@article {pmid40571317,
year = {2025},
author = {Karinen, S and Forero-Rodríguez, J and Järvinen, A and Eklund, KK and Barreto, G and Salem, A},
title = {CRISPR/Cas9-mediated Knockout of LYVE1 In Human Tongue Cancer Cells Reveals Transcriptomic Changes in Metastasis-associated Pathways.},
journal = {Cancer genomics & proteomics},
volume = {22},
number = {4},
pages = {525-537},
doi = {10.21873/cgp.20519},
pmid = {40571317},
issn = {1790-6245},
mesh = {Humans ; *Tongue Neoplasms/genetics/pathology/metabolism ; *CRISPR-Cas Systems ; *Transcriptome ; *Vesicular Transport Proteins/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; Epithelial-Mesenchymal Transition ; Cell Line, Tumor ; Gene Knockout Techniques ; Neoplasm Metastasis ; *Squamous Cell Carcinoma of Head and Neck/genetics ; },
abstract = {BACKGROUND/AIM: Tongue squamous cell carcinoma (TSCC), a highly aggressive subtype of head and neck cancers, is characterized by frequent lymphatic metastasis and poor prognosis. Recently, we showed that lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) is involved in TSCC progression, yet the underlying molecular mechanisms remain unclear.

MATERIALS AND METHODS: CRISPR/Cas9 gene editing was employed to generate LYVE1 knockout (KO) TSCC cell lines. Single-cell clones were isolated, screened, and validated through sequencing and Inference of CRISPR Edits (ICE) analysis and qRT-PCR. RNA sequencing was performed on LYVE1 KO and wild-type (WT) cells to identify differentially expressed genes (DEGs). Bioinformatic analyses, including Gene Ontology (GO) enrichment and protein-protein interaction (PPI) network mapping, were conducted to explore affected pathways. Finally, network topology was examined using NetworkAnalyzer and cytoHubba plugins.

RESULTS: Transcriptomic analysis revealed significant down-regulation of pro-metastatic pathways, including epithelial-mesenchymal transition (EMT), extracellular matrix remodeling, and immune modulation. DEG analysis identified 263 genes, with key down-regulated targets such as WNT5A, TGFB2, and MMP2, and up-regulation of tumor-suppressive genes including PTGS2. GO and PPI analyses highlighted LYVE1's pivotal role in regulating cell adhesion, migration, and immune response.

CONCLUSION: LYVE1 KO reduces TSCC invasive potential by disrupting EMT and tumor-stroma interactions, aligning with previous experimental findings. These results suggest LYVE1 as a critical driver of metastasis, highlighting its potential as a therapeutic target.},
}

Humans
*Tongue Neoplasms/genetics/pathology/metabolism
*CRISPR-Cas Systems
*Transcriptome
*Vesicular Transport Proteins/genetics/metabolism
Gene Expression Regulation, Neoplastic
Epithelial-Mesenchymal Transition
Cell Line, Tumor
Gene Knockout Techniques
Neoplasm Metastasis
*Squamous Cell Carcinoma of Head and Neck/genetics

@article {pmid40571168,
year = {2025},
author = {Jeong, JH and Kim, SH and Kim, JY},
title = {Empowering Agrobacterium: Ternary vector systems as a new arsenal for plant transformation and genome editing.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108631},
doi = {10.1016/j.biotechadv.2025.108631},
pmid = {40571168},
issn = {1873-1899},
abstract = {The continuous evolution of plant transformation technologies is pivotal for accelerating genetic advancements in agriculture. Among these, ternary vector systems have emerged as a transformative innovation, significantly enhancing Agrobacterium-mediated plant transformation by overcoming critical biological barriers. Unlike traditional binary vectors, ternary vector systems incorporate accessory virulence genes and immune suppressors that overcome the intrinsic transformation barriers of recalcitrant crops. This has enabled 1.5- to 21.5-fold increases in stable transformation efficiency in species previously resistant to Agrobacterium-mediated transformation, such as maize, sorghum, and soybean, thereby expanding the effective host range of plant genetic engineering. Furthermore, the fusion of ternary vectors with advanced genome editing technologies like CRISPR/Cas is revolutionizing precision breeding, facilitating unprecedented control over genetic modifications. Future innovations are likely to focus on expanding the capabilities of ternary vectors, including transient delivery of morphogenic factors to enhance regeneration and organelle-targeted transformation for broader genetic modifications. Additionally, refining Agrobacterium engineering, such as developing auxotrophic strains for improved biosafety and optimizing secretion systems for enhanced protein delivery, presents exciting opportunities for further advancements. This review highlights the recent breakthroughs in ternary vector engineering, including its potential to optimize regeneration pathways via morphogenic factors and extend genetic transformation to previously unamenable plant species. By bridging the gap between transformation efficiency and targeted genome modifications, these advancements are reshaping the landscape of plant biotechnology, driving more resilient and high-performing crops in an era of global agricultural challenges.},
}

@article {pmid40569097,
year = {2025},
author = {Jenkins, K and Layton, D and Gough, T and O'Neil, T and Malaver Otega, L and Mishra, K and Bruce, K and Morris, K and Wise, T and Challagulla, A and Doran, T and Bean, A},
title = {Production of immune receptor knockout chickens via direct in vivo transfection of primordial germ cells.},
journal = {Animal biotechnology},
volume = {36},
number = {1},
pages = {2523027},
doi = {10.1080/10495398.2025.2523027},
pmid = {40569097},
issn = {1532-2378},
mesh = {Animals ; *Chickens/genetics ; *Receptor, Interferon alpha-beta/genetics ; *Gene Knockout Techniques/veterinary/methods ; *Transfection/veterinary/methods ; *Animals, Genetically Modified/genetics ; *Germ Cells ; *Receptors, Interleukin-1 Type I/genetics ; CRISPR-Cas Systems ; Chick Embryo ; Male ; Female ; },
abstract = {The advancement of genetic engineering in chickens has enabled significant advancement in developmental biology, bioreactors, and disease resilience. The development of CRISPR/Cas9 genome engineering technology has further expanded the potential applications of genetic engineering in poultry. In this study we aimed to evaluate the efficacy of a direct in vivo transfection method, previously demonstrated to produce transgenic chickens, in generating gene knockout (KO) chickens. Specifically, we targeted the Interferon-α/β Receptor 1 (IFNAR1) and Interleukin 1 receptor, type I (IL1R1), both critical pathways in the inflammatory and antiviral responses. We designed guide RNAs targeting the genes and validated their efficiency in vivo via microinjection into the developing embryos. PCR analysis confirmed the presence of gene deletions in chimeric roosters, which were subsequently bred to produce G1 germline heterozygote KO offspring. Homozygous KO chickens were generated and subjected to phenotypic and functional analyses. Our results demonstrated successful generation of functional knockouts of both IFNAR1 and IL1R1 using a direct in vivo transfection. Overall, this study demonstrates that direct in vivo transfection provides a robust and predictable method for generating KO chickens, facilitating further research into avian immune responses and the development of antiviral strategies.},
}

Animals
*Chickens/genetics
*Receptor, Interferon alpha-beta/genetics
*Gene Knockout Techniques/veterinary/methods
*Transfection/veterinary/methods
*Animals, Genetically Modified/genetics
*Germ Cells
*Receptors, Interleukin-1 Type I/genetics
CRISPR-Cas Systems
Chick Embryo
Male
Female

