective cultures, bacteriophages, and CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated)-based tools as alternatives to synthetic preservatives; and (iii) the development of functional foods containing probiotics, prebiotics, synbiotics, and postbiotics, with the potential to modulate the gut microbiota and promote metabolic, immune, and cognitive health. In addition to reviewing the microbiological and technological mechanisms involved, the paper discusses international regulatory milestones, scalability challenges, and market trends related to consumer acceptance and clean labeling. Finally, emerging trends and research gaps are addressed, including the use of omics technologies, artificial intelligence, and unexplored microbial resources. Food microbiology, by incorporating sustainable practices and advanced technologies, is positioned as a strategic pillar for building a healthy, circular, science-based food model.},
}

@article {pmid40646930,
year = {2025},
author = {Han, P and Wang, Y and Sun, H},
title = {Impact of Temperature Stresses on Wheat Quality: A Focus on Starch and Protein Composition.},
journal = {Foods (Basel, Switzerland)},
volume = {14},
number = {13},
pages = {},
doi = {10.3390/foods14132178},
pmid = {40646930},
issn = {2304-8158},
abstract = {With climate change, maintaining wheat quality has become essential for the functional properties, end-use, commodity value, and nutritional benefits of wheat flour. Temperature indirectly influences wheat quality by modulating grain size, starch and protein content, and the balance between these components. This review systematically analyzes temperature-mediated alterations in wheat grain quality, with particular emphasis on the two core components: starch and protein. Specifically, daytime warming generally increases protein content while reducing starch accumulation; however, temperatures exceeding 30 °C diminish key protein quality parameters (UPP%, Glu/Gli ratio, HMW-GS/LMW-GS ratio). Nighttime warming enhances protein quality but compromises starch content and yield potential. Conversely, under low-temperature conditions, starch content declines, whereas protein content is primarily influenced by genotypes and treated temperatures. Furthermore, the underlying mechanisms driving temperature-induced changes in wheat quality traits are discussed. However, the mechanisms of temperature effects have not been fully elucidated, and the results often vary between regions or over years. Thus, identifying conserved high/low-temperature resistance genes, QTLs, epialleles, and epiQTL, as well as developing corresponding molecular markers and epi-markers, is an urgent priority. Meanwhile, genome-editing tools such as CRISPR/Cas could serve as a powerful approach for creating new wheat germplasm with durable high/low-temperature resistance.},
}

@article {pmid40646801,
year = {2025},
author = {Han, H and Zhang, D and Hao, W and Liu, A and Xia, N and Cui, M and Luo, J and Jiang, S and Zheng, W and Chen, N and Gu, J and Bai, J and Zhu, J},
title = {Parallel and Visual Detections of ASFV by CRISPR-Cas12a and CRISPR-Cas13a Systems Targeting the Viral S273R Gene.},
journal = {Animals : an open access journal from MDPI},
volume = {15},
number = {13},
pages = {},
doi = {10.3390/ani15131902},
pmid = {40646801},
issn = {2076-2615},
support = {32473040; 32172867//Natural Science Foundation of China/ ; },
abstract = {African swine fever virus (ASFV) causes a highly contagious and lethal hemorrhagic disease and significantly threatens the pig industry. There is no commercially effective vaccine available currently, making the detection of ASFV critical for control and prevention. Previously, we established the CRISPR-LbCas12a and LwCRSIRP-Cas13a visual detections of ASFV, separately, targeting the structural p17 gene D117L. In this study, we performed the parallel detections of ASFV based on the conserved viral protease gene S273R using CRISPR-LbCas12a and CRISPR-LbuCas13a systems. Our results showed that both systems are able to specifically detect ASFV as low as two copies of the S273R gene, and effectively detect clinical samples with minimal DNA purification. The work promotes CRISPR-Cas systems for the application of on-site detection in the field.},
}

@article {pmid40646370,
year = {2025},
author = {Liu, Y and Liu, H and Wang, G and Jia, X and Zhou, J and Dong, X and Li, H and Chang, H and Li, Z and Jin, Z and Li, K},
title = {Dual-Check CRISPR-SERS strategy for sensitively detecting Monkeypox DNA and its single-base mutated DNA.},
journal = {Mikrochimica acta},
volume = {192},
number = {8},
pages = {497},
pmid = {40646370},
issn = {1436-5073},
support = {2025GXNSFHA069082//Guangxi Natural Science Foundation Project/ ; AD22035215//Guangxi Science and Technology Project/ ; 2022YFE0134600//National Key Research and Development Program of China/ ; AB22080055//Key Research and Development Program of Guangxi/ ; },
mesh = {*Spectrum Analysis, Raman/methods ; Metal Nanoparticles/chemistry ; Gold/chemistry ; *CRISPR-Cas Systems ; *DNA, Viral/genetics/analysis ; DNA, Single-Stranded/chemistry/genetics ; Limit of Detection ; Plasmids/genetics ; CRISPR-Associated Proteins/metabolism ; Endodeoxyribonucleases/metabolism ; Carbocyanines/chemistry ; Silver/chemistry ; Bacterial Proteins/metabolism ; },
abstract = {This study presents a convenient and efficient dual-check strategy for detecting Monkeypox DNA utilizing the SERS (Surface-enhanced Raman Spectroscopy)-CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system. The Monkeypox plasmid DNA (mpDNA) is recognized by the CRISPR RNA (crRNA)-Cas12a protein complex, where crRNA encompasses a targeting complementary sequence. Upon recognition, the trans-cleavage activity of Cas12a is activated and trans-cleaves the probe DNA (Cy3-ssDNA) which is modified on Au nanoparticles (AuNPs). As the ssDNA strand is cleaved, Cy3 molecules are released in the solution, while the amount of Cy3 modified on the AuNPs decreases. The free Cy3 molecules are collected from the supernatant, and their SERS intensities are measured using the silver nanopillar (AgNRs) substrate. The mpDNA with varying concentration from 5 nM to 0.5 fM can be quantitatively determined based on the SERS signals of free Cy3 and the collected nanotag. This strategy allows the detection of mpDNA with a concentration of 50 fM within 60 min. Moreover, the strategy can successfully detect single-base mutated mpDNA. Owing to the non-specific trans-cleavage activity of the protein, this strategy can be adapted to various nucleic acid detection scenarios by designing complementary RNA and DNA sequences.},
}

*Spectrum Analysis, Raman/methods
Metal Nanoparticles/chemistry
Gold/chemistry
*CRISPR-Cas Systems
*DNA, Viral/genetics/analysis
DNA, Single-Stranded/chemistry/genetics
Limit of Detection
Plasmids/genetics
CRISPR-Associated Proteins/metabolism
Endodeoxyribonucleases/metabolism
Carbocyanines/chemistry
Silver/chemistry
Bacterial Proteins/metabolism

@article {pmid40646080,
year = {2025},
author = {Jung, Y and Seo, E and Yang, S and Bae, S and Kim, JH and Jang, HK and Jo, DH},
title = {Establishment and rescue of fibroblast cell lines carrying a nonsense mutation of RB1 by CRISPR-based base editing.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {25074},
pmid = {40646080},
issn = {2045-2322},
support = {18-2023-0010//Seoul National University Hospital/ ; GTL24021-000//Ministry of Science and ICT, South Korea/ ; RS-2024-00400829//Ministry of Science and ICT, South Korea/ ; RS-2023-00260351//Ministry of Science and ICT, South Korea/ ; RS-2023-00301850//Ministry of Education/ ; RS-2024-00438476//Ministry of Health & Welfare/ ; },
mesh = {*Codon, Nonsense/genetics ; Humans ; *Gene Editing/methods ; *Fibroblasts/metabolism/cytology ; *CRISPR-Cas Systems ; *Retinoblastoma Binding Proteins/genetics/metabolism ; Cell Line ; Cell Proliferation/genetics ; Retinoblastoma/genetics/pathology ; Ubiquitin-Protein Ligases ; },
abstract = {Pathogenic variants of the RB1 gene have commonly been found in many cancer types, including retinoblastoma. Nonsense mutations are the most common mutation type in retinoblastoma; however, few cell lines mimic nonsense mutations in the RB1 gene that are commonly observed in patients. Here, we established retinoblastoma-like cell lines carrying mono- and bi-allelic nonsense mutations in the RB1 gene. We introduced the R552X mutation using target activation-induced cytidine deaminase base editing and successfully constructed cell lines carrying mono- and bi-allelic mutations. The model cell lines showed decreased RB1 expression at both the mRNA and protein levels, and increased cell proliferation. Furthermore, we rescued the nonsense mutation in the RB1 gene in model cell lines by converting stop codon 552 to tryptophan using an adenine base editor. This approach may be applicable for establishing cell lines with pathogenic variants found in patients and suggests a strong potential for the application of gene editing as a therapeutic strategy.},
}

*Codon, Nonsense/genetics
Humans
*Gene Editing/methods
*Fibroblasts/metabolism/cytology
*CRISPR-Cas Systems
*Retinoblastoma Binding Proteins/genetics/metabolism
Cell Line
Cell Proliferation/genetics
Retinoblastoma/genetics/pathology
Ubiquitin-Protein Ligases

@article {pmid40645983,
year = {2025},
author = {Ching, RW and Świst-Rosowska, KM and Erikson, G and Koschorz, B and Engist, B and Jenuwein, T},
title = {Forced expression of MSR repeat transcripts above a threshold limit breaks heterochromatin organisation.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6420},
pmid = {40645983},
issn = {2041-1723},
support = {CRC992 'MEDEP'//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Heterochromatin/metabolism/genetics ; Animals ; Mice ; *DNA, Satellite/genetics/metabolism ; Fibroblasts/metabolism ; Transcription, Genetic ; CRISPR-Cas Systems ; Chromosome Segregation/genetics ; },
abstract = {Mouse heterochromatin is characterised by transcriptionally competent major satellite repeat (MSR) sequences and it has been proposed that MSR RNA contributes to the integrity of heterochromatin. We establish an inducible dCas9-effector system in mouse embryonic fibroblasts, where we can modulate MSR transcription through the targeting of a dCas9-Repressor or a dCas9-Activator. With this system, we can define a threshold limit of >300-fold deregulation of MSR transcript levels, above which the structural organisation of heterochromatin becomes disrupted. MEF cells expressing MSR RNA above this threshold limit are not viable and the defects in heterochromatin organisation and chromosome segregation cannot be reverted. This study highlights the importance of restricting MSR RNA output to maintain heterochromatin integrity and relates MSR transcript levels to either physiological or pathological conditions. It also reveals that the structural organisation of heterochromatin is governed by the transcriptional chromatin state and associated MSR RNA of the MSR repeats.},
}

*Heterochromatin/metabolism/genetics
Animals
Mice
*DNA, Satellite/genetics/metabolism
Fibroblasts/metabolism
Transcription, Genetic
CRISPR-Cas Systems
Chromosome Segregation/genetics

@article {pmid40600900,
year = {2025},
author = {Hou, Y and Li, Y and Zheng, R and Zhang, F and Guo, F and Li, M and Zeng, M},
title = {Leveraging protein language models for cross-variant CRISPR/Cas9 sgRNA activity prediction.},
journal = {Bioinformatics (Oxford, England)},
volume = {41},
number = {7},
pages = {},
doi = {10.1093/bioinformatics/btaf385},
pmid = {40600900},
issn = {1367-4811},
support = {2022YFC3400300//National Key Research and Development Program of China/ ; 62472445//National Natural Science Foundation of China/ ; 2023JJ40763//Hunan Provincial Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics/metabolism ; Deep Learning ; Computational Biology/methods ; },
abstract = {MOTIVATION: Accurate prediction of single-guide RNA (sgRNA) activity is crucial for optimizing the CRISPR/Cas9 gene-editing system, as it directly influences the efficiency and accuracy of genome modifications. However, existing prediction methods mainly rely on large-scale experimental data of a single Cas9 variant to construct Cas9 protein (variants)-specific sgRNA activity prediction models, which limits their generalization ability and prediction performance across different Cas9 protein (variants), as well as their scalability to the continuously discovered new variants.

RESULTS: In this study, we proposed PLM-CRISPR, a novel deep learning-based model that leverages protein language models to capture Cas9 protein (variants) representations for cross-variant sgRNA activity prediction. PLM-CRISPR uses tailored feature extraction modules for both sgRNA and protein sequences, incorporating a cross-variant training strategy and a dynamic feature fusion mechanism to effectively model their interactions. Extensive experiments demonstrate that PLM-CRISPR outperforms existing methods across datasets spanning seven Cas9 protein (variants) in three real-world scenarios, demonstrating its superior performance in handling data-scarce situations, including cases with few or no samples for novel variants. Comparative analyses with traditional machine learning and deep learning models further confirm the effectiveness of PLM-CRISPR. Additionally, motif analysis reveals that PLM-CRISPR accurately identifies high-activity sgRNA sequence patterns across diverse Cas9 protein (variants). Overall, PLM-CRISPR provides a robust, scalable, and generalizable solution for sgRNA activity prediction across diverse Cas9 protein (variants).

The source code can be obtained from https://github.com/CSUBioGroup/PLM-CRISPR.},
}

*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Gene Editing/methods
*CRISPR-Associated Protein 9/genetics/metabolism
Deep Learning
Computational Biology/methods

@article {pmid40480611,
year = {2025},
author = {Huang, L and Song, Y and Li, N and Gao, J and Zhang, B and Liu, Z and Zheng, Y},
title = {Deletion of bikaverin and fusarubin biosynthesis gene clusters via CRISPR/Cas9 system in Fusarium fujikuroi and its effect on GA3 biosynthesis.},
journal = {Journal of biotechnology},
volume = {405},
number = {},
pages = {229-237},
doi = {10.1016/j.jbiotec.2025.06.004},
pmid = {40480611},
issn = {1873-4863},
mesh = {*Fusarium/genetics/metabolism ; *Gibberellins/metabolism ; *CRISPR-Cas Systems/genetics ; Multigene Family/genetics ; Gene Deletion ; Fungal Proteins/genetics/metabolism ; Xanthones ; },
abstract = {Gibberellic acid (GA3) is a critical plant hormone with significant agricultural applications, yet its production in Fusarium fujikuroi is constrained by competition for metabolic precursors, particularly acetyl-CoA, which is essential for GA3 biosynthesis. The genome of F. fujikuroi harbors numerous secondary metabolite biosynthetic gene clusters that divert acetyl-CoA away from the GA3 pathway, thereby limiting its yield. To address this challenge, we employed the CRISPR/Cas9 system to delete the bikaverin and fusarubin biosynthesis gene clusters, which are known to compete with GA3 biosynthesis for acetyl-CoA. This genetic intervention resulted in a substantial increase in GA3 production, with the ΔBIKΔFSR strain yielding 31.67 % more GA3 compared to the wild-type strain. Notably, the deletion of these gene clusters not only enhanced GA3 biosynthesis but also improved mycelial growth and carbon assimilation, as evidenced by increased consumption of reducing sugars during fermentation. We further employed qRT-PCR to assess comparative expression levels of genes associated with the glycohydrolysis, glycolysis, and the TCA pathway in engineered strain. Results indicated that removing by-product gene clusters enhances the glycohydrolase system, accelerating carbon assimilation. Given the presence of dozens of secondary metabolite biosynthetic gene clusters in the F. fujikuroi genome, the strategy reported here offers a promising avenue for further enhancing GA3 production by targeting additional non-essential gene clusters.},
}

*Fusarium/genetics/metabolism
*Gibberellins/metabolism
*CRISPR-Cas Systems/genetics
Multigene Family/genetics
Gene Deletion
Fungal Proteins/genetics/metabolism
Xanthones

@article {pmid40472724,
year = {2025},
author = {Shimon, O and Dean, AM and Cohen, S and Moser, AL and Dacso, CC and Gilad, Y and Lonard, DM and O'Malley, BW},
title = {CRISPR-Cas9 engineering of human T regulatory cells - Design and optimization of a manufacturing process.},
journal = {Molecular immunology},
volume = {184},
number = {},
pages = {13-21},
doi = {10.1016/j.molimm.2025.05.019},
pmid = {40472724},
issn = {1872-9142},
mesh = {Humans ; *T-Lymphocytes, Regulatory/immunology ; *CRISPR-Cas Systems/genetics/immunology ; Animals ; *Gene Editing/methods ; Mice ; Immunotherapy, Adoptive/methods ; },
abstract = {Regulatory T cells (Tregs) are a subset of CD4 + T cells that comprise 5-10 % of the total CD4 + T cell population. Tregs, which are critically important for the maintenance of immune tolerance and immune homeostasis, are distinguished from other subtypes of CD4 + T cells by the expression of the transcription factor FOXP3. Because of the centrality to immunoregulation, Tregs have gained increasing attention as promising targets for clinical applications in autoimmune diseases, transplant rejection and graft-versus-host disease (GvHD). However, the essential role of Tregs in the complex network of the immune system implies their targeting as a promising therapeutic approach also in other medical indications, such as neurodegenerative diseases and cancer. Our group recently published a study showing that genetically modified Tregs are capable of clearing solid malignancies in various mice models, including an aggressive triple negative breast cancer (TNBC) and prostate cancer, which provides the impetus to develop an adoptive cell therapy using Steroid Receptor Coactivator 3 (SRC-3) knock out (KO) Tregs. It is well known that isolation, genetic editing and the expansion of Tregs as a homogenous and healthy population present specific technical challenges. In this context, here we outline the development of a process for the production of SRC-3 KO human Tregs (hTregs), which can subsequently be adapted for Current Good Manufacturing Practice (cGMP) settings to facilitate clinical-scale production.},
}

Humans
*T-Lymphocytes, Regulatory/immunology
*CRISPR-Cas Systems/genetics/immunology
Animals
*Gene Editing/methods
Mice
Immunotherapy, Adoptive/methods

@article {pmid40449665,
year = {2025},
author = {Gomes, D and Rodrigues, JL and Scrutton, NS and Rodrigues, LR},
title = {De novo production of prenylnaringenin compounds by a metabolically engineered Escherichia coli.},
journal = {Journal of biotechnology},
volume = {405},
number = {},
pages = {215-228},
doi = {10.1016/j.jbiotec.2025.05.017},
pmid = {40449665},
issn = {1873-4863},
mesh = {*Escherichia coli/genetics/metabolism ; *Metabolic Engineering/methods ; *Flavanones/metabolism ; Dimethylallyltranstransferase/metabolism/genetics ; Biosynthetic Pathways/genetics ; CRISPR-Cas Systems ; Organophosphorus Compounds/metabolism ; Hemiterpenes ; },
abstract = {Prenylnaringenin (PN) compounds, namely 8-prenylnaringenin (8-PN), 3'-prenylnaringenin (3'-PN), and 6-prenylnaringenin (6-PN), are reported to have several interesting bioactivities. This study aimed to validate a biosynthetic pathway for de novo production of PN in Escherichia coli. A previously optimized E. coli chassis capable of efficiently de novo producing naringenin was used to evaluate eleven prenyltransferases (PTs) for the production of PN compounds. As PT reaction requires dimethylallyl pyrophosphate (DMAPP) as extended substrate that has limited availability inside the cells, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 12a (Cas12a) (CRISPR-Cas12a) was used to construct ten boosted DMAPP-E. coli strains. All the PTs, in combination with the naringenin biosynthetic pathway, were tested in these strains. Experiments in 96-well deep well plates identified twelve strains capable of producing PN. E. coli M-PAR-121 with the integration of the 1-deoxy-D-xylulose-5-phosphate synthase (DXS) gene from E. coli (EcDXS) into the lacZ locus of the genome (E. coli M-PAR-121:EcDXS) expressing the soluble aromatic PT from Streptomyces roseochromogenes (CloQ) and the naringenin biosynthetic pathway was selected as the best producer strain. After optimizing the production media in shake flasks, 160.57 µM of 3'-PN, 4.4 µM of 6-PN, and 2.66 µM of 8-PN were obtained. The production was then evaluated at the bioreactor scale and 397.57 µM of 3'-PN (135.33 mg/L) and 25.61 µM of 6-PN (8.72 mg/L) were obtained. To the best of our knowledge, this work represents the first report of de novo production of PN compounds using E. coli as a chassis.},
}

*Escherichia coli/genetics/metabolism
*Metabolic Engineering/methods
*Flavanones/metabolism
Dimethylallyltranstransferase/metabolism/genetics
Biosynthetic Pathways/genetics
CRISPR-Cas Systems
Organophosphorus Compounds/metabolism
Hemiterpenes

@article {pmid40393529,
year = {2025},
author = {Hegeman, CV and Elsharkasy, OM and Driedonks, TAP and Friesen, KRJ and Vader, P and de Jong, OG},
title = {Modulating binding affinity of aptamer-based loading constructs enhances extracellular vesicle-mediated CRISPR/Cas9 delivery.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {384},
number = {},
pages = {113853},
doi = {10.1016/j.jconrel.2025.113853},
pmid = {40393529},
issn = {1873-4995},
mesh = {*Extracellular Vesicles/metabolism ; *CRISPR-Cas Systems ; *Aptamers, Nucleotide/administration & dosage/genetics/metabolism ; Humans ; *Ribonucleoproteins/administration & dosage ; RNA, Guide, CRISPR-Cas Systems/genetics/administration & dosage ; *CRISPR-Associated Protein 9 ; HEK293 Cells ; Gene Editing ; },
abstract = {The CRISPR/Cas9 toolbox consists of modular nucleases that can be employed to efficiently modify genomic sequences with high specificity. However, delivery of the large Cas9-sgRNA ribonucleoprotein (RNP) complexes remains challenging due to their immunogenicity, size, and overall negative charge. An approach to overcome these limitations is the use of extracellular vesicles (EVs) as intracellular delivery vehicles. EVs exhibit the natural ability to carry and deliver RNA and proteins across biological barriers, and can be engineered to load and deliver a variety of biotherapeutic molecules. Previous studies have shown that efficient EV-mediated cargo delivery does not only require active loading strategies, but also benefits from strategies to release cargo from the EV membrane. Here, we load Cas9 RNP complexes into EVs by expressing sgRNAs containing MS2 aptamers (MS2-sgRNAs), alongside Cas9 and a fusion protein of CD63 and tandem MS2 coat proteins (MCPs). We demonstrate that efficient Cas9 RNP delivery can also be facilitated by modulating the binding affinity between MS2 aptamers and the MCPs. To study the effect of altering the binding affinity between the MS2 hairpin and the MCP on Cas9 RNP delivery, various mutations affecting the binding affinity were made in both the interacting MS2-hairpin and the RNA-binding domain of the MCPs. Comparing Cas9 RNP delivery of the modulated MS2-sgRNAs revealed that adapting binding affinity highly affects functional RNP delivery. Mutations resulting in high affinity did not facilitate efficient RNP delivery unless combined with a photo-inducible release strategy, showing that cargo release was a limiting factor in RNP delivery. Mutations that decreased affinity resolved this issue, resulting in Cas9 RNP delivery without the requirement of additional release strategies. However, further decreasing affinity resulted in decreased Cas9 gene-editing efficiency due to decreased levels of Cas9 RNP loading into EVs. A similar effect on functional delivery was seen after modification of the RNA-binding domain of the MCPs. Our results demonstrate that EVs are capable of functional Cas9-sgRNA complex delivery, and that modulation of binding affinity can be used to increase efficient functional delivery with non-covalent loading constructs, without the need for additional engineering strategies for cargo release.},
}

*Extracellular Vesicles/metabolism
*CRISPR-Cas Systems
*Aptamers, Nucleotide/administration & dosage/genetics/metabolism
Humans
*Ribonucleoproteins/administration & dosage
RNA, Guide, CRISPR-Cas Systems/genetics/administration & dosage
*CRISPR-Associated Protein 9
HEK293 Cells
Gene Editing

@article {pmid40644543,
year = {2025},
author = {Cao, D and Zhu, J and Guo, Y and Zhou, Y and Zeng, J and Tu, Y and Zhao, Z and Xie, L and Song, E and Zhu, M and Yin, L},
title = {Dynamically covalent lipid nanoparticles mediate CRISPR-Cas9 genome editing against choroidal neovascularization in mice.},
journal = {Science advances},
volume = {11},
number = {28},
pages = {eadj0006},
doi = {10.1126/sciadv.adj0006},
pmid = {40644543},
issn = {2375-2548},
mesh = {Animals ; *Choroidal Neovascularization/genetics/therapy/pathology ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Mice ; *Nanoparticles/chemistry ; Vascular Endothelial Growth Factor A/genetics/antagonists & inhibitors ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Lipids/chemistry ; Disease Models, Animal ; Humans ; RNA, Messenger/genetics ; Retinal Pigment Epithelium/metabolism ; CRISPR-Associated Protein 9 ; Liposomes ; },
abstract = {As an important modality for choroidal neovascularization (CNV) treatment, intravitreal injection of vascular endothelial growth factor A (VEGFA) inhibitors suffers from undesired response rate, low patient compliance, and ocular damage. Here, dynamically covalent lipid nanoparticles (LNPs) were engineered to mediate VEGFA gene editing and CNV treatment by codelivering Cas9 mRNA (mCas9) and single guide RNA (sgRNA) targeting VEGFA (sgVEGFA). A library of lipidoids bearing iminoboronate ester linkage was developed via facile "one-pot" synthesis, and the top-performing lipidoid-A4B3C7 was formulated into LNP-A4B3C7 with the highest mRNA transfection efficiency. Inside the diseased retinal pigment epithelial cells, LNPs were dissociated upon H2O2-triggered lipidoid degradation, facilitating mRNA/sgRNA release to potentiate the gene editing efficiency. In laser-induced CNV mice, mCas9/sgVEGFA@LNP-A4B3C7 after single intravitreal injection led to pronounced VEGFA disruption and CNV area reduction, outperforming the clinical anti-VEGF drug in eliciting sustained therapeutic effect. This study establishes a robust nonviral platform for mRNA delivery and genome editing and renders a promising strategy for CNV treatment.},
}