@article {pmid40568938,
year = {2025},
author = {Liu, X and Tan, H and Wang, J and Cao, Y and Li, P and Fan, X and Wang, Q and Zhang, H and Zhang, J and Yang, T and Zhao, G and Zhang, X and Duan, X and Zi, L and Liu, L and Ma, L and Chen, Z and Liang, L and Liu, R},
title = {SELECT: high-precision genome editing strategy via integration of CRISPR-Cas and DNA damage response for cross-species applications.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf595},
pmid = {40568938},
issn = {1362-4962},
support = {2023YFC3402300//National Key R&D Program of China/ ; 22208044//National Natural Science Foundation of China/ ; 22278058//National Natural Science Foundation of China/ ; XLYC2203075//Xingliao Talent Plan/ ; 2023JJ12SN030//Science and Technology Innovation Foundation of Dalian/ ; 2024-MSBA-09//Natural Science Foundation of Liaoning Province/ ; DUT24YG131//Fundamental Research Funds for the Central Universities/ ; DUT23YG219//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Saccharomyces cerevisiae/genetics ; Escherichia coli/genetics ; *DNA Damage ; Promoter Regions, Genetic ; DNA Breaks, Double-Stranded ; },
abstract = {CRISPR-based methods enable genome modifications for diverse applications but often face challenges, such as inconsistent efficiencies, reduced performance in iterative modifications, and difficulties generating high-quality datasets for high-throughput genome engineering. Here, we present SELECT (SOS Enhanced programmabLE CRISPR-Cas ediTing), a novel strategy integrating the CRISPR-Cas system with the DNA damage response. By employing designed and optimized double-strand break induced promoters that are activated upon genome editing, SELECT enables a counter-selection process to eliminate unedited cells, ensuring high-fidelity editing. This approach achieves up to 100% efficiency for point mutations, iterative knockouts, and insertions. In high-throughput library editing, SELECT achieved up to 94.2% efficiency and preserved higher library diversity compared with conventional methods. Application of SELECT in flaviolin biosynthesis resulted in a 3.97-fold increase in production. Furthermore, integration with machine learning tools allowed rapid mapping of genotype-phenotype relationships. SELECT provides a versatile platform for precision genome engineering in Escherichia coli and Saccharomyces cerevisiae.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
Saccharomyces cerevisiae/genetics
Escherichia coli/genetics
*DNA Damage
Promoter Regions, Genetic
DNA Breaks, Double-Stranded

@article {pmid40567005,
year = {2025},
author = {Zeng, Z and Wang, H and Luo, Y and Chen, W and Xu, M and Wei, H and Chen, Z and Xiang, T and Wang, L and Han, N and Huang, X and Bian, H},
title = {CRISPR/Cas9-mediated editing of barley lipoxygenase genes promotes grain fatty acid accumulation and storability.},
journal = {GM crops & food},
volume = {16},
number = {1},
pages = {482-497},
doi = {10.1080/21645698.2025.2523069},
pmid = {40567005},
issn = {2164-5701},
mesh = {*Hordeum/genetics/metabolism/enzymology ; *CRISPR-Cas Systems ; Gene Editing ; *Lipoxygenase/genetics/metabolism ; *Fatty Acids/metabolism ; *Edible Grain/genetics/metabolism ; Plants, Genetically Modified/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Seeds/genetics/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {Plant lipoxygenases (LOXs) catalyze the oxidation of polyunsaturated fatty acids, which can adversely affect grain storability. Although the genetic engineering of LOXs holds great potential for improving grain storage quality, this approach remains largely unexplored in barley. In this study, we identified five LOX genes in the barley genome: HvLOXA, HvLOXB, and HvLOXC1-3. HvLOXC1 exhibited the highest expression in early developing grains, roots, and shoots; HvLOXA was predominantly expressed in embryos, whereas HvLOXB and HvLOXC3 were weakly expressed across tissues. Transgene-free homozygous barley mutants of loxB, loxC1, and loxAloxC1 were generated using CRISPR/Cas9-mediated genome editing. Compared to the wild-type, all mutants displayed normal plant height, tiller number, and grain size, although the loxC1 and loxAloxC1 mutants exhibited significantly lower thousand grain weights. Notably, the total LOX activity in mature grains decreased by 36-42% in loxC1 mutants and by 94% in loxAloxC1 mutants, with no significant change observed in loxB mutants. Additionally, the loxAloxC1 double mutants had a significantly lower malondialdehyde content and accumulated 10-21% more fatty acids than the wild-type. Artificial aging treatment experiments revealed that loxAloxC1 mutants had enhanced grain storability, demonstrated by significantly higher germination rates, reduced lipid peroxidation, and improved seedling growth. Our findings highlight that the targeted knockout of LOX genes, particularly the double mutation of HvLOXA and HvLOXC1, represents a promising genetic strategy for improving grain storability and nutritional value in barley.},
}

*Hordeum/genetics/metabolism/enzymology
*CRISPR-Cas Systems
Gene Editing
*Lipoxygenase/genetics/metabolism
*Fatty Acids/metabolism
*Edible Grain/genetics/metabolism
Plants, Genetically Modified/genetics/metabolism
*Plant Proteins/genetics/metabolism
Seeds/genetics/metabolism
Gene Expression Regulation, Plant

@article {pmid40566742,
year = {2025},
author = {Ni, H and Kelley, K and Xie, N and Zou, H and Friedel, RH},
title = {Generation of Plexin-B1 Conditional Knockout Mouse With CRISPR/Cas9 Technology.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {63},
number = {3},
pages = {e70019},
doi = {10.1002/dvg.70019},
pmid = {40566742},
issn = {1526-968X},
support = {NS092735/NS/NINDS NIH HHS/United States ; NS134159/NS/NINDS NIH HHS/United States ; AG077828/AG/NIA NIH HHS/United States ; CA196521/CA/NCI NIH HHS/United States ; DOH01-C39068GG//New York State Department of Health/ ; DOH01-C38330GG//New York State Department of Health/ ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; Mice, Knockout ; *Nerve Tissue Proteins/genetics ; Alleles ; Gene Targeting ; *Receptors, Cell Surface/genetics ; Gene Editing/methods ; Female ; },
abstract = {Plexins are axon guidance transmembrane receptors that control cytoskeleton and membrane dynamics in development and adult physiology. As plexins are expressed in multiple cell types in various tissues, floxed alleles that enable conditional deletion are needed to facilitate cell type-specific functional analysis. We report here the generation of a conditional floxed allele of Plexin-B1 (gene symbol Plxnb1) in mouse using CRISPR/Cas9 technology to insert two loxP sites flanking critical exons. Targeting reagents (Cas9 protein, sgRNAs, ssODNs) were delivered into single-cell embryos by electroporation. After screening a total of 128 mouse pups by PCR and Sanger sequencing, two mice were identified carrying both loxP sites in the targeted Plxnb1 locus (success rate ~ 1.6%). The usage of Alt-R modified ssODNs increased targeting frequencies at one loxP site, but not the other. We also tested homology directed repair (HDR) enhancer V2 reagent, but addition of the enhancer reduced the viability of mouse embryos. The Plxnb1[flox] allele was successfully transmitted through the germline in Mendelian ratios, and effective excision of the floxed region was confirmed by breeding with Cre recombinase strains.},
}

Animals
Mice
*CRISPR-Cas Systems
Mice, Knockout
*Nerve Tissue Proteins/genetics
Alleles
Gene Targeting
*Receptors, Cell Surface/genetics
Gene Editing/methods
Female