Animals
*Choroidal Neovascularization/genetics/therapy/pathology
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Mice
*Nanoparticles/chemistry
Vascular Endothelial Growth Factor A/genetics/antagonists & inhibitors
RNA, Guide, CRISPR-Cas Systems/genetics
*Lipids/chemistry
Disease Models, Animal
Humans
RNA, Messenger/genetics
Retinal Pigment Epithelium/metabolism
CRISPR-Associated Protein 9
Liposomes

@article {pmid40644365,
year = {2025},
author = {Yan, J and Dong, H and Tian, T and Xiao, C and Sun, Y and Gong, J and Xia, Q and Hou, Y},
title = {Doublesex and GATAβ4 synergistically regulate the sex-dimorphic expression of storage protein 1 in Bombyx mori.},
journal = {PLoS genetics},
volume = {21},
number = {7},
pages = {e1011762},
doi = {10.1371/journal.pgen.1011762},
pmid = {40644365},
issn = {1553-7404},
mesh = {Animals ; *Bombyx/genetics ; Female ; Male ; *Insect Proteins/genetics/metabolism ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Sex Characteristics ; *GATA Transcription Factors/genetics/metabolism ; Gene Expression Regulation ; Transcription Factors/genetics ; DNA-Binding Proteins ; },
abstract = {Sexually dimorphic traits are widespread in organisms and are crucial for reproduction and behavior. These traits are typically controlled by sex-specific genes. However, their regulatory mechanisms are complex and incompletely understood. In Bombyx mori, a group of sex-differential storage proteins (SPs) exists, with storage protein 1 (SP1) expressed exclusively in females. In this study, we used the CRISPR/Cas9 system to knock out the doublesex gene and found that SP1 expression was sharply upregulated in male doublesex mutants and downregulated in female doublesex mutants, which suggests that doublesex is a key factor in the sex-differential expression of SP1. Then, we revealed that the female-specific doublesex isoform (dsxF) bound to and activated the SP1 promoter more strongly than the male-specific isoform (dsxM). Meanwhile, a transcription factor named GATAβ4 was found to be involved in the regulation by doublesex. Overexpression of GATAβ4 in Bombyx mori larvae affected adult reproductive behavior and dramatically upregulated SP1 expression in males. Furthermore, GATAβ4 interacted with both dsxF and dsxM, promoting nuclear translocation of dsxM, which in turn inhibited GATAβ4 binding to the SP1 promoter. In total, we found that dsxM did not directly repress SP1 expression in males but instead cooperated with other transcription factors to regulate downstream gene expression. These findings provide new insights into the regulation of sex-specific genes and the mechanisms controlling dimorphic traits.},
}

Animals
*Bombyx/genetics
Female
Male
*Insect Proteins/genetics/metabolism
Promoter Regions, Genetic
CRISPR-Cas Systems
Sex Characteristics
*GATA Transcription Factors/genetics/metabolism
Gene Expression Regulation
Transcription Factors/genetics
DNA-Binding Proteins

@article {pmid40643795,
year = {2025},
author = {Kim, YM and Na, HJ and Kwon, DH and Lee, JH and Park, BM and Lee, S and Nam, TW and Park, MY and Park, SH and Kim, SJ and Choi, B and Lee, HW},
title = {Generation of NOD SCID mice with near-complete deletions of Il2rg and Prkdc for human cancer and HSC engraftment.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {35},
pmid = {40643795},
issn = {1573-9368},
support = {RS-2024-00359509//National Research Foundation of Republic of Korea and Ministry of Science and ICT/ ; RS-2023-00224201//National Research Foundation of Republic of Korea and Ministry of Science and ICT/ ; RS-2023-00224201//National Research Foundation of Republic of Korea and Ministry of Science and ICT/ ; RS-2023-00224201//National Research Foundation of Republic of Korea and Ministry of Science and ICT/ ; RS-2023-00224201//National Research Foundation of Republic of Korea and Ministry of Science and ICT/ ; 20012378//the Ministry of Trade, Industry & Energy (MOTIE, Korea)/ ; 20012378//the Ministry of Trade, Industry & Energy (MOTIE, Korea)/ ; 20012378//the Ministry of Trade, Industry & Energy (MOTIE, Korea)/ ; 20012378//the Ministry of Trade, Industry & Energy (MOTIE, Korea)/ ; 20012378//the Ministry of Trade, Industry & Energy (MOTIE, Korea)/ ; 20012378//the Ministry of Trade, Industry & Energy (MOTIE, Korea)/ ; S3142545//the Ministry of SMEs and Startups (MSS, Korea)/ ; S3142545//the Ministry of SMEs and Startups (MSS, Korea)/ ; S3142545//the Ministry of SMEs and Startups (MSS, Korea)/ ; S3142545//the Ministry of SMEs and Startups (MSS, Korea)/ ; S3142545//the Ministry of SMEs and Startups (MSS, Korea)/ ; },
mesh = {Animals ; Humans ; Mice ; *Interleukin Receptor Common gamma Subunit/genetics ; Mice, Inbred NOD ; Mice, SCID ; CRISPR-Cas Systems ; *DNA-Activated Protein Kinase/genetics ; *Neoplasms/genetics/therapy/pathology ; *Hematopoietic Stem Cell Transplantation ; Disease Models, Animal ; Mice, Knockout ; },
abstract = {Immunodeficient mouse models are invaluable tools for preclinical research, particularly for cancer therapies and studies of the human immune system. Notably, strains with combined Prkdc (scid) and Il2rg (null) mutations-such as NOG and NSG mice- are widely used due to their profound immunodeficiency, allowing efficient engraftment of various human cells. However, these models were generated by disrupting the Il2rg gene through replacement with a neomycin resistance (Neo) cassette in embryonic stem cells. Incomplete excision of this cassette can inadvertently alter the expression of neighboring genes, thereby introducing potential confounding variables. In addition, they may still express mutant mRNAs that escape nonsense-mediated decay (NMD) and/or produce truncated proteins with residual activity, potentially compromising the interpretation of experimental outcomes. To address this, we developed the N2G mouse strain (NOD-2-Genes KO) where almost all genomic loci of both Prkdc and Il2rg genes are deleted via CRISPR/Cas9 genome editing. N2G mice exhibited tumor growth comparable to NOG mice following the transplantation with several human cancer cell lines. Moreover, human CD34[+] cord blood (CB) cells engrafted into N2G mice showed robust reconstitution of human immune cells, especially T cells in peripheral blood, spleen and bone marrow, compared to NSG mice. These results suggest that N2G mice, lacking residual mutant mRNA and the exogenous Neo resistant gene, offer an advanced model for preclinical studies.},
}

Animals
Humans
Mice
*Interleukin Receptor Common gamma Subunit/genetics
Mice, Inbred NOD
Mice, SCID
CRISPR-Cas Systems
*DNA-Activated Protein Kinase/genetics
*Neoplasms/genetics/therapy/pathology
*Hematopoietic Stem Cell Transplantation
Disease Models, Animal
Mice, Knockout

@article {pmid40643739,
year = {2025},
author = {Friberg, M and Sharma, S and Sitbon, F and Andersson, M and Hofvander, P},
title = {Modifying the potato tuber storage protein patatin targeting improved thermal stability.},
journal = {Planta},
volume = {262},
number = {2},
pages = {46},
pmid = {40643739},
issn = {1432-2048},
support = {2018-01846//Svenska Forskningsrådet Formas/ ; "Breeding of starch potatoes"//SLU Grogrund/ ; "Potato tuber//C4F/ ; sink//C4F/ ; starch development"//C4F/ ; },
mesh = {*Solanum tuberosum/genetics/metabolism ; *Plant Proteins/genetics/metabolism/chemistry ; *Plant Tubers/genetics/metabolism ; Gene Editing/methods ; Protein Stability ; CRISPR-Cas Systems ; Multigene Family ; Carboxylic Ester Hydrolases ; },
abstract = {Gene editing of the patatin gene cluster using a single-guide RNA sequence consistently modifies over 10% of the targeted genes in modified individuals. Patatins have gained recent attention, as a group of highly nutritious proteins with excellent functional properties. Some techniques have been suggested for industrial-scale patatin purification, mostly as a by-product from potato starch processing. The purification process has proved to be a challenge due to the low thermostability of patatins, especially under acidic conditions. One strategy to make patatin more accessible for extraction would be to stabilize the protein structure through the introduction of point mutations. Here, we show that the tuber expression of patatin genes is dominated by a few genes from the extended gene family, most of which were predicted to be catalytically inactive. We have further evaluated the suitability of the patatin gene cluster as a target for clustered regularly interspaced repeat (CRISPR)/Cas9-based mutagenesis. In the mutation study, we show that targeting using a single single-stranded guide RNA (sgRNA) can lead to mutations in over 10% of all alleles. Finally, four patatin variants with amino acid substitutions were designed based on in silico analysis of patatin protein structure. These modified patatins were then heterologously expressed in bacteria and evaluated for increased thermostability. While none of the mutant proteins performed better than a wild-type variant, with regard to their thermal properties, one candidate proved to be less sensitive to shifting pH, making it an interesting candidate for further optimizations.},
}

*Solanum tuberosum/genetics/metabolism
*Plant Proteins/genetics/metabolism/chemistry
*Plant Tubers/genetics/metabolism
Gene Editing/methods
Protein Stability
CRISPR-Cas Systems
Multigene Family
Carboxylic Ester Hydrolases

@article {pmid40642869,
year = {2025},
author = {Li, Y and Tang, Y and Li, X and Høyland-Kroghsbo, NM and Ingmer, H and Jiao, X and Li, Q},
title = {Quorum Sensing Inhibits Type III-A CRISPR-Cas System Activity Through Repressing Positive Regulators SarA and ArcR in Staphylococcus Aureus.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e06049},
doi = {10.1002/advs.202506049},
pmid = {40642869},
issn = {2198-3844},
support = {2024YFE0198800//National Key Research and Development Program of China/ ; 2023YFD1800503//National Key Research and Development Program of China/ ; 2022YFC2604200//National Key Research and Development Program of China/ ; BK20190883//Natural Science Foundation of Jiangsu Province/ ; 32072821//National Natural Science Foundation of China/ ; 31730094//National Natural Science Foundation of China/ ; KYCX21_3267//Postgraduate Research & Practice Innovation Program of Jiangsu Province/ ; R264-2017-3936//Lundbeck Foundation/ ; 2024ZB284//Jiangsu Funding Program for Excellent Postdoctoral Talent/ ; JX103SYL2024060102//Open Co-operation Project of Center for Global Health/ ; //Priority Academic Program Development of Jiangsu Higher Education Institutions/ ; },
abstract = {CRISPR-Cas is an adaptive immune system that protects prokaryotes from the invasion of foreign genetic elements. The components and immunity mechanisms of CRISPR-Cas have been extensively studied, but the regulation of this system in Staphylococci remains unclear. Here, it is shown that the cell-cell communication, known as quorum sensing (QS), inhibits the expression and activity of the type III-A CRISPR-Cas system in S. aureus. The QS regulator, AgrA, directly binds to the promoters of two transcriptional regulators encoding the genes sarA and arcR to inhibit their expression. However, both SarA and ArcR act as positive regulators that promote the transcription of cas genes by directly binding to a novel promoter Pcas. Furthermore, the Pcas of 300 bp located in cas1 displays as a critical regulatory node to initiate the transcription of cas10 and csm3. Our data reveal a new regulatory mechanism for QS-mediated repression of the Type III-A CRISPR-Cas system, which may allow S. aureus to acquire foreign genetic elements encoding antibiotic resistance or virulence factors specifically at high cell density.},
}

@article {pmid40641638,
year = {2025},
author = {Hua, G and He, C and Zuo, E},
title = {SuperDecode: A versatile toolkit for mutation analysis in genome editing.},
journal = {aBIOTECH},
volume = {6},
number = {2},
pages = {377-380},
pmid = {40641638},
issn = {2662-1738},
abstract = {The CRISPR-Cas system has revolutionized modern life sciences, enabling groundbreaking applications ranging from functional genomics to therapeutic development. Despite its transformative potential, significant technical limitations persist in current computational tools for quantifying editing efficiency - particularly concerning data processing capabilities, analytical throughput, and operational flexibility. This research presents SuperDecode, a novel computational framework designed to address these methodological constraints. The SuperDecode offers key advantages, including local processing capabilities, large-size sequencing files, batch-processing, and diversified operational functions.},
}

@article {pmid40641119,
year = {2025},
author = {Du, J and Gong, X and Huang, R and Zheng, B and Chen, C and Yang, Z},
title = {Harnessing CRISPR/Cas9 to overcome targeted therapy resistance in non‑small cell lung cancer: Advances and challenges (Review).},
journal = {Oncology reports},
volume = {54},
number = {3},
pages = {},
doi = {10.3892/or.2025.8944},
pmid = {40641119},
issn = {1791-2431},
mesh = {Humans ; *Carcinoma, Non-Small-Cell Lung/genetics/drug therapy/therapy ; *CRISPR-Cas Systems/genetics ; *Drug Resistance, Neoplasm/genetics ; *Lung Neoplasms/genetics/drug therapy/therapy ; *Gene Editing/methods ; Molecular Targeted Therapy/methods ; Genetic Therapy/methods ; },
abstract = {Targeted therapy has markedly improved outcomes for patients with non‑small cell lung cancer (NSCLC). However, the emergence of drug resistance remains a major clinical challenge, limiting long‑term treatment efficacy. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, a revolutionary gene‑editing technology, offers precise and efficient genetic modifications, providing new insights into the mechanisms of drug resistance in NSCLC. The present review explored the application of CRISPR/Cas9 in overcoming resistance associated with key oncogenic drivers, including EGFR, KRAS, ALK, ROS1, MET and BRAF. It summarized recent advances in CRISPR‑based strategies to reverse resistance, enhance targeted therapy effectiveness and develop potential therapeutic interventions. Additionally, it discussed current limitations, including off‑target effects, delivery challenges and ethical concerns, while highlighting future directions for clinical translation. Using CRISPR/Cas9 technology may pave the way for novel, personalized treatment approaches in NSCLC, ultimately improving patient outcome.},
}

Humans
*Carcinoma, Non-Small-Cell Lung/genetics/drug therapy/therapy
*CRISPR-Cas Systems/genetics
*Drug Resistance, Neoplasm/genetics
*Lung Neoplasms/genetics/drug therapy/therapy
*Gene Editing/methods
Molecular Targeted Therapy/methods
Genetic Therapy/methods

@article {pmid40457715,
year = {2025},
author = {Bourel, C and Souza-Fonseca-Guimaraes, F and Lesage, S},
title = {Highlights of 2024: unleashing the power of NK cells-cancer's worst nightmare.},
journal = {Immunology and cell biology},
volume = {103},
number = {6},
pages = {526-529},
doi = {10.1111/imcb.70037},
pmid = {40457715},
issn = {1440-1711},
mesh = {Humans ; *Killer Cells, Natural/immunology ; *Neoplasms/therapy/immunology ; *Immunotherapy, Adoptive/methods ; Animals ; CRISPR-Cas Systems ; Cytokines/metabolism ; Lymphocyte Activation ; Cytotoxicity, Immunologic ; },
abstract = {In this article for the Highlights of 2024 Series, we discuss strategies to enhance NK cell-based cancer therapies. These include (1) cytokine expression on bacterial membranes to boost NK cell activation in tumors, (2) optimizing CAR-NK cell manufacturing for improved efficacy, (3) using CRISPR-Cas9 to identify and target inhibitory genes, and (4) using tetraspecific engagers to enhance cytotoxicity and cytokine memory-like NK cells strengthening anti-tumor responses. This year's progress holds much promise for cancer treatments exploiting NK cells.},
}

Humans
*Killer Cells, Natural/immunology
*Neoplasms/therapy/immunology
*Immunotherapy, Adoptive/methods
Animals
CRISPR-Cas Systems
Cytokines/metabolism
Lymphocyte Activation
Cytotoxicity, Immunologic

@article {pmid40640739,
year = {2025},
author = {Dai, P and Huang, T and Ye, X and Mi, S and Zhang, J and Luo, X and Hu, D and Zhang, H},
title = {K1 Klebsiella pneumoniae is more conserved than K2 for both virulence plasmid and chromosome.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {652},
pmid = {40640739},
issn = {1471-2164},
support = {2023KY1326//Zhejiang Provincial Health Commission/ ; 82172332//National Natural Science Foundation of China/ ; GSWS2019039//Gusu Health Youth Talent of Suzhou/ ; SKY2021007//Science and Technology Program of Suzhou/ ; XKTJ-TD202001//Discipline Construction of the Second Affiliated Hospital of Soochow University/ ; },
abstract = {OBJECTIVE: To illustrate the differences between K1 and K2 Klebsiella pneumoniae strains.

METHODS: Totally 68 K1 and 99 K2 K. pneumoniae strains from GenBank were analyzed for virulence genes, sequence types (STs), restriction-modification (R-M) systems, and clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems. Phylogenetic trees of the virulence plasmids and chromosomes in the strains were built using kSNP4.

RESULTS: Virulence genes peg-344, allS, p-rmpA, p-rmpA2, c-rmpA, iroN, and iucA were more prevalent in K1 strains than K2. K1 strains were categorized into 7 STs with 79.41% being ST23 while K2 strains were categorized into 14 STs with 38.38% being ST14. K1 strains showed higher rates of CRISPR-Cas systems than K2 while lower rates of Type I and II R-M systems were found in K1 strains than K2. More rates of virulence plasmids (52/68 vs. 24/99) were found in K1 strains than K2. Based upon the phylogenetic tree of virulence plasmids, 46 in K1 strains belonged to the same clade while 11 and 7 virulence plasmids in K2 strains constituted the 2 major clades. For the chromosomes, 61 K1 strains belonged to the same clade while 99 K2 strains could be categorized into 4 major clades.

CONCLUSIONS: K1 K. pneumoniae strains are more conserved than K2 for both virulence plasmids and chromosomes. K1 strains are deficient in R-M systems but rich in CRISPR-Cas, which is contrary to K2.},
}

@article {pmid40640156,
year = {2025},
author = {Imburgia, C and Organick, L and Zhang, K and Cardozo, N and McBride, J and Bee, C and Wilde, D and Roote, G and Jorgensen, S and Ward, D and Anderson, C and Strauss, K and Ceze, L and Nivala, J},
title = {Random access and semantic search in DNA data storage enabled by Cas9 and machine-guided design.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6388},
pmid = {40640156},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/genetics ; *Information Storage and Retrieval/methods ; *CRISPR-Associated Protein 9/metabolism/genetics ; Machine Learning ; Semantics ; Sequence Analysis, DNA/methods ; },
abstract = {DNA is a promising medium for digital data storage due to its exceptional data density and longevity. Practical DNA-based storage systems require selective data retrieval to minimize decoding time and costs. In this work, we introduce CRISPR-Cas9 as a user-friendly tool for multiplexed, low-latency molecular data extraction. We first present a one-pot, multiplexed random access method in which specific data files are selectively cleaved using a CRISPR-Cas9 addressing system and then sequenced via nanopore technology. This approach was validated on a pool of 1.6 million DNA sequences, comprising 25 unique data files. We then developed a molecular similarity-search approach combining machine learning with Cas9-based retrieval. Using a deep neural network, we mapped a database of 1.74 million images into a reduced-dimensional embedding, encoding each embedding as a Cas9 target sequence. These target sequences act as molecular addresses, capturing clusters of semantically related images. By leveraging Cas9's off-target cleavage activity, query sequences cleave both exact and closely related targets, enabling high-fidelity retrieval of molecular addresses corresponding to in silico image clusters similar to the query. These approaches move towards addressing key challenges in molecular data retrieval by offering simplified, rapid isothermal protocols and new DNA data access capabilities.},
}

*CRISPR-Cas Systems/genetics
*DNA/genetics
*Information Storage and Retrieval/methods
*CRISPR-Associated Protein 9/metabolism/genetics
Machine Learning
Semantics
Sequence Analysis, DNA/methods

@article {pmid40640155,
year = {2025},
author = {Wang, J and Ye, X and Liu, Y and Li, W and Zhang, X and Zhang, W and Yi, C and Liu, C},
title = {Regulating cleavage activity and enabling microRNA detection with split sgRNA in Cas12b.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {6392},
pmid = {40640155},
issn = {2041-1723},
support = {22307150//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *MicroRNAs/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Herpesvirus 4, Human/genetics/isolation & purification ; Colorectal Neoplasms/blood/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; HEK293 Cells ; },
abstract = {The CRISPR-Cas12b system has revolutionized molecular diagnostics, yet its reliance on single guide RNAs (sgRNAs) exceeding 100 nt limits precise regulation and applications. We present a split sgRNA strategy for Cas12b, utilizing universal components with customizable Spacer to detect various nucleic acid targets by simply replacing Spacer. Glyoxal labeling of the universal split direct repeat (DR) region represses Cas12b activity, which is restored by elevated temperatures or prolonged incubation, enabling dynamic regulation. Incorporating a photo-cleavable linker into the DR allows UV-mediated modulation, ensuring compatibility with recombinase polymerase amplification. Successful detection of Epstein-Barr virus in clinical plasma samples matched the sensitivity of traditional qPCR. Importantly, microRNAs can replace the Spacer, enabling direct detection without reverse transcription or amplification. Supported by evidence from cultured cells and plasma from healthy individuals and colorectal cancer patients, this method yields consistent results with RT-qPCR while simplifying protocols. This split strategy enhances Cas12b systems, offering a promising approach for clinical nucleic acid analysis.},
}

Humans
*MicroRNAs/genetics/metabolism
*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Herpesvirus 4, Human/genetics/isolation & purification
Colorectal Neoplasms/blood/genetics
*CRISPR-Associated Proteins/genetics/metabolism
HEK293 Cells

@article {pmid40639408,
year = {2025},
author = {Ille, K and Melzer, S},
title = {Efficient and versatile rapeseed transformation for new breeding technologies.},
journal = {The Plant journal : for cell and molecular biology},
volume = {123},
number = {1},
pages = {e70330},
doi = {10.1111/tpj.70330},
pmid = {40639408},
issn = {1365-313X},
support = {Enable FKZ: 031B0801D//Bundesministerium für Bildung und Forschung/ ; Epibrass FKZ:031B1223B//Bundesministerium für Bildung und Forschung/ ; },
mesh = {*Brassica napus/genetics ; *Transformation, Genetic ; Plants, Genetically Modified/genetics ; *Plant Breeding/methods ; CRISPR-Cas Systems ; Gene Editing ; Plant Proteins/genetics/metabolism ; Phenotype ; },
abstract = {Many gene functions are widely studied and understood in Arabidopsis; however, the lack of efficient transformation systems often limits the application and verification of this knowledge in crop plants. Brassica napus L., a member of the Brassicaceae family, is usually transformed by Agrobacterium-mediated hypocotyl transformation, but not all growth types are equally amenable to transformation. In particular, winter rapeseed, which requires vernalization to initiate flowering, is recalcitrant to in vitro regeneration and transformation. The analysis of gene functions in rapeseed is further complicated by the allotetraploid nature of its genome and the genome triplication within the Brassica genus, which has led to the presence of a large number of gene homologs for each Arabidopsis ortholog. We have established a transformation method that facilitates the regeneration of winter rapeseed by using the WUSCHEL gene from Beta vulgaris. This allowed us to efficiently transform a winter and spring rapeseed genotype in small-scale experiments. As proof of principle, we targeted BnCLV3 and BnSPL9/15 with CRISPR/Cas9 and showed that entire gene families are effectively edited using this transformation protocol. This allowed us to simultaneously study many redundantly acting homologous genes in rapeseed. We observed mutant phenotypes for BnCLV3 and BnSPL9/15 in primary transformants, indicating that biallelic knockouts were obtained for up to eight genes. This allowed an initial phenotypic characterization to be performed already a few months after starting the experiment.},
}

*Brassica napus/genetics
*Transformation, Genetic
Plants, Genetically Modified/genetics
*Plant Breeding/methods
CRISPR-Cas Systems
Gene Editing
Plant Proteins/genetics/metabolism
Phenotype