@article {pmid40566685,
year = {2025},
author = {Liu, J and Zhang, R and Chai, N and Su, L and Zheng, Z and Liu, T and Guo, Z and Ma, Y and Xie, Y and Xie, X and Lin, Q and Chen, L and Liu, YG and Zhu, Q},
title = {Programmable genome engineering and gene modifications for plant biodesign.},
journal = {Plant communications},
volume = {},
number = {},
pages = {101427},
doi = {10.1016/j.xplc.2025.101427},
pmid = {40566685},
issn = {2590-3462},
abstract = {Plant science has entered a transformative era with genome editing by enabling precise DNA alterations to address global challenges such as climate adaptability and food safety. These alterations are primarily driven by the integration of three modular components that can be activated or suppressed: DNA-targeting modules, effector modules, and control modules. The field has evolved from protein-centric systems (zinc finger nucleases and transcription activator-like effector nucleases) to RNA-focused platforms (CRISPR-Cas and other nucleases), which facilitate diverse control over genetic and epigenetic contexts. The modular design of DNA-targeting modules paired with effector domains, with or without inducible systems, provides scientists with superior precision in regulating transcription and altering chromatin states. The present review article examines these three modules and highlights various optimization methods. Additionally, it outlines innovative tools such as optogenetic systems and receptor-integrated systems that enable spatiotemporal control of genome editor expression. These modular instruments overcome traditional boundaries and allow scientists to create plants with favorable characteristics, decipher complex gene networks, and adopt sustainable farming practices.},
}

@article {pmid40565548,
year = {2025},
author = {Xavier, KVM and Silva, AMA and Luz, ACO and da Silva, FSC and de Melo, BST and Pitta, JLLP and Leal-Balbino, TC},
title = {Diversity and Role of Prophages in Pseudomonas aeruginosa: Resistance Genes and Bacterial Interactions.},
journal = {Genes},
volume = {16},
number = {6},
pages = {},
doi = {10.3390/genes16060656},
pmid = {40565548},
issn = {2073-4425},
mesh = {*Pseudomonas aeruginosa/genetics/virology/drug effects/pathogenicity ; *Prophages/genetics ; CRISPR-Cas Systems/genetics ; *Drug Resistance, Bacterial/genetics ; Humans ; Pseudomonas Infections/microbiology ; Genome, Bacterial ; Brazil ; },
abstract = {Pseudomonas aeruginosa is a major pathogen associated with hospital-acquired infections, and the spread of carbapenem-resistant isolates highlights the urgency of developing non-conventional therapies, such as phage therapy. For this alternative to be effective, understanding phage-host interactions is crucial for the selection of candidate phages and offers new insights into these dynamics. Background/Objectives: This study aimed to characterize prophage diversity in clinical P. aeruginosa genomes, assess the relationship between phages and the CRISPR/Cas system, and investigate the potential role of prophages in disseminating resistance genes. Methods: A total of 141 genomes from Brazilian hospitals were analyzed. Prophage detection was performed using VIBRANT, and in silico analyses were conducted to evaluate taxonomic diversity, the presence of resistance genes, phage life cycle, genomic distribution, and the presence of the CRISPR/Cas system. Results: A total of 841 viral sequences were identified by the VIBRANT tool, of which 498 were confirmed by CheckV, with a predominance of the class Caudoviricetes and high overall phage diversity. No statistically significant difference was observed in the number of prophages between isolates with and without CRISPR/Cas systems. Prophages carrying resistance genes such as rsmA, OXA-56, SPM-1, and others were detected in isolates harboring the type I-C CRISPR/Cas system. Additionally, prophages showed no preference for specific insertion sites along the bacterial genome. Conclusions: These findings provide evidence of a well-established phage-host relationship. The dual role of prophages-as vectors of antimicrobial resistance and as potential therapeutic agents-reflects their dynamic impact on bacterial communities and reinforces their importance in developing new strategies to combat antimicrobial resistance.},
}

*Pseudomonas aeruginosa/genetics/virology/drug effects/pathogenicity
*Prophages/genetics
CRISPR-Cas Systems/genetics
*Drug Resistance, Bacterial/genetics
Humans
Pseudomonas Infections/microbiology
Genome, Bacterial
Brazil

@article {pmid40565267,
year = {2025},
author = {Chakraborty, M and Nielsen, L and Nash, D and Bruder, MR and Nissimov, JI and Charles, TC and Aucoin, MG},
title = {Baculovirus Variant Detection from Transient CRISPR-Cas9-Mediated Disruption of gp64 at Different Gene Locations.},
journal = {International journal of molecular sciences},
volume = {26},
number = {12},
pages = {},
doi = {10.3390/ijms26125805},
pmid = {40565267},
issn = {1422-0067},
support = {RGPIN 355513-2017/RGPIN-2023-03666//Natural Sciences and Engineering Research Council/ ; },
mesh = {*CRISPR-Cas Systems ; Animals ; *Baculoviridae/genetics ; *Viral Envelope Proteins/genetics ; Genetic Vectors/genetics ; Genome, Viral ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; Sf9 Cells ; Mutation ; },
abstract = {The Baculovirus Expression Vector System (BEVS) is an important protein and complex biologics production platform. The baculovirus GP64 protein is the major envelope glycoprotein that aids in virus entry and is required for cell-to-cell transmission in cell culture. Several studies have developed strategies around gp64 gene disruption in an attempt to minimize baculovirus co-production. Here, we investigate the result of transiently targeting the baculovirus gp64 gene with CRISPR-Cas9 during infection. Because not all genomes are effectively disrupted, we describe a variant calling methodology that allows the detection of the targeted mutations in gp64 even though these mutations are not the dominant sequences. Using a transfection-infection assay (T-I assay), the AcMNPV gp64 gene was targeted at six different locations to evaluate the effects of single and multiple targeting sites, and we demonstrated a reduction in the levels of baculovirus vectors while maintaining or enhancing foreign protein production when protein was driven by a p6.9 promoter. Viral genomes were subsequently isolated from the supernatant and cell pellet fractions, and our sequencing pipeline successfully detected indel mutations within gp64 for most of the single-guide RNA (sgRNA) targets. We also observed that 68.8% of variants found in the virus stock were conserved upon virus propagation in cell culture, thus indicating that they are not detrimental to viral fitness. This work provides a comprehensive assessment of CRISPR-Cas9 genome editing of baculovirus vectors, with potential applications in enhancing the efficiency of the BEVS.},
}

*CRISPR-Cas Systems
Animals
*Baculoviridae/genetics
*Viral Envelope Proteins/genetics
Genetic Vectors/genetics
Genome, Viral
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing
Sf9 Cells
Mutation