@article {pmid40637794,
year = {2025},
author = {Hajian, M and Pirali, A and Moghaddam, SHH and Moradi-Hajidavaloo, R and Varnosfaderani, SR and Jozi, M and Izadi, T and Tanhaie-Vash, N and Kues, W and Nasr-Esfahani, MH and Jafarpour, F and Eghbalsaied, S},
title = {Efficient gene editing of BMP15, GDF9, and MSTN-but not the imprinted CLPG gene-in goat embryos via electrotransfection and handmade cloning.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {150},
pmid = {40637794},
issn = {1438-7948},
support = {CT1402091612//Iranian Vice President for Science/ ; Ref 3.4-IRN-1191261-GF-E//Alexander von Humboldt-Stiftung/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Myostatin/genetics ; *Bone Morphogenetic Protein 15/genetics ; *Goats/genetics/embryology ; *Growth Differentiation Factor 9/genetics/metabolism ; CRISPR-Cas Systems ; Electroporation ; Nuclear Transfer Techniques ; Genomic Imprinting ; Female ; Cloning, Organism ; Embryo, Mammalian/metabolism ; },
abstract = {CRISPR/Cas9 technology represents a powerful tool for advancing livestock breeding by enabling precise, on-target gene edits without the genomic mixing associated with traditional introgression methods. In this study, we employed a dual gRNA-based CRISPR/Cas9 strategy to induce targeted deletions and indel mutations in both reproductive and growth-related genes. These included the metacentric genes bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), which are associated with increased ovulation rate and litter size, as well as the telomeric genes myostatin (MSTN) and callipyge (CLPG), which are linked to muscle development and enhanced meat production. We employed an optimized electrotransfection protocol consisting of 10-20 µg of each plasmid DNA, 250 µL OptiMEM-GlutaMAX, and one million goat fibroblast cells. The electroporation was performed using a Bio-Rad system in a 4-mm cuvette, with two 10-millisecond pulses at 270 volts, separated by a 10-second interval. This protocol enabled efficient genome editing of goat embryonic fibroblast cells, which were subsequently used to generate cloned embryos via handmade somatic cell nuclear transfer (SCNT), involving manual enucleation and cell-oocyte fusion steps. Sequencing revealed high mutation rates (78-97%) and a predominance of biallelic edits in BMP15, GDF9, and MSTN. Notably, MSTN gRNAs with a 7-bp overlapping sequence at their 3' ends showed a high editing efficiency. In contrast, the imprinted CLPG gene exhibited a significantly lower mutation rate (~ 30%), likely due to epigenetic constraints. While overall mutation rates did not differ significantly between metacentric and telomeric genes, on-target deletions were more frequent in metacentric genes (43%) than in telomeric ones (20%). Embryo development rates from gene-edited cells were comparable to those from non-edited controls. These findings underscore the utility of combining electrotransfection with SCNT for efficient editing of non-imprinted genes and highlight the need for improved strategies to overcome barriers in editing imprinted loci.},
}

Animals
*Gene Editing/methods
*Myostatin/genetics
*Bone Morphogenetic Protein 15/genetics
*Goats/genetics/embryology
*Growth Differentiation Factor 9/genetics/metabolism
CRISPR-Cas Systems
Electroporation
Nuclear Transfer Techniques
Genomic Imprinting
Female
Cloning, Organism
Embryo, Mammalian/metabolism

@article {pmid40637230,
year = {2025},
author = {Amistadi, S and Fontana, L and Magnoni, C and Felix, T and Charvin, MK and Martinucci, P and Gautier, C and Greau, L and Bessières, B and Antoniou, P and Romano, O and Allemand, E and Mussolino, C and Miccio, A},
title = {Dissecting the epigenetic regulation of the fetal hemoglobin genes to unravel a novel therapeutic approach for β-hemoglobinopathies.},
journal = {Nucleic acids research},
volume = {53},
number = {13},
pages = {},
doi = {10.1093/nar/gkaf637},
pmid = {40637230},
issn = {1362-4962},
support = {//French National Research Agency/ ; ANR-10-IAHU-01//Agence Nationale de la Recherches/ ; ANR-21-CE18-0066-01//Agence Nationale de la Recherches/ ; CA21113//European Cooperation in Science and Technology/ ; //Institut Imagine/ ; PLP202110014595//Fondation pour la Recherche Médicale/ ; },
mesh = {Humans ; *Fetal Hemoglobin/genetics/metabolism ; *Epigenesis, Genetic ; DNA Methylation ; gamma-Globins/genetics ; *beta-Globins/genetics ; *Anemia, Sickle Cell/genetics/therapy ; Promoter Regions, Genetic ; Gene Editing/methods ; *Hemoglobinopathies/genetics/therapy ; CRISPR-Cas Systems ; Erythroid Cells/metabolism ; },
abstract = {Beta-hemoglobinopathies are severe genetic diseases caused by mutations affecting the production of the adult β-globin chain. The clinical severity is mitigated by the co-inheritance of mutations that reactivate the production of the fetal β-like γ-globin in adults. However, the epigenetic mechanisms underlying the adult-to-fetal hemoglobin (HbA-to-HbF) switching are still not fully understood. Here, we used epigenome editing technologies to dissect the molecular mechanisms underlying γ- and β-globin gene regulation and to develop novel potential therapeutics for β-hemoglobinopathies. Targeted removal of DNA methylation by dCas9-Tet1 (alone or together with the deposition of histone acetylation by CBP-dCas9) at the fetal promoters led to efficient and durable γ-globin reactivation, demonstrating that DNA methylation is a driver for HbF repression. This strategy, characterized by high specificity and a good safety profile, led to a substantial correction of the pathological phenotype in erythroid cells from patients with sickle cell disease.},
}

Humans
*Fetal Hemoglobin/genetics/metabolism
*Epigenesis, Genetic
DNA Methylation
gamma-Globins/genetics
*beta-Globins/genetics
*Anemia, Sickle Cell/genetics/therapy
Promoter Regions, Genetic
Gene Editing/methods
*Hemoglobinopathies/genetics/therapy
CRISPR-Cas Systems
Erythroid Cells/metabolism

@article {pmid40636259,
year = {2025},
author = {Zheng, H and Wang, B and Dong, X and Wu, J and Shi, L and Zhang, J and Chen, H and Zhou, A},
title = {SIRT7 deletion inhibits Glaesserella parasuis-mediated inflammatory responses in porcine alveolar macrophages.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1589199},
pmid = {40636259},
issn = {2235-2988},
mesh = {Animals ; Swine ; *Sirtuins/genetics/metabolism/deficiency ; *Macrophages, Alveolar/microbiology/immunology ; *Haemophilus parasuis/immunology/pathogenicity ; *Inflammation/microbiology ; *Haemophilus Infections/veterinary/immunology/microbiology/pathology ; Signal Transduction ; *Swine Diseases/microbiology/immunology ; Gene Deletion ; CRISPR-Cas Systems ; },
abstract = {Glaesserella parasuis (GPS) infection causes severe inflammatory disorder, resulting in lung injury. SIRT7 is an NAD[+]-dependent deacetylase known to regulate inflammatory responses, but its role in GPS infection remains unclear. Here we found that GPS infection increased SIRT7 expression and induced inflammatory responses. Deficiency of SIRT7 by CRISPR/Cas9 technology significantly inhibited GPS-induced cytopathic effects and inflammatory responses. In addition, RNA-seq analysis showed that differentially expressed genes(DEGs) induced by SIRT7 deficiency were enriched in biological processes such as cell proliferation, actin cytoskeleton formation, lipid synthesis, protein kinase activation regulation, and GTPase activity regulation. Functional enrichment analysis further indicated the involvement of these DEGs in tight junction pathway, PI3K-Akt signaling pathway, actin cytoskeleton regulation, cGMP-PKG signaling pathway, Hippo signaling pathway, and TNF signaling pathway. Finally, we identified some hub genes (GNAI3, GNAI1, JAK1, NDUFS8, CYC1) related to oxidative phosphorylation. In summary, our results demonstrate that SIRT7 is pivotal for GPS-induced inflammatory responses, which represents a promising target resistant to GPS infection.},
}

Animals
Swine
*Sirtuins/genetics/metabolism/deficiency
*Macrophages, Alveolar/microbiology/immunology
*Haemophilus parasuis/immunology/pathogenicity
*Inflammation/microbiology
*Haemophilus Infections/veterinary/immunology/microbiology/pathology
Signal Transduction
*Swine Diseases/microbiology/immunology
Gene Deletion
CRISPR-Cas Systems

@article {pmid40635088,
year = {2025},
author = {Wulff, JP and Laminack, RK and Scott, MJ},
title = {Genetic and behavioral analyses suggest that larval and adult stages of Lucilia cuprina employ different sensory systems to detect rotten beef.},
journal = {Parasites & vectors},
volume = {18},
number = {1},
pages = {270},
pmid = {40635088},
issn = {1756-3305},
support = {DEB-2030345//National Science Foundation/ ; },
mesh = {Animals ; Larva/genetics/physiology/growth & development ; Female ; Cattle ; Receptors, Odorant/genetics ; *Calliphoridae/genetics/physiology/growth & development ; Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Lucilia cuprina ; },
abstract = {BACKGROUND: The blowfly Lucilia cuprina is a destructive parasite of sheep that causes flystrike or myiasis. Larvae consume the animal's living flesh, producing large wounds that can lead to death. The main aim of this study was to identify genes that may play important roles in the behavior and physiology of L. cuprina larvae.

METHODS: An RNA-Seq analysis of RNA from whole larvae at different developmental stages and third-instar head and gut tissues was used to identify sensory receptors and other genes relevant to the physiology of L. cuprina larvae. In addition, CRISPR/Cas9 gene editing was used to obtain a loss-of-function mutation for the L. cuprina odorant coreceptor gene (LcupOrco). The response of mutant larvae and adult females to fresh and rotten meat at different temperatures was evaluated.

RESULTS: The RNA-Seq analysis suggested that odorant (OR), gustatory, ionotropic, and Pickpocket receptors may not play a central role in the L. cuprina larval sensory signaling and digestive systems. Rather, ATP-binding cassettes (ABCs) were highly enriched in head and gut RNA, and odorant-binding proteins (OBPs) only in the head. To confirm that ORs are not essential for larval detection of rotten beef, diet-choice assays were performed including larvae and adults homozygous for a null mutation in LcupOrco. While the attraction of adult females to rotten beef was disrupted, LcupOrco mutant larvae showed no change in diet preference.

CONCLUSIONS: The expression pattern of the ABC and OBP gene families suggests a central role in the sensory system of the L. cuprina larva for these receptors. Behavioral assays showed that ORs are essential for the adult female response to rotten beef, but not for larval behavior. These findings are consistent with high levels of expression of LcupOrco in the adult female antenna but very low expression in larvae.},
}

Animals
Larva/genetics/physiology/growth & development
Female
Cattle
Receptors, Odorant/genetics
*Calliphoridae/genetics/physiology/growth & development
Insect Proteins/genetics/metabolism
CRISPR-Cas Systems
Lucilia cuprina

@article {pmid40634958,
year = {2025},
author = {Tian, Y and Bao, X and Lei, S and Huang, Y and Wang, X and Tu, Y and He, Q and Zhang, F and Xu, H and Ashrafizadeh, M and Sethi, G and Wang, F and Zeng, Z},
title = {In vivo CRISPR screening identifies POU3F3 as a novel regulator of ferroptosis resistance in hepatocellular carcinoma via retinoic acid signaling.},
journal = {Cell communication and signaling : CCS},
volume = {23},
number = {1},
pages = {329},
pmid = {40634958},
issn = {1478-811X},
support = {32260169//National Natural Science Foundation of China/ ; 82303903//National Natural Science Foundation of China/ ; BSH-Q-2023-06//the Affiliated Hospital of Guizhou Medical University Postdoctoral fellow Start-up fund/ ; },
mesh = {*Ferroptosis/drug effects/genetics ; Humans ; *Carcinoma, Hepatocellular/genetics/pathology/metabolism/drug therapy ; *Liver Neoplasms/genetics/pathology/metabolism/drug therapy ; Animals ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Signal Transduction/drug effects ; *Tretinoin/metabolism ; Sorafenib/pharmacology ; Mice ; *Octamer Transcription Factor-3/metabolism/genetics ; Cell Line, Tumor ; Mice, Nude ; *CRISPR-Cas Systems/genetics ; },
abstract = {BACKGROUND: Sorafenib, a ferroptosis agonist, is a first-line treatment for advanced hepatocellular carcinoma (HCC). However, its clinical efficacy is limited due to drug resistance, resulting in modest improvements in patient survival. Hence, the present study has been designed to identify critical molecular targets associated with sorafenib resistance and investigate the potential inhibitors in overcoming this therapeutic challenge.

METHODS: In vivo whole-genome CRISPR/Cas9 library screens were conducted to identify resistance factors to ferroptosis agonists, such as RSL3 and sorafenib, in HCC. The effects and underlying molecular mechanisms of these resistance factors were investigated in HCC cells using ferroptosis detection assays, xenograft tumor models, chromatin immunoprecipitation (ChIP), and dual-luciferase reporter assays. Potential inhibitors targeting these factors were evaluated through computer-aided virtual screening, molecular dynamics simulations, surface plasmon resonance analysis, and functional evaluations.

RESULTS: A retinoic acid metabolism gene cluster, including ADH4, ALDH1A1, ALDH1A3, FABP5, RBP1, and RDH10, was found demonstrating upregulation in HCC cells treated with ferroptosis agonist, sorafenib. This gene cluster contributes to the ferroptosis resistance by producing the strong reducing agent retinoic acid. The transcription factor POU3F3 was identified as a key regulator for the retinoic acid metabolism gene cluster, which simultaneously binds to their promoters, increasing their transcription and promoting retinoic acid production. Knockdown of POU3F3 significantly enhanced the pro-ferroptotic and inhibitory effects of sorafenib on HCC cells by suppressing retinoic acid metabolism. Furthermore, rosarin was identified as a POU3F3 inhibitor, with an equilibrium dissociation constant of 7.57 µM, and demonstrated a synergistic effect with sorafenib against HCC cells both in vitro and in vivo.

CONCLUSIONS: According to the results, POU3F3 acts as a protective regulator against sorafenib-induced ferroptosis in HCC cells by enhancing the transcription of multiple retinoic acid metabolism genes and promoting retinoic acid production. The POU3F3 inhibitor, rosarin, shows potential as an ideal candidate for overcoming sorafenib resistance in HCC.},
}

*Ferroptosis/drug effects/genetics
Humans
*Carcinoma, Hepatocellular/genetics/pathology/metabolism/drug therapy
*Liver Neoplasms/genetics/pathology/metabolism/drug therapy
Animals
*Drug Resistance, Neoplasm/genetics/drug effects
*Signal Transduction/drug effects
*Tretinoin/metabolism
Sorafenib/pharmacology
Mice
*Octamer Transcription Factor-3/metabolism/genetics
Cell Line, Tumor
Mice, Nude
*CRISPR-Cas Systems/genetics

@article {pmid40516842,
year = {2025},
author = {Sun, M and Ni, C and Li, A and Liu, J and Guo, H and Xu, F and Li, K and Cao, X and Shi, X and Guo, R},
title = {A biomimetic nanoplatform mediates hypoxia-adenosine axis disruption and PD-L1 knockout for enhanced MRI-guided chemodynamic-immunotherapy.},
journal = {Acta biomaterialia},
volume = {201},
number = {},
pages = {618-632},
doi = {10.1016/j.actbio.2025.06.021},
pmid = {40516842},
issn = {1878-7568},
mesh = {Animals ; *Immunotherapy ; *B7-H1 Antigen/genetics/metabolism/deficiency ; *Nanoparticles/chemistry/therapeutic use ; *Magnetic Resonance Imaging ; Mice ; Cell Line, Tumor ; Manganese Compounds/chemistry/pharmacology ; *Adenosine/metabolism ; Humans ; *Biomimetic Materials/chemistry/pharmacology ; *Gene Knockout Techniques ; CRISPR-Cas Systems ; Oxides/chemistry/pharmacology ; Tumor Microenvironment ; Melanoma, Experimental/therapy/pathology/diagnostic imaging ; Biomimetics ; },
abstract = {Malignant melanoma is an extremely aggressive and fatal form of skin cancer due to the limited efficacy of conventional therapies. While immune checkpoint blockade therapy and chemodynamic therapy (CDT) have emerged as promising strategies for melanoma treatment, their effectiveness is compromised by the immunosuppressive and complex tumor microenvironment (TME). Here, cancer cell membrane-camouflaged nanoplatforms (PPMC@CM) were developed to co-deliver the CRISPR/Cas9-PD-L1 system and manganese dioxide nanoparticles (MnO2 NPs) for magnetic resonance imaging (MRI)-guided CDT and enhanced immunotherapy. The formed PPMC@CM could efficiently accumulate at tumor sites by homologous targeting, generate O2 to relieve hypoxia, and deplete glutathione (GSH) to enhance Mn[2+]-mediated Fenton-like reactions for enhanced CDT. Meanwhile, CRISPR/Cas9-mediated PD-L1 knockout effectively suppressed the PD-L1 expression, while hypoxia relief attenuated the immunosuppressive hypoxia-CD39/CD73-adenosine (ADO) pathway, thereby boosting the PD-L1-mediated immunotherapy. In vivo experimental results demonstrated that PPMC@CM nanoplatform could efficiently inhibit the growth and metastasis of melanoma by enhanced CDT and amplified immunotherapy, and provide targeted MRI of tumors. This work presents a novelty strategy to design biomimetic theranostic nanoplatform for melanoma by the combination of CDT and improved immunotherapy with CRISPR/Cas9-PD-L1 system and hypoxia-ADO axis inhibition. STATEMENT OF SIGNIFICANCE: Malignant melanoma is a highly aggressive and treatment-refractory skin cancer, where conventional therapies exhibit limited efficacy and immune checkpoint blockade (ICB) is often compromised by the immunosuppressive tumor microenvironment (TME). To address these challenges, we developed a biomimetic nanoplatform (PPMC@CM) to codeliver MnO2 nanoparticles and the CRISPR/Cas9-PD-L1 gene-editing system for MRI-guided chemodynamic therapy and enhanced immunotherapy. The PPMC@CM nanoplatform could efficiently accumulate at tumor sites by homologous targeting and relieve hypoxia to suppress the hypoxia-CD39/CD73-adenosine immunosuppressive axis. Additionally, the CRISPR/Cas9-mediated PD-L1 knockout significantly suppresses PD-L1 expression, thereby boosting ICB efficacy. Moreover, PPMC@CM could deplete glutathione in the TME to amplify Mn[2+]-mediated Fenton-like reactions for enhanced chemodynamic therapy. This research represents a promising theranostic nanoplatform for melanoma by combining chemodynamic therapy and immunotherapy.},
}

Animals
*Immunotherapy
*B7-H1 Antigen/genetics/metabolism/deficiency
*Nanoparticles/chemistry/therapeutic use
*Magnetic Resonance Imaging
Mice
Cell Line, Tumor
Manganese Compounds/chemistry/pharmacology
*Adenosine/metabolism
Humans
*Biomimetic Materials/chemistry/pharmacology
*Gene Knockout Techniques
CRISPR-Cas Systems
Oxides/chemistry/pharmacology
Tumor Microenvironment
Melanoma, Experimental/therapy/pathology/diagnostic imaging
Biomimetics

@article {pmid40499611,
year = {2025},
author = {Wang, C and Wang, Q and Jin, Y and Li, C and Xin, M and Jiang, X and Wan, J},
title = {Lambda exonuclease assisted helicase-dependent amplification CRISPR/Cas12a detection of Listeria monocytogenes.},
journal = {Biochimie},
volume = {235},
number = {},
pages = {106-112},
doi = {10.1016/j.biochi.2025.06.002},
pmid = {40499611},
issn = {1638-6183},
mesh = {*Listeria monocytogenes/genetics/isolation & purification ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *DNA Helicases/metabolism ; DNA, Single-Stranded/genetics ; *Bacterial Proteins/genetics/metabolism ; *Nucleic Acid Amplification Techniques/methods ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {We describe the construction of a protospacer adjacent motif-free CRISPR/Cas12a fluorescent biosensor based on lambda exonuclease (λ-exo) and helicase-dependent amplification (HDA) to detect Listeria monocytogenes(L. monocytogenes). The hlyA gene of L. monocytogenes was amplified by HDA. After λ-exo catalyzed cleavage of 5' phosphorylated single-stranded DNA of amplification product double-stranded DNA, the double-stranded DNA formed single-stranded DNA (ssDNA). The ssDNA as a substrate activated the trans-cleavage capability of CRISPR/Cas12a to cleave the reporter gene to produce fluorescence signals. Under optimized experimental conditions, the lower limit of L. monocytogenes detection by the fluorescent biosensor was 11.5 CFU/mL, with a linear range of detection from 10[1] to 10[7] CFU/mL. The fluorescent biosensor permits simple and sensitive detection of L. monocytogenes and provides a promising analysis platform for clinical diagnosis and biomedical research without protospacer adjacent motif sequence ssDNA.},
}

*Listeria monocytogenes/genetics/isolation & purification
*Biosensing Techniques/methods
*CRISPR-Cas Systems
*DNA Helicases/metabolism
DNA, Single-Stranded/genetics
*Bacterial Proteins/genetics/metabolism
*Nucleic Acid Amplification Techniques/methods
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40634673,
year = {2025},
author = {Asumadu, P and Guo, Z and Qi, S and Liu, C and Li, Y and Shi, Q and Kong, D and Ye, H and Fu, C and Wang, Z},
title = {Programmable DNA aptamer logic gates: from structural design to integrated systems for intelligent nanoscale biosensors.},
journal = {Analytical and bioanalytical chemistry},
volume = {},
number = {},
pages = {},
pmid = {40634673},
issn = {1618-2650},
support = {BZ2022056//Caili Fu/ ; Science//Caili Fu/ ; Technology Project of Jiangsu Province//Caili Fu/ ; 32372417//Hua Ye/ ; National Natural Science Foundation of China//Hua Ye/ ; 1182921902//Dezhao Kong/ ; 32001804//Dezhao Kong/ ; Emerging Science//Dezhao Kong/ ; Technology Innovation Team Fu//Dezhao Kong/ ; National Natural Science Foundation of China//Dezhao Kong/ ; 22406069//Chang Liu/ ; National Natural Science Foundation of China//Chang Liu/ ; 1184882301//Qiaoqiao Shi/ ; Natural Science Foundation of the Jiangsu Higher E//Qiaoqiao Shi/ ; },
abstract = {DNA aptamer-based logic gates represent significant advances in molecular computing, enabling complex biological computations at the nanoscale. These systems leverage the unique programmable properties of DNA aptamers-short, single-stranded oligonucleotides with high specificity and binding affinity for diverse applications across fields such as clinical diagnostics, food/environmental monitoring, and targeted therapeutic delivery, garnering significant research interest in the past few decades. In this review, we first expand on the fundamentals of aptamers, including its SELEX process and post-SELEX modifications. We systematically examine the design principles and operation mechanisms of DNA aptamer-based logic gates, mainly AND, OR, INHIBIT and NOT as reported by researchers. Then, we highlight various logic gates based on different oligonucleotides spanning from intact and split aptamers to DNA origami architectures, DNA nanorobots, and G-quadruplex structural switches, bringing to light their applications across various fields. Recent innovations in multi-input/output gate cascades, CRISPR-Cas-integrated systems and signal amplification approaches are highlighted as key developments in DNA aptamer-based logic gates. Finally, we elucidate challenges relating to DNA aptamer-based systems such as aptamer performance, cross-reactivity in complex multi-input systems and the complexities of merging other systems to amplify output readability, among others, to the end that in addressing these challenges, we will be able to unlock the full potential of this system.},
}

@article {pmid40632881,
year = {2025},
author = {Thi Pham, N and Wang, CH and Chen, CH and Tseng, YT and Guthula, LS and Chau, LK and Yu, CY and Chen, YL},
title = {Integration of CRISPR/Cas12a and a Fiber Optic Particle Plasmon Resonance Sensor for Single Nucleotide Polymorphism Detection in an Aldehyde Dehydrogenase 2 Gene.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c01035},
pmid = {40632881},
issn = {2379-3694},
abstract = {The highly prevalent single nucleotide polymorphism (SNP, rs671) of the aldehyde dehydrogenase (ALDH2) gene in Asian populations instigates various human pathologies and thus accentuates the urgent need for effective diagnostic tools. In this study, we present an ultrasensitive biosensing method by a combination of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a with the fiber optic nanogold-linked sorbent assay (FONLISA) for precise SNP identification. This method leverages the sequence-specific recognition capability of the CRISPR/Cas system and the ultrahigh sensitivity via the dual signal enhancement mechanisms by integrating the trans-cleavage mechanism of Cas12a to amplify the signal from an activity reporter and the subsequent waveguide-enhanced nanoplasmonic absorption by a signaling reporter. In this method, Cas12a targets a double-stranded DNA from the ALDH2 SNP and then activates the degradation of the activity reporter, a free biotin-labeled single-stranded DNA probe (ssDNA[b]), by trans-cleavage. An unhybridized complementary single-stranded DNA probe (ssDNA[c]) labeled with a gold nanoparticle (AuNP) as the signaling reporter (AuNP@ssDNA[c]) is subsequently released and captured by the immobilized ssDNA[b] on the fiber core surface, resulting in a detectable nanoplasmonic absorption signal. The method also utilized an indispensable nanoplasmonic signal generator, carboxymethyl dextran-coated AuNP, to improve the preparation and bioconjugation processes. The CRISPR-FONLISA system demonstrates the ability to analyze the ALDH2 rs671 SNP from double-stranded DNA with a limit of detection of 71 aM. Furthermore, both cell lines and unamplified DNA extracted from blood samples were conducted to verify the system accuracy for ALDH2 rs671 SNP detection.},
}