@article {pmid40565111,
year = {2025},
author = {Bouchard, C and Rousseau, J and Lamothe, G and Dubost, M and Bastrenta, L and Ramezani, S and Tremblay, JP},
title = {In Vitro Correction of Point Mutations in the DYSF Gene Using Prime Editing.},
journal = {International journal of molecular sciences},
volume = {26},
number = {12},
pages = {},
doi = {10.3390/ijms26125647},
pmid = {40565111},
issn = {1422-0067},
support = {TREM-01//Jain Foundation/ ; },
mesh = {Humans ; *Point Mutation ; *Gene Editing/methods ; HEK293 Cells ; *Dysferlin/genetics ; Genetic Therapy/methods ; Myoblasts/metabolism ; Fibroblasts/metabolism ; CRISPR-Cas Systems ; },
abstract = {Dysferlinopathy is caused by over 500 mutations in the gene encoding dysferlin, including close to 300 point mutations. One option to cure the disease is to use a gene therapy to correct these mutations at the root. Prime editing is a technique which can replace the mutated nucleotide with the wild-type nucleotide. In this article, prime editing is used to correct several point mutations in the DYSF gene responsible for dysferlinopathy. In vitro editing of HEK293T cells reaches up to 31%. Notably, editing was more efficient in myoblasts than in patient-derived fibroblasts. The prime editing technique was also used to create a new myoblast clone containing a patient mutation from a healthy myoblast cell line.},
}

Humans
*Point Mutation
*Gene Editing/methods
HEK293 Cells
*Dysferlin/genetics
Genetic Therapy/methods
Myoblasts/metabolism
Fibroblasts/metabolism
CRISPR-Cas Systems

@article {pmid40521923,
year = {2025},
author = {Cao, Y and Huang, C and Li, K and Zhang, X and Tong, M and Tu, J and Yang, K and Yuan, S and Zhang, H},
title = {CRISPR/Cas12a-assisted electrochemiluminescent detection of ochratoxin A based on COF@Ru coupled with a DNA tetrahedral scaffold.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {25},
pages = {5298-5307},
doi = {10.1039/d5ay00427f},
pmid = {40521923},
issn = {1759-9679},
mesh = {*Ochratoxins/analysis ; *CRISPR-Cas Systems ; *Electrochemical Techniques/methods ; *Biosensing Techniques/methods ; *Luminescent Measurements/methods ; *Metal-Organic Frameworks/chemistry ; Limit of Detection ; Gold/chemistry ; *DNA/chemistry ; Metal Nanoparticles/chemistry ; Aptamers, Nucleotide/chemistry ; DNA Probes/chemistry ; },
abstract = {To sensitively detect ochratoxin A (OTA), a CRISPR/Cas12a-assisted electrochemiluminescence (ECL) aptasensor based on COF@Ru coupled with a DNA tetrahedral scaffold (DTS) was successfully fabricated. The covalent organic framework (COF) acted as a confined micro-reactor for the tris(2,2'-bipyridyl)ruthenium(II)/tri-n-propylamine (Ru(bpy)3[2+]/TPrA) system and hence, evidently enhanced ECL signals. Au nanoparticles (AuNPs) coupled with DTS caused more ferrocene (Fc)-modified quenching DNA probe molecules to be attached to the surface of the sensing electrode, which could reduce the detection background. Activated CRISPR/Cas12a could effectively amplify the ECL signal since it could cut off a considerable amount of quenching DNA probe molecules and make them move away from the electrode. Owing to these strategies, the fabricated ECL aptasensor could detect OTA as low as 3.5 fg mL[-1] in a linear detection range of 10[-5]-100 ng mL[-1]. Furthermore, the CRISPR/Cas12a and COF@Ru coupled with DTS-based ECL biosensor possessed high stability and specificity. More importantly, the ECL biosensor could effectively detect OTA in food samples, indicating that it can play a role in monitoring OTA levels in the field of food safety.},
}

*Ochratoxins/analysis
*CRISPR-Cas Systems
*Electrochemical Techniques/methods
*Biosensing Techniques/methods
*Luminescent Measurements/methods
*Metal-Organic Frameworks/chemistry
Limit of Detection
Gold/chemistry
*DNA/chemistry
Metal Nanoparticles/chemistry
Aptamers, Nucleotide/chemistry
DNA Probes/chemistry

@article {pmid40518166,
year = {2025},
author = {Ge, L and Xiong, H},
title = {[Advances in CRISPR-Cas9 genome editing for the treatment of muscular dystrophies].},
journal = {Zhonghua er ke za zhi = Chinese journal of pediatrics},
volume = {63},
number = {7},
pages = {808-811},
doi = {10.3760/cma.j.cn112140-20250421-00356},
pmid = {40518166},
issn = {0578-1310},
mesh = {Humans ; *Genetic Therapy/methods ; *CRISPR-Cas Systems ; *Muscular Dystrophies/therapy/genetics ; Animals ; *Gene Editing/methods ; Muscular Dystrophy, Duchenne/therapy/genetics ; Disease Models, Animal ; },
}

Humans
*Genetic Therapy/methods
*CRISPR-Cas Systems
*Muscular Dystrophies/therapy/genetics
Animals
*Gene Editing/methods
Muscular Dystrophy, Duchenne/therapy/genetics
Disease Models, Animal

@article {pmid40501421,
year = {2025},
author = {Zheng, Y and Liu, B and Zuo, Q and Deng, F and Lan, C},
title = {Detection of Streptococcus anginosus in fecal samples using PCR-CRISPR /Cas12a system.},
journal = {Bioanalysis},
volume = {17},
number = {11},
pages = {737-745},
doi = {10.1080/17576180.2025.2518042},
pmid = {40501421},
issn = {1757-6199},
mesh = {*Feces/microbiology ; Humans ; *CRISPR-Cas Systems/genetics ; *Polymerase Chain Reaction/methods ; *Streptococcus anginosus/genetics/isolation & purification ; DNA, Bacterial/genetics ; RNA, Ribosomal, 16S/genetics ; },
abstract = {OBJECTIVE: To develop a highly sensitive and specific detection method based on PCR-CRISPR/Cas12a for the detection of Streptococcus anginosus (S. anginosus) in feces and to evaluate its detection rate in the general population as well as its potential as a gastrointestinal tumor marker.

MATERIALS AND METHODS: Specific primers and crRNA targeting the 16S rDNA of S. anginosus were designed to construct a PCR-CRISPR/Cas12a detection system. A total of 230 fecal samples were collected from the general population, and bacterial DNA was extracted. The target gene was detected using this system to verify its sensitivity, specificity, and stability.

RESULTS: The established detection system demonstrated strong specificity, with stable recognition of S. anginosus, and a minimum detection limit of 10[-7] ng/μL. The detection rate of S. anginosus in fecal samples from the general population was 51.7%.

CONCLUSION: The PCR-CRISPR/Cas12a system can efficiently detect S. anginosus in feces, providing a reliable technical tool for exploring its association with gastrointestinal tumors.},
}

*Feces/microbiology
Humans
*CRISPR-Cas Systems/genetics
*Polymerase Chain Reaction/methods
*Streptococcus anginosus/genetics/isolation & purification
DNA, Bacterial/genetics
RNA, Ribosomal, 16S/genetics