@article {pmid40632864,
year = {2025},
author = {Yi, JY and Choi, H and Kim, M and Jeong, Y and Hahn, JS and Son, B and Park, HH and Sung, C},
title = {High-throughput multiplexed gene and cell doping analysis through CRISPR-Cas12a system integrated with blood direct PCR.},
journal = {Science advances},
volume = {11},
number = {28},
pages = {eadv7234},
doi = {10.1126/sciadv.adv7234},
pmid = {40632864},
issn = {2375-2548},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Doping in Sports ; *Polymerase Chain Reaction/methods ; *High-Throughput Screening Assays ; },
abstract = {Advancements in gene and cell therapies introduce "gene and cell doping," requiring efficient and sensitive detection methods. Here, we report a high-throughput multiplexed gene and cell doping analysis (HiMDA) using CRISPR-Cas12a system integrated with blood direct polymerase chain reaction (PCR). Blood direct PCR enables simultaneous amplification of multiple exogenous genes directly from whole-blood samples. Coupled with sequence-specific DNA recognition and fluorescence reporter system, HiMDA achieves multiplexed, on-target detection of doping genes and cells. Our results demonstrate HiMDA's feasibility with only 5 microliters of blood required for the entire 90-minute process. HiMDA exhibits exceptional sensitivity, detecting as few as 2.5 copies of doping target genes from blood-four times more sensitive than current anti-doping standards-and identifying in vivo doping up to 10 days. These findings highlight HiMDA's robust high-throughput, multiplexed capabilities, satisfying the sensitivity and selectivity demands of anti-doping research. HiMDA offers a flexible solution to meet future doping detection challenges.},
}

*CRISPR-Cas Systems/genetics
Humans
*Doping in Sports
*Polymerase Chain Reaction/methods
*High-Throughput Screening Assays

@article {pmid40632850,
year = {2025},
author = {Pal, P and Gao, S and Gao, H and Qin, X and Cella, M and Wang, Q and Shan, L},
title = {Establishment of a reverse genetics system for studying human immune functions in mice.},
journal = {Science advances},
volume = {11},
number = {28},
pages = {eadu1561},
doi = {10.1126/sciadv.adu1561},
pmid = {40632850},
issn = {2375-2548},
mesh = {Animals ; Humans ; Mice ; *Reverse Genetics/methods ; Antigens, CD34/metabolism/genetics ; CRISPR-Cas Systems ; Gene Editing ; Fetal Blood/cytology ; Disease Models, Animal ; },
abstract = {Reverse genetics approaches in mice are widely used to understand gene functions and their aberrations in diseases. However, limitations exist in translating findings from animal models to human physiology. Humanized mice provide a powerful bridge to understanding human physiology and mechanisms of disease pathogenesis while maintaining the feasibility of working with small animals. Methods for generating humanized mouse models that allow scientists to probe contributions of particular genes have been rudimentary. Here, we established an efficient method for generating genetically modified human cord blood-derived CD34[+] cells for transplantation, resulting in humanized mice with near-complete loss of specific gene expression by the human immune system. Mice transplanted with Cas9-edited human CD34[+] cells recapitulate functional consequences of specific gene losses in the human immune system. Our approach enables targeted gene knockouts in humanized mice, offering a valuable tool for human gene function studies in vivo.},
}

Animals
Humans
Mice
*Reverse Genetics/methods
Antigens, CD34/metabolism/genetics
CRISPR-Cas Systems
Gene Editing
Fetal Blood/cytology
Disease Models, Animal

@article {pmid40632760,
year = {2025},
author = {Chao, A and Wang, J and Xiu, L and Bold, B and Ghonaim, AH and Chen, J and Hu, Q and Yin, K},
title = {CRISPR/Cas-Based Biosensing Strategies for Non-Nucleic Acid Contaminants in Food Safety: Status, Challenges, and Perspectives.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c04162},
pmid = {40632760},
issn = {1520-5118},
abstract = {Non-nucleic acid targets (non-NATs), such as heavy metals, toxins, and pesticide residues, pose critical threats to food safety. Although CRISPR/Cas systems were initially developed for nucleic acid detection, recent advances have enabled their adaptation to non-NATs analysis by transducing target recognition into nucleic acid signals. Unlike previous reviews categorized by target type, this work establishes a mechanism-centric framework, systematically classifying non-NAT-to-nucleic acid signal conversion methodologies into three paradigms: (1) aptamer-based systems, (2) catalytic nucleic acid-based methods (e.g., DNAzymes), and (3) protein-mediated strategies (e.g., antibodies, transcription factors). When integrated with CRISPR/Cas, these systems achieve rapid, sensitive detection at picomolar (pM) levels without relying on chromatographic or spectroscopic instruments. Furthermore, we critically discuss challenges, including the limited diversity of recognition elements, inefficient signal conversion, and inflexible signal outputs, proposing solutions including synthetic-biology-driven bioreceptor design and artificial-intelligence-based data analysis. By bridging mechanistic principles with applications in complex food matrices, this review provides actionable insights to advance CRISPR-based tools for rapid, on-site, food safety monitoring.},
}

@article {pmid40632661,
year = {2025},
author = {Wang, S and Zheng, J and Zhang, X and Zhao, M and Li, J and Su, M and Qiu, W},
title = {Genome-Wide CRISPR-Cas9 Knockout Screening Identifies Genes Modulating Cisplatin-Induced Cytotoxicity in Renal Proximal Tubule Epithelial Cells.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {39},
number = {13},
pages = {e70780},
doi = {10.1096/fj.202402401RR},
pmid = {40632661},
issn = {1530-6860},
support = {5202009//Natural Science Foundation of Beijing Municipality/ ; 5192022//Natural Science Foundation of Beijing Municipality/ ; 81870196//MOST | National Natural Science Foundation of China (NSFC)/ ; },
mesh = {*Cisplatin/toxicity/pharmacology/adverse effects ; *Kidney Tubules, Proximal/drug effects/metabolism/cytology/pathology ; *CRISPR-Cas Systems/genetics ; *Epithelial Cells/drug effects/metabolism/pathology ; Humans ; *Antineoplastic Agents/toxicity ; Animals ; Gene Knockout Techniques ; },
abstract = {Cisplatin is widely used as a first-line chemotherapy drug for various cancers. However, cisplatin-induced nephrotoxicity (CIN) greatly restricts its application. Renal proximal tubular epithelial cells (RPTECs) can be extensively damaged during CIN. However, it still lacks an ideal method to prevent CIN, because the mechanism and therapeutic targets of CIN remain largely unclear. In the present study, we used a genome-scale CRISPR-Cas9 knock-out method to functionally screen key genes of cisplatin-induced RPTEC injury. We found 815 genes significantly enriched (p < 0.05) from positive selection screening strategy, which may synergistically enhance cisplatin cytotoxicity in RPTECs. Importantly, we identified ERAP2 as a novel molecule associated with CIN. We found that the expression of ERAP2 in RPTECs was significantly up-regulated by cisplatin. Data from CCK-8 assay and flow cytometry showed that inhibition of ERAP2 alleviated cisplatin-induced RPTEC injury. Furthermore, RNA-seq and qPCR results revealed that three necroptosis-associated genes, PLA2G4C, HIST1H2AC, and HIST1H2AM, were downregulated following ERAP2 inhibition, suggesting that ERAP2 may be a novel therapeutic target of CIN through the modulation of necroptosis pathway.},
}

*Cisplatin/toxicity/pharmacology/adverse effects
*Kidney Tubules, Proximal/drug effects/metabolism/cytology/pathology
*CRISPR-Cas Systems/genetics
*Epithelial Cells/drug effects/metabolism/pathology
Humans
*Antineoplastic Agents/toxicity
Animals
Gene Knockout Techniques

@article {pmid40632351,
year = {2025},
author = {Lokya, V and Singh, S and Chaudhary, R and Jangra, A and Tiwari, S},
title = {Emerging trends in transgene-free crop development: insights into genome editing and its regulatory overview.},
journal = {Plant molecular biology},
volume = {115},
number = {4},
pages = {84},
pmid = {40632351},
issn = {1573-5028},
support = {BIRAC/TechTransfer/08/I2/QUT-BBF//Biotechnology Industry Research Assistance Council/ ; Core research grant//BRIC-National Agri-Food and Biomanufacturing Institute (BRIC-NABI)/ ; },
mesh = {*Gene Editing/methods/trends ; *Crops, Agricultural/genetics/growth & development ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; *Genome, Plant ; Transgenes ; },
abstract = {Genome editing tools have revolutionized plant biology research offering unparalleled applications for genome manipulation and trait improvement in crops. Adopting such advanced biotechnological tools is inevitable to meet increasing global food demand and address challenges in food production, including (a)biotic stresses and inadequate nutritional value. Despite reliance on conventional genetic manipulation methods, the CRISPR-Cas-mediated genome editing toolbox allows precise modification of DNA/RNA in a target organism's genome. So far, CRISPR-Cas has been widely used to enhance yield, quality, stress tolerance, and nutritional value in various food crops. However, challenges such as reagent delivery in suitable explants, precise editing with minimal off-target effect, and generating transgene-free plants persist as major bottlenecks in most plant species. Components of CRISPR-Cas construct mainly Cas, guide RNA (gRNA), and selectable marker genes are often integrated into the host genome, which raises regulatory concerns. However, adapting advanced gene-editing strategies, including high-efficiency Cas endonucleases, DNA-independent RNP delivery, morphogenetic regulators, and grafting-mediated editing, are paving the way for transgene-free crop improvement while easing biosafety regulations. Further, regulatory frameworks for genome-edited crops vary globally, with several countries accepting them and others debating their legal status. Hence, the disparity in global regulatory guidelines of genome editing curbs commercialization. The current review highlights the emerging CRISPR-mediated tools or methods and their applications in developing transgene-free designer crops to harness the benefits of advanced genome manipulation.},
}

*Gene Editing/methods/trends
*Crops, Agricultural/genetics/growth & development
CRISPR-Cas Systems
Plants, Genetically Modified/genetics
*Genome, Plant
Transgenes

@article {pmid40632173,
year = {2025},
author = {Thomas, L and Abraham, T},
title = {Disabling iron uptake and pilus assembly in uropathogenic Escherichia coli using CRISPR-Cas9: a step towards antivirulence therapy.},
journal = {Antonie van Leeuwenhoek},
volume = {118},
number = {8},
pages = {110},
pmid = {40632173},
issn = {1572-9699},
support = {363661//University Grants Commission/ ; },
mesh = {*Uropathogenic Escherichia coli/genetics/metabolism/pathogenicity ; *CRISPR-Cas Systems ; *Iron/metabolism ; *Fimbriae, Bacterial/metabolism/genetics ; Escherichia coli Proteins/genetics/metabolism ; Escherichia coli Infections/microbiology ; Virulence Factors/genetics/metabolism ; Virulence/genetics ; Molecular Docking Simulation ; Humans ; Fimbriae Proteins/genetics/metabolism ; Gene Editing ; },
abstract = {Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs), driven by virulence factors such as iron acquisition systems and adhesive pili. In this study, we employed CRISPR-Cas9-mediated genome editing to functionally inactivate two critical virulence genes-iucD, involved in aerobactin-mediated iron uptake, and papC, encoding the outer membrane usher protein essential for P pilus assembly. Using a clinical UPEC isolate, we introduced premature stop codons via homologous repair templates guided by gene-specific single-guide RNAs. Colony PCR and Sanger sequencing confirmed precise site-specific editing, leading to truncated protein variants. In silico analyses using InterPro and Swiss-Model revealed a complete loss of essential domains in both proteins. Molecular docking studies demonstrated a marked reduction in binding affinities of truncated iucD for NAD(P)H and impaired protein-protein interaction between truncated PapC and PapG. This study highlights the utility of CRISPR-Cas9 as a powerful tool for dissecting bacterial pathogenesis and supports the potential of targeting virulence determinants like iucD and papC as part of an antivirulence strategy for managing UPEC infections.},
}

*Uropathogenic Escherichia coli/genetics/metabolism/pathogenicity
*CRISPR-Cas Systems
*Iron/metabolism
*Fimbriae, Bacterial/metabolism/genetics
Escherichia coli Proteins/genetics/metabolism
Escherichia coli Infections/microbiology
Virulence Factors/genetics/metabolism
Virulence/genetics
Molecular Docking Simulation
Humans
Fimbriae Proteins/genetics/metabolism
Gene Editing