@article {pmid40490360,
year = {2025},
author = {Kagawa, N and Umesono, Y and Suzuki, KT and Mochii, M},
title = {Step-by-Step Protocol for Making a Knock-In Xenopus laevis to Visualize Endogenous Gene Expression.},
journal = {Development, growth & differentiation},
volume = {67},
number = {5},
pages = {293-302},
doi = {10.1111/dgd.70011},
pmid = {40490360},
issn = {1440-169X},
support = {//University of Hyogo/ ; JP18K06266//Japan Society for the Promotion of Science/ ; JP21H03829//Japan Society for the Promotion of Science/ ; 22NIBB331//NIBB Collaborative Research Program/ ; 23NIBB340//NIBB Collaborative Research Program/ ; JPMJCR 2025//JST, CREST/ ; },
mesh = {Animals ; *Xenopus laevis/genetics ; *Gene Knock-In Techniques/methods ; CRISPR-Cas Systems/genetics ; Green Fluorescent Proteins/genetics/metabolism ; },
abstract = {We established a novel knock-in technique, New and Easy Xenopus Targeted integration (NEXTi), to recapitulate endogenous gene expression by reporter expression. NEXTi is a CRISPR-Cas9-based method to integrate a donor DNA containing a reporter gene (egfp) into the target 5' untranslated region (UTR) of the Xenopus laevis genome. It enables us to track eGFP expression under the regulation of endogenous promoter/enhancer activities. We obtained about 2% to 13% of knock-in vector-injected embryos showing eGFP signal in a tissue-specific manner, targeting krt.12.2.L, myod1.S, sox2.L, and bcan.S loci, as previously reported. In addition, F1 embryos which show stable eGFP signals were obtained by outcrossing the matured injected frogs with wild-type animals. Integrations of donor DNAs into target 5' UTRs were confirmed by PCR amplification and sequencing. Here, we describe the step-by-step protocol for preparation of donor DNA and single guide RNA, microinjection, and genotyping of F1 animals for the NEXTi procedure.},
}

Animals
*Xenopus laevis/genetics
*Gene Knock-In Techniques/methods
CRISPR-Cas Systems/genetics
Green Fluorescent Proteins/genetics/metabolism

@article {pmid39883121,
year = {2025},
author = {Paryani, M and Gupta, N and Jain, SK and Butani, S},
title = {Lowering LDL cholesterol by PCSK9 inhibition: a new era of gene silencing, RNA, and alternative therapies.},
journal = {Naunyn-Schmiedeberg's archives of pharmacology},
volume = {398},
number = {6},
pages = {6597-6615},
pmid = {39883121},
issn = {1432-1912},
mesh = {Humans ; *Antibodies, Monoclonal/pharmacology/therapeutic use ; Cholesterol, LDL/blood ; CRISPR-Cas Systems ; Gene Silencing ; Hypercholesterolemia/drug therapy/economics ; *Oligonucleotides, Antisense/pharmacology/therapeutic use ; *Plant Extracts/pharmacology/therapeutic use ; Proprotein Convertase 9/genetics/metabolism ; *Proprotein Convertases/antagonists & inhibitors ; Protein Subunit Vaccines/pharmacology/therapeutic use ; *RNA, Small Interfering/pharmacology/therapeutic use ; *Anticholesteremic Agents/pharmacology/therapeutic use ; Fibronectin Type III Domain ; Recombinant Proteins ; },
abstract = {Proprotein convertase subtilisin/kexin type 9 (PCSK9) discovery has added a new paradigm to our understanding of cholesterol homeostasis and lipid metabolism. Since its discovery, PCSK9 inhibitors have become a widely investigated therapeutic class for lipid management in cardiovascular diseases and hypercholesterolemia. Scientists have explored different approaches for PCSK9 inhibition, such as monoclonal antibodies (mAbs), gene silencing and gene editing techniques, vaccines, mimetic peptides, and small molecules. European Medicines Agency (EMA) and United States Food and Drug Administration (US FDA) have approved only three PCSK9 inhibitors, including two monoclonal antibodies and one small interfering ribonucleic acid (siRNA). Despite the efficacy of approved large molecules, high costs and the need for regular injection have limited their adherence to the patient. This review aims to provide an understanding of PCSK9's function in Low-Density Lipoprotein Cholesterol (LDL-C) management, its current treatment, recent advancements, and potential future development of small molecules in the class of PCSK9 inhibitors.},
}

Humans
*Antibodies, Monoclonal/pharmacology/therapeutic use
Cholesterol, LDL/blood
CRISPR-Cas Systems
Gene Silencing
Hypercholesterolemia/drug therapy/economics
*Oligonucleotides, Antisense/pharmacology/therapeutic use
*Plant Extracts/pharmacology/therapeutic use
Proprotein Convertase 9/genetics/metabolism
*Proprotein Convertases/antagonists & inhibitors
Protein Subunit Vaccines/pharmacology/therapeutic use
*RNA, Small Interfering/pharmacology/therapeutic use
*Anticholesteremic Agents/pharmacology/therapeutic use
Fibronectin Type III Domain
Recombinant Proteins

@article {pmid30965672,
year = {2019},
author = {Song, HY and Chien, CS and Yarmishyn, AA and Chou, SJ and Yang, YP and Wang, ML and Wang, CY and Leu, HB and Yu, WC and Chang, YL and Chiou, SH},
title = {Generation of GLA-Knockout Human Embryonic Stem Cell Lines to Model Autophagic Dysfunction and Exosome Secretion in Fabry Disease-Associated Hypertrophic Cardiomyopathy.},
journal = {Cells},
volume = {8},
number = {4},
pages = {},
pmid = {30965672},
issn = {2073-4409},
mesh = {Apoptosis ; *Autophagy ; CRISPR-Cas Systems ; Cardiomyopathy, Hypertrophic/etiology/*genetics/*pathology ; Cell Line ; Exosomes ; Fabry Disease/*complications ; Gene Knockout Techniques ; Human Embryonic Stem Cells/metabolism ; Humans ; *Models, Biological ; Myocytes, Cardiac/metabolism ; Reactive Oxygen Species/metabolism ; Trihexosylceramides/metabolism ; alpha-Galactosidase/*genetics ; },
abstract = {Fabry disease (FD) is a rare inherited disorder characterized by a wide range of systemic symptoms; it is particularly associated with cardiovascular and renal problems. Enzyme replacement therapy and pharmacological chaperone migalastat are the only approved and effective treatment strategies for FD patients. It is well documented that alpha-galactosidase A (GLA) enzyme activity deficiency causes globotriaosylceramide (Gb3) accumulation, which plays a crucial role in the etiology of FD. However, the detailed mechanisms remain unclear, and the lack of a reliable and powerful disease model is an obstacle. In this study, we created such a model by using CRISPR/Cas9-mediated editing of GLA gene to knockout its expression in human embryonic stem cells (hESCs). The cardiomyocytes differentiated from these hESCs (GLA-null CMs) were characterized by the accumulation of Gb3 and significant increases of cell surface area, the landmarks of FD-associated cardiomyopathy. Furthermore, we used mass spectrometry to compare the proteomes of GLA-null CMs and parental wild type CMs and found that the Rab GTPases involved in exocytotic vesicle release were significantly downregulated. This caused impairment of autophagic flux and protein turnover, resulting in an increase of reactive oxygen species and apoptosis. To summarize, we established a FD model which can be used as a promising tool to study human hypertrophic cardiomyopathy in a physiologically and pathologically relevant manner and to develop new therapies by targeting Rab GTPases signaling-related exosomal vesicles transportation.},
}

Apoptosis
*Autophagy
CRISPR-Cas Systems
Cardiomyopathy, Hypertrophic/etiology/*genetics/*pathology
Cell Line
Exosomes
Fabry Disease/*complications
Gene Knockout Techniques
Human Embryonic Stem Cells/metabolism
Humans
*Models, Biological
Myocytes, Cardiac/metabolism
Reactive Oxygen Species/metabolism
Trihexosylceramides/metabolism
alpha-Galactosidase/*genetics

@article {pmid28565744,
year = {2017},
author = {Cheng, LH and Liu, Y and Niu, T},
title = {[Chromosomal large fragment deletion induced by CRISPR/Cas9 gene editing system].},
journal = {Zhonghua xue ye xue za zhi = Zhonghua xueyexue zazhi},
volume = {38},
number = {5},
pages = {427-431},
pmid = {28565744},
issn = {0253-2727},
mesh = {Animals ; *CRISPR-Cas Systems ; Chromosome Deletion ; DNA ; Gene Deletion ; *Gene Editing ; HEK293 Cells ; Humans ; Mice ; Multigene Family ; Mutagenesis, Insertional ; NIH 3T3 Cells ; Plasmids ; },
abstract = {Objective: Using CRISPR-Cas9 gene editing technology to achieve a number of genes co-deletion on the same chromosome. Methods: CRISPR-Cas9 lentiviral plasmid that could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse 11B3 chromosome was constructed via molecular clone. HEK293T cells were transfected to package lentivirus of CRISPR or Cas9 cDNA, then mouse NIH3T3 cells were infected by lentivirus and genomic DNA of these cells was extracted. The deleted fragment was amplified by PCR, TA clone, Sanger sequencing and other techniques were used to confirm the deletion of Aloxe3-Alox12b-Alox8 cluster genes. Results: The CRISPR-Cas9 lentiviral plasmid, which could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes, was successfully constructed. Deletion of target chromosome fragment (Aloxe3-Alox12b-Alox8 cluster genes) was verified by PCR. The deletion of Aloxe3-Alox12b-Alox8 cluster genes was affirmed by TA clone, Sanger sequencing, and the breakpoint junctions of the CRISPR-Cas9 system mediate cutting events were accurately recombined, insertion mutation did not occur between two cleavage sites at all. Conclusion: Large fragment deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse chromosome 11B3 was successfully induced by CRISPR-Cas9 gene editing system.},
}

Animals
*CRISPR-Cas Systems
Chromosome Deletion
DNA
Gene Deletion
*Gene Editing
HEK293 Cells
Humans
Mice
Multigene Family
Mutagenesis, Insertional
NIH 3T3 Cells
Plasmids

@article {pmid28549865,
year = {2017},
author = {Powell, SK and Gregory, J and Akbarian, S and Brennand, KJ},
title = {Application of CRISPR/Cas9 to the study of brain development and neuropsychiatric disease.},
journal = {Molecular and cellular neurosciences},
volume = {82},
number = {},
pages = {157-166},
pmid = {28549865},
issn = {1095-9327},
support = {P50 MH096890/MH/NIMH NIH HHS/United States ; R01 MH101454/MH/NIMH NIH HHS/United States ; R01 MH106056/MH/NIMH NIH HHS/United States ; },
mesh = {Brain/*growth & development ; Brain Diseases/therapy ; CRISPR-Cas Systems/*genetics ; *Gene Editing/methods ; Gene Expression/*genetics ; Humans ; Induced Pluripotent Stem Cells/*cytology ; },
abstract = {CRISPR/Cas9 technology has transformed our ability to manipulate the genome and epigenome, from efficient genomic editing to targeted localization of effectors to specific loci. Through the manipulation of DNA- and histone-modifying enzyme activities, activation or repression of gene expression, and targeting of transcriptional regulators, the role of gene-regulatory and epigenetic pathways in basic biology and disease processes can be directly queried. Here, we discuss emerging CRISPR-based methodologies, with specific consideration of neurobiological applications of human induced pluripotent stem cell (hiPSC)-based models.},
}

Brain/*growth & development
Brain Diseases/therapy
CRISPR-Cas Systems/*genetics
*Gene Editing/methods
Gene Expression/*genetics
Humans
Induced Pluripotent Stem Cells/*cytology

@article {pmid28549739,
year = {2017},
author = {Deng, H and Gao, R and Liao, X and Cai, Y},
title = {Characterization of a major facilitator superfamily transporter in Shiraia bambusicola.},
journal = {Research in microbiology},
volume = {168},
number = {7},
pages = {664-672},
doi = {10.1016/j.resmic.2017.05.002},
pmid = {28549739},
issn = {1769-7123},
mesh = {Ascomycota/chemistry/drug effects/*genetics/pathogenicity ; CRISPR-Cas Systems ; Membrane Transport Proteins/*genetics/*metabolism ; Mutation ; Perylene/*analogs & derivatives/metabolism/pharmacology ; Phenol ; Quinones/*metabolism/pharmacology ; Reactive Oxygen Species/metabolism ; Sasa/microbiology ; },
abstract = {Reactive oxygen species (ROS) generated by photo-activated hypocrellin from Shiraia bambusicola are detrimental to cellular macromolecules. However, S. bambusicola can still maintain excellent morphology during continuous hypocrellin production, indicating an extraordinary autoresistance system that protects against the harmful ROS. In this study, a major facilitator superfamily transporter (MFS) was isolated from S. bambusicola and deleted using the clustered regularly interspaced short palindromic repeat sequences (CRISPR)/Cas9 system. The ΔMFS mutant abolished hypocrellin production and was slightly sensitive to 40-μM hypocrellin, while the ΔMFS compliment strain restored hypocrellin production and resistance. Hypocrellin treatment also enhanced the relative expression of MFS in wild-type S. bambusicola. Subsequent pathogenicity assays showed that MFS deletion reduced damage to bamboo leaves. By contrast, restoration of hypocrellin production in the MFS compliment strain generated similar necrotic lesions on bamboo leaves to those observed with the wild-type strain. These results revealed that the identified MFS is involved in efflux of hypocrellin from cells, which reduces the hypocrellin toxicity. Furthermore, hypocrellin contributed to the virulence of S. bambusicola on bamboo leaves. These findings could help to reduce plant loss by disrupting hypocrellin biosynthesis in S. bambusicola, or overexpressing the associated resistance gene in transgenic plants.},
}

Ascomycota/chemistry/drug effects/*genetics/pathogenicity
CRISPR-Cas Systems
Membrane Transport Proteins/*genetics/*metabolism
Mutation
Perylene/*analogs & derivatives/metabolism/pharmacology
Phenol
Quinones/*metabolism/pharmacology
Reactive Oxygen Species/metabolism
Sasa/microbiology

@article {pmid40562814,
year = {2025},
author = {Qiao, JH and Zang, Y and Gao, Q and Liu, S and Zhang, XW and Hu, W and Wang, Y and Han, C and Li, D and Wang, XB},
title = {Transgene- and tissue culture-free heritable genome editing using RNA virus-based delivery in wheat.},
journal = {Nature plants},
volume = {},
number = {},
pages = {},
pmid = {40562814},
issn = {2055-0278},
support = {32425046//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {CRISPR-Cas genome editing technology is a cutting-edge strategy for crop breeding. However, the delivery of genome-editing reagents remains to be a technological bottleneck in monocot plants[1]. Here we engineered barley yellow striate mosaic virus (BYSMV) into a negative-strand RNA virus-based vector system[2] for delivery of both Cas9 and single guide RNA to achieve heritable gene editing in different wheat cultivars. We found that fusion of a mobile transfer RNA sequence[3] to the Cas9 messenger RNA and single guide RNAs could deliver them into the growth points of axillary meristems to achieve gene editing before tiller generation. The resulting nascent tillers contained simultaneous mutations in the three homoeoalleles. Moreover, the progeny seedlings are virus-free and harbour bi-allelic or homozygous mutations. Given BYSMV infects 26 monocot species[4], the BYSMV delivery system could have wide applicability for achieving highly efficient, non-transgenic and less genotype-dependent heritable genome editing, thereby facilitating genomic studies and crops breeding.},
}