@article {pmid40631558,
year = {2025},
author = {He, C and Zhu, W and Zhang, X and Wu, W and Tan, J and Li, B and Huang, D and Chen, Y and Xiang, Z and Huang, L and Gong, L},
title = {Sensitive and Visualized Detection of Hantavirus Using CRISPR/Cas12a Based on AutoCORDSv2 Design.},
journal = {Journal of medical virology},
volume = {97},
number = {7},
pages = {e70460},
doi = {10.1002/jmv.70460},
pmid = {40631558},
issn = {1096-9071},
support = {//This study is financially supported by The President Foundation of Nanfang Hospital, Southern Medical University (grant number 2023B036); National Science and Technology Infrastructure of China (Project No. National Pathogen Resource Center-NPRC-32); The CAMS Innovation Fund for Medical Sciences (grant number 2021-I2M-1-039); National Key R&D Program of China, grant number (grant number 2022YFC2303400); The Guangdong Basic and Applied Basic Research Foundation (grant number 2023A1515140080); Dongguan Social development technology projects Science and Technology Planning Project (grant number 20231800940792)./ ; },
mesh = {*CRISPR-Cas Systems ; Sensitivity and Specificity ; Humans ; *Molecular Diagnostic Techniques/methods ; *Orthohantavirus/isolation & purification/genetics ; RNA, Viral/genetics ; *Hantavirus Infections/diagnosis/virology ; *Hantaan virus/genetics/isolation & purification ; DNA Primers/genetics ; },
abstract = {In recent years, detection technologies based on the CRISPR/Cas12a method have been extensively utilized in the fields of nucleic acid, enzyme, and macromolecule detection, thereby reinforcing their significant role in the detection landscape. Enhancing the simplicity of design, efficiency, and automation of the CRISPR/Cas12a detection system is essential for advancing its application in diagnostics. Recently, we developed an automated CRISPR/Cas12a design system named AutoCORDSv2. This system can process published genomic sequences of pathogenic bacteria in a high-throughput manner and automatically generate conserved and highly specific crRNA sequences, along with primer sequences for target amplification. This capability facilitates the specific and precise design of the CRISPR/Cas12a detection system. In this study, crRNAs targeting the Hantaan virus (HTNV) and Seoul virus (SEOV), as well as RT-PCR primers and RT-RPA primers, were designed using AutoCORDSv2. The experimental results demonstrated that the CRISPR/Cas12a system, automatically designed by AutoCORDSv2, was specific for the detection of both the HTNV and SEOV, with no cross-reactivity observed with other pathogens. The detection sensitivity reached 6 copies/μL (equivalent to 111 copies per amplification reaction), whether measured by a microplate reader or directly observed with the naked eye. The detection results for 50 samples were consistent with those obtained from commercial RT-qPCR kits, indicating high precision. Furthermore, the CRISPR/Cas12a system designed by AutoCORDSv2 can also be utilized for the development of a single-tube detection system with a sensitivity of 42 copies per reaction. This system combined with a 5-min extraction step and RT-RPA, further underscoring its potential for application.},
}

*CRISPR-Cas Systems
Sensitivity and Specificity
Humans
*Molecular Diagnostic Techniques/methods
*Orthohantavirus/isolation & purification/genetics
RNA, Viral/genetics
*Hantavirus Infections/diagnosis/virology
*Hantaan virus/genetics/isolation & purification
DNA Primers/genetics

@article {pmid40630641,
year = {2025},
author = {Pan, K and Zeng, A and Ruan, X and Mo, X and Shen, B and Zhao, J and Zhou, Y},
title = {The apicoplast localized isocitrate dehydrogenase is needed for de novo fatty acid synthesis in the apicoplast of Toxoplasma gondii.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1542122},
pmid = {40630641},
issn = {2235-2988},
mesh = {*Toxoplasma/enzymology/growth & development/genetics/metabolism ; *Isocitrate Dehydrogenase/metabolism/genetics ; Animals ; *Fatty Acids/biosynthesis ; *Apicoplasts/enzymology/metabolism/genetics ; Mice ; Virulence ; CRISPR-Cas Systems ; Gene Deletion ; NADP/metabolism ; Glucose/metabolism ; },
abstract = {Toxoplasma gondii (T. gondii), an apicomplexan parasite, infects a wide range of warm-blooded animals and poses significant risks to human health. The fatty acid synthesis II (FASII) pathway in the apicoplast, which is the major source of fatty acids in parasites, is considered a potential drug target. The apicoplast also harbors some enzymes of central carbon metabolism, which are crucial for its survival, but their biological roles remain unclear. In this study, we focused on apicoplast-localized isocitrate dehydrogenase 1 (ICDH1) and deleted it using CRISPR-Cas9 technology. The Δicdh1 mutant tachyzoites displayed markedly impaired growth kinetics, with further suppression under serum-deprived conditions. However, this deletion did not affect the viability or virulence of the Δicdh1 mutant in mice. NADPH, a product of ICDH1-mediated decarboxylation of isocitrate, is an essential cofactor for fatty acid synthesis. Using [13]C6 glucose as a metabolic carbon source, we showed that the mutant strains had reduced incorporation of glucose-derived carbons into medium-chain length fatty acids (C14:0 and C16:0). Additionally, the growth of the mutant was partially restored by supplementation with exogenous C14:0 and C16:0 fatty acids. These results indicate that ICDH1 deletion affects the FASII pathway and parasite growth. Consistent with previous studies, this study confirms that T. gondii has metabolic flexibility in the apicoplast that allows it to acquire fatty acids through various pathways.},
}

*Toxoplasma/enzymology/growth & development/genetics/metabolism
*Isocitrate Dehydrogenase/metabolism/genetics
Animals
*Fatty Acids/biosynthesis
*Apicoplasts/enzymology/metabolism/genetics
Mice
Virulence
CRISPR-Cas Systems
Gene Deletion
NADP/metabolism
Glucose/metabolism

@article {pmid40629383,
year = {2025},
author = {Gou, F and Liu, D and Gong, C and Wang, K and Wang, X and Chen, Y and Liu, Q and Tian, C},
title = {Development of an efficient heterologous protein expression platform in Aspergillus niger through genetic modification of a glucoamylase hyperproducing industrial strain.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {160},
pmid = {40629383},
issn = {1475-2859},
support = {2023YFC3402300//National Key Research & Developmental Program of China/ ; U22A20441//National Natural Science Foundation of China/ ; XDA0510300//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; },
mesh = {*Aspergillus niger/genetics/metabolism/enzymology ; *Glucan 1,4-alpha-Glucosidase/genetics/biosynthesis/metabolism ; Recombinant Proteins/genetics/biosynthesis/metabolism ; CRISPR-Cas Systems ; Industrial Microbiology ; Genetic Engineering ; },
abstract = {BACKGROUND: Aspergillus niger is widely used in industrial enzyme production due to its strong secretion capacity and the status of generally recognized as safe (GRAS). However, heterologous protein expression in A. niger is frequently constrained by high levels of background endogenous protein secretion, limited access to native high transcription loci, and limitations in the efficiency of the secretory machinery. To address these limitations, this study genetically engineered a chassis strain based on an industrial glucoamylase-producing A. niger strain AnN1 for constructing the improved heterologous protein expression.

RESULTS: In this study, by using CRISPR/Cas9-assisted marker recycling, we deleted 13 of the 20 copies of the heterologous glucoamylase TeGlaA gene and disrupted the major extracellular protease gene PepA, resulting in the low-background strain AnN2. Compared to the parental strain AnN1, AnN2 exhibited 61% less extracellular protein and significantly reduced glucoamylase activity, while retaining multiple transcriptionally active integration loci. Four diverse proteins were integrated into the high-expression loci originally occupied by the TeGlaA gene in the chassis AnN2. These recombinant protein included a homologous glucose oxidase (AnGoxM), a thermostable pectate lyase A (MtPlyA), a bacterial triose phosphate isomerase (TPI), and a medical protein Lingzhi-8 (LZ8). All target proteins were successfully expressed and secreted within 48-72 h, with yields ranging from 110.8 to 416.8 mg/L in 50 mL shake-flasks cultivation. The enzyme activities of AnGoxM, MtPlyA and TPI reached ~ 1276 - 1328 U/mL, ~ 1627. 43 - 2105.69 U/mL, and ~ 1751.02 to 1906.81 U/mg after 48 h, respectively. Additionally, Overexpression of Cvc2, a COPI vesicle trafficking component, further enhanced MtPlyA production by 18%, highlighting the benefit of combining transcriptional and secretory pathway engineering.

CONCLUSIONS: Our results demonstrated that the chassis AnN2 served as a robust, modular, and time-efficient platform for heterologous protein expression in A. niger. Through site-specific integration of target genes into native high-expression loci and strategic modulation of the secretory pathway, we successfully enabled the rapid production of functional enzymes and bioactive proteins from diverse origins. This dual-level optimization strategy, which integrates rational genomic engineering with targeted enhancement of the secretory pathway, enabled high-yield expression while minimizing background interference. Together, these findings offer a practical framework for constructing versatile fungal expression systems and highlight the potential of combining genetic and cellular engineering to improve recombinant protein production in filamentous fungi.},
}

*Aspergillus niger/genetics/metabolism/enzymology
*Glucan 1,4-alpha-Glucosidase/genetics/biosynthesis/metabolism
Recombinant Proteins/genetics/biosynthesis/metabolism
CRISPR-Cas Systems
Industrial Microbiology
Genetic Engineering

@article {pmid40586242,
year = {2025},
author = {Sreekanth, V and Jan, M and Zhao, KT and Lim, D and Siriwongsup, S and Davis, JR and McConkey, M and Kovalcik, V and Barkal, S and Law, BK and Fife, J and Tian, R and Vinyard, ME and Becerra, B and Kampmann, M and Sherwood, RI and Pinello, L and Liu, DR and Ebert, BL and Choudhary, A},
title = {A Molecular Glue Approach to Control the Half-Life of CRISPR-Based Technologies.},
journal = {Journal of the American Chemical Society},
volume = {147},
number = {27},
pages = {23844-23856},
doi = {10.1021/jacs.5c06230},
pmid = {40586242},
issn = {1520-5126},
mesh = {Half-Life ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Thalidomide/analogs & derivatives/chemistry/pharmacology/metabolism ; Ubiquitination ; },
abstract = {Cas9 is a programmable nuclease that has furnished transformative technologies, including base editors and transcription modulators (e.g., CRISPRi/a), but several applications of these technologies, including therapeutics, mandatorily require precision control of their half-life. For example, such control can help avert any potential immunological and adverse events in clinical trials. Current genome editing technologies to control the half-life of Cas9 are slow, have lower activity, involve fusion of large response elements (>230 amino acids), utilize expensive controllers with poor pharmacological attributes, and cannot be implemented in vivo on several CRISPR-based technologies. We report a general platform for half-life control using the molecular glue, pomalidomide, that binds to a ubiquitin ligase complex and a response-element bearing CRISPR-based technology, thereby causing the latter's rapid ubiquitination and degradation. Using pomalidomide, we were able to control the half-life of large CRISPR-based technologies (e.g., base editors and CRISPRi) and small anti-CRISPRs that inhibit such technologies, allowing us to build the first examples of on-switch for base editors. The ability to switch on, fine-tune, and switch-off CRISPR-based technologies with pomalidomide allowed complete control over their activity, specificity, and genome editing outcome. Importantly, the miniature size of the response element and favorable pharmacological attributes of the drug pomalidomide allowed control of activity of base editor in vivo using AAV as the delivery vehicle. These studies provide methods and reagents to precisely control the dosage and half-life of CRISPR-based technologies, propelling their therapeutic development.},
}

Half-Life
Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*Thalidomide/analogs & derivatives/chemistry/pharmacology/metabolism
Ubiquitination

@article {pmid40577080,
year = {2025},
author = {Butti, P and Bellusci, F and Brambilla, E and Branduardi, P},
title = {Genomically integrated cassettes swapping: bringing modularity to the strain level in Saccharomyces cerevisiae.},
journal = {FEMS yeast research},
volume = {25},
number = {},
pages = {},
doi = {10.1093/femsyr/foaf032},
pmid = {40577080},
issn = {1567-1364},
support = {//University of Milano-Bicocca/ ; CN_00000033//Italian Ministry of University and Research/ ; H43C22000530001//Italian Ministry of University and Research/ ; },
mesh = {*Saccharomyces cerevisiae/genetics ; CRISPR-Cas Systems ; *Synthetic Biology/methods ; *Gene Editing/methods ; *Genome, Fungal ; },
abstract = {A large variety of synthetic biology toolkits for the introduction of multiple expression cassettes is available for Saccharomyces cerevisiae. Unfortunately, none of these tools is designed to allow the modification - exchange or removal - of the cassettes already integrated into the genome in a standardized way. The application of the modularity principle therefore ends to the steps preceding the final host engineering, making microbial cell factories construction stiff and strictly sequential. In this work, we describe a system that easily allows CRISPR-mediated swapping or removal of previously integrated cassettes, thus bringing the modularity to the strain level, enhancing the possibility of modifying existing strains with a reduced number of steps. In the system, each cassette is tagged with specific barcodes, which can be used as targets for CRISPR nucleases (Cas9 and Cas12a), allowing the excision of the construct from the genome and its substitution with another expression cassette or the restoration of the wild type locus in one single standardized step. The system has been applied to the previously developed Easy-MISE toolkit and tested by swapping fluorescent protein expression cassettes with an efficiency of ∼90% quantified by PCR and flow cytometry.},
}

*Saccharomyces cerevisiae/genetics
CRISPR-Cas Systems
*Synthetic Biology/methods
*Gene Editing/methods
*Genome, Fungal