@article {pmid40560955,
year = {2025},
author = {Leprince, A and Moineau, S},
title = {CRISPR-Cas in the Cheese Industry.},
journal = {Annual review of virology},
volume = {},
number = {},
pages = {},
doi = {10.1146/annurev-virology-092623-111016},
pmid = {40560955},
issn = {2327-0578},
abstract = {Bacteria have evolved a wide range of defense systems to combat phage infections. In the cheese industry, lactic acid bacteria (LAB) used for milk fermentation continuously face threats from phages. Therefore, selecting or developing industrial strains with enhanced phage resistance requires a focus on robust defense systems. Among these systems, the clustered regularly interspaced short palindromic repeats (CRISPR) and their CRISPR-associated proteins (Cas) are notably prevalent in LAB. The early characterization of this adaptive immune system was closely tied to the cheese industry, particularly with Streptococcus thermophilus in which CRISPR-Cas systems are ubiquitous and highly active. This review underscores the contributions of S. thermophilus and its virulent phages to our understanding of the function and mechanisms of CRISPR-Cas systems. Additionally, we review the diversity of CRISPR-Cas systems in LAB used in the cheese industry, the counter-defense strategies employed by dairy phages, and the applications of CRISPR-Cas systems within this sector.},
}

@article {pmid40560862,
year = {2025},
author = {Nakamura, K and Ito, S and Ohguchi, Y and Jimbo, T and Wada, Y and Nakajima, R and Kanou, M and Yamana, K and Ueda, H},
title = {Establishment of Cre/LoxP-mediated multifunctional reporter knock-in rats with the CRISPR system.},
journal = {PloS one},
volume = {20},
number = {6},
pages = {e0325444},
doi = {10.1371/journal.pone.0325444},
pmid = {40560862},
issn = {1932-6203},
mesh = {Animals ; Rats ; *Integrases/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *Genes, Reporter ; Rats, Transgenic ; Male ; },
abstract = {Rats and mice are essential experimental animals in preclinical research, serving as models for various human diseases and contributing significantly to drug development. Rats offer distinct advantages over mice due to their larger size, which allows for more complex surgical procedures, repeated blood sampling, or sophisticated behavioral analysis. However, unlike the case with mice, genetically modified rat lines for achieving complex experimental objectives-such as tissue-specific gene knockout or visualization of specific cell populations-are still limited. We here established LoxP-mediated multifunctional reporter KI rats, enabling us to evaluate fluorescence, bioluminescence, and cell-killing assays simultaneously with only one gene-modified rat line. CRISPR/Cas12a, also known as CRISPR/Cpf1, was successfully used to insert the Cre sequence into a target locus to generate Cre driver rats. These results will contribute to the application of gene-modified rats for a more comprehensive understanding of physiology, and for extrapolation of their capabilities in preclinical research.},
}

Animals
Rats
*Integrases/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Gene Knock-In Techniques/methods
*Genes, Reporter
Rats, Transgenic
Male

@article {pmid40560492,
year = {2025},
author = {Lv, D and Xu, Y and Wang, ZX and Zhang, QL and Yan, JP and Xu, JW},
title = {CRISPR/Cas9-mediated genome editing in Ganoderma lucidum: recent advances and biotechnological opportunities.},
journal = {World journal of microbiology & biotechnology},
volume = {41},
number = {7},
pages = {223},
pmid = {40560492},
issn = {1573-0972},
support = {32360014//National Natural Science Foundation of China/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Reishi/genetics/metabolism ; *Biotechnology/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Fungal ; },
abstract = {Ganoderma lucidum is a well-known traditional medicinal mushroom that has attracted considerable attention due to its potential as a promising cell factory for producing high-value bioactive compounds. However, conventional methods for the genetic manipulation of G. lucidum are often time-consuming and labor-intensive, hindering research into the biosynthesis and regulatory mechanisms of its valuable natural products. In recent years, the clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) system has emerged as a powerful genome editing tool, extensively utilized in life sciences research due to its high efficiency and user-friendliness. This review provides a structured overview of advancements in CRISPR/Cas9-mediated genome editing technology in G. lucidum. We first discuss the development and optimization of the CRISPR system, focusing on the various expression strategies for Cas9 and guide RNA established in G. lucidum. Furthermore, we highlight the application of this system for targeted gene deletion, insertion, and replacement in genome editing, as well as its use in the functional analysis of genes in G. lucidum. In addition, we discuss the limitations and challenges associated with employing CRISPR/Cas9 tools in G. lucidum and provide an outlook on the future development of the CRISPR/Cas9 system and its applications in this organism.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Reishi/genetics/metabolism
*Biotechnology/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Genome, Fungal

@article {pmid40560319,
year = {2025},
author = {Peer, LA and Mir, BA},
title = {Molecular mechanisms and genetic regulation of self-incompatibility in flowering plants: implications for crop improvement and evolutionary biology.},
journal = {Plant molecular biology},
volume = {115},
number = {4},
pages = {76},
pmid = {40560319},
issn = {1573-5028},
mesh = {*Self-Incompatibility in Flowering Plants/genetics ; *Crops, Agricultural/genetics ; Biological Evolution ; *Gene Expression Regulation, Plant ; Evolution, Molecular ; Pollination/genetics ; Self-Fertilization/genetics ; Plant Proteins/genetics/metabolism ; },
abstract = {Self-incompatibility is a fundamental biological mechanism in flowering plants that prevents self-fertilization, thereby promoting outcrossing and enhancing genetic diversity. This complex system has independently evolved across multiple angiosperm lineages and is crucial in maintaining plant reproductive success. Recent research has expanded our understanding of self-incompatibility's molecular basis and uncovered key genes and signaling pathways involved in self-incompatibility responses, such as S-RNase in Solanaceae and PrsS-PrpS in Papaveraceae, as well as the SRK-SCR interaction in Brassicaceae. However, despite significant advances, many aspects of self-incompatibility, such as the interplay between gene duplications, polyploidization, and the evolution of novel self-incompatibility mechanisms, remain underexplored. This review integrates findings from various plant families, including Solanaceae, Rosaceae, Papaveraceae, and Brassicaceae, and discusses the evolutionary dynamics of self-incompatibility systems, highlighting the role of gene duplication, recombination, and translocation events in shaping self-incompatibility diversity. Special emphasis is placed on understanding how modern molecular techniques, such as CRISPR/Cas9 and marker-assisted selection, can be employed to transition self-incompatibility to self-compatibility in economically significant crops. Additionally, the role of epigenetic changes and modifier genes in mediating transitions from self-incompatibility to self-compatibility is addressed, offering insights into how these mechanisms can be leveraged for crop breeding and hybrid seed production. Future research should focus on elucidating the molecular mechanisms underlying self-incompatibility responses, exploring the potential of targeted gene editing to overcome reproductive barriers, and understanding the evolutionary resilience of self-incompatibility systems to environmental changes.},
}

*Self-Incompatibility in Flowering Plants/genetics
*Crops, Agricultural/genetics
Biological Evolution
*Gene Expression Regulation, Plant
Evolution, Molecular
Pollination/genetics
Self-Fertilization/genetics
Plant Proteins/genetics/metabolism

@article {pmid40559612,
year = {2025},
author = {Jurić, I and Jelić, M and Markanović, M and Kanižaj, L and Bošnjak, Z and Budimir, A and Kuliš, T and Tambić-Andrašević, A and Ivančić-Baće, I and Mareković, I},
title = {CRISPR-Cas Dynamics in Carbapenem-Resistant and Carbapenem-Susceptible Klebsiella pneumoniae Clinical Isolates from a Croatian Tertiary Hospital.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {6},
pages = {},
doi = {10.3390/pathogens14060604},
pmid = {40559612},
issn = {2076-0817},
support = {10106-24-1295 to I.M. and 20286539 to I.I.B.//School od Medicine, University of Zagreb and Faculty of Science, University of Zagreb/ ; },
mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation & purification/classification ; Humans ; *CRISPR-Cas Systems/genetics ; *Carbapenems/pharmacology ; *Klebsiella Infections/microbiology ; beta-Lactamases/genetics ; Croatia ; Tertiary Care Centers ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; Bacterial Proteins/genetics ; Multilocus Sequence Typing ; Drug Resistance, Multiple, Bacterial/genetics ; *Carbapenem-Resistant Enterobacteriaceae/genetics/drug effects/isolation & purification ; },
abstract = {(1) Background: CRISPR-Cas systems provide adaptive immunity against mobile genetic elements (MGEs) carrying antimicrobial resistance (AMR) genes. Carbapenem-resistant (CR) Klebsiella pneumoniae is a major public health concern, and the role of CRISPR-Cas in its resistance is understudied. This study explored CRISPR-Cas associations with multidrug resistance in clinical K. pneumoniae. (2) Methods: 400 K. pneumoniae isolates (200 CR and 200 carbapenem susceptible (CS)) were analyzed. Carbapenemase genes (blaOXA-48, blaNDM-1, blaKPC-2), cas1, rpoB, and CRISPR1-3 loci were identified by PCR, while only CRISPR loci were sequenced. Genetic relatedness was assessed via PFGE, MLST, and spacer analysis. Statistical analysis utilized chi-squared and Fisher's exact tests. (3) Results: CRISPR-Cas was present in 15.8% of isolates, mainly subtypes I-E and I-E* (93.3%), with CRISPR3 loci showing the greatest spacer diversity. Clonal complexes ST14/15/101 (CR) and ST35 (CS) were identified. blaOXA-48 was linked to CRISPR-Cas-negative strains, while blaNDM-1 and blaKPC-2 were more frequent in CRISPR-Cas-positive strains (p < 0.0001). Imipenem/relebactam resistance was higher in CRISPR-Cas-negative isolates. (4) Conclusions: K. pneumoniae CRISPR-Cas systems correlate with specific carbapenemase profiles, suggesting pressure against blaOXA-48 acquisition. The coexistence of I-E and I-E* subtypes highlight synergies in targeting MGEs. CRISPR loci could be tools for subtyping organisms following MLST.},
}

*Klebsiella pneumoniae/genetics/drug effects/isolation & purification/classification
Humans
*CRISPR-Cas Systems/genetics
*Carbapenems/pharmacology
*Klebsiella Infections/microbiology
beta-Lactamases/genetics
Croatia
Tertiary Care Centers
Anti-Bacterial Agents/pharmacology
Microbial Sensitivity Tests
Bacterial Proteins/genetics
Multilocus Sequence Typing
Drug Resistance, Multiple, Bacterial/genetics
*Carbapenem-Resistant Enterobacteriaceae/genetics/drug effects/isolation & purification

@article {pmid40558493,
year = {2025},
author = {Parikh, SJ and Terron, HM and Burgard, LA and Butler, DD and LaFerla, FM and Lane, S and Leissring, MA},
title = {5' DREDGE: Direct Repeat-Enabled Downregulation of Gene Expression via the 5' UTR of Target Genes.},
journal = {Cells},
volume = {14},
number = {12},
pages = {},
doi = {10.3390/cells14120866},
pmid = {40558493},
issn = {2073-4409},
support = {1R01AG066928-04A1/NH/NIH HHS/United States ; },
mesh = {*5' Untranslated Regions/genetics ; *Down-Regulation/genetics ; Humans ; CRISPR-Cas Systems/genetics ; RNA, Messenger/genetics/metabolism ; },
abstract = {Despite the availability of numerous methods for controlling gene expression, there remains a strong need for technologies that maximize two key properties: selectivity and reversibility. To this end, we developed a novel approach that exploits the highly sequence-specific nature of CRISPR-associated endoribonucleases (Cas RNases), which recognize and cleave short RNA sequences known as direct repeats (DRs). In this approach, referred to as DREDGE (direct repeat-enabled downregulation of gene expression), selective control of gene expression is enabled by introducing one or more DRs into the untranslated regions (UTRs) of target mRNAs, which can then be cleaved upon expression of the cognate Cas RNase. We previously demonstrated that the expression of target genes with DRs in their 3' UTRs are efficiently controlled by the DNase-dead version of Cas12a (dCas12a) with a high degree of selectivity and complete reversibility. Here, we assess the feasibility of using DREDGE to regulate the expression of genes with DRs inserted in their 5' UTRs. Among the five different Cas RNases tested, Csy4 was found to be the most efficient in this context, yielding robust downregulation with rapid onset in doxycycline-regulatable systems targeting either a stably expressed fluorescent protein or an endogenous gene, both in a fully reversible manner. Unexpectedly, dCas12a was also found to be modestly effective despite binding essentially irreversibly to the cut mRNA on its 5' end and thereby boosting mRNA levels. Our results expand the utility of DREDGE as an attractive method for regulating gene expression in a targeted, highly selective, and fully reversible manner.},
}

*5' Untranslated Regions/genetics
*Down-Regulation/genetics
Humans
CRISPR-Cas Systems/genetics
RNA, Messenger/genetics/metabolism

@article {pmid40558461,
year = {2025},
author = {Jeddoub, O and Touil, N and Nyabi, O and El Fahime, E and Ennibi, K and Gala, JL and Benjouad, A and Belayachi, L},
title = {The Use of CRISPR-Cas Systems for Viral Detection: A Bibliometric Analysis and Systematic Review.},
journal = {Biosensors},
volume = {15},
number = {6},
pages = {},
doi = {10.3390/bios15060379},
pmid = {40558461},
issn = {2079-6374},
mesh = {*CRISPR-Cas Systems ; Bibliometrics ; Humans ; *Viruses/isolation & purification/genetics ; *Virus Diseases/diagnosis ; },
abstract = {Viral infections impose a significant burden on global public health and the economy. This study examines the current state of CRISPR-Cas system research, focusing on their applications in viral detection and their evolution over recent years. A bibliometric analysis and systematic review were conducted using articles published between 2019 and 2024, retrieved from Web of Science, Scopus, and PubMed databases. Out of 2713 identified articles, 194 were included in the analysis. The findings reveal substantial growth in scientific output related to CRISPR-Cas systems, with the United States leading in research and development in this field. The rapid increase in CRISPR-Cas research during this period underscores its immense potential to transform viral diagnostics. With advantages such as speed, precision, and suitability for deployment in resource-limited settings, CRISPR-Cas systems outperform many traditional diagnostic methods. The concerted efforts of scientists worldwide further highlight the promising future of this technology. CRISPR-Cas systems are emerging as a powerful alternative, offering the possibility of expedited and accessible point-of-care testing and paving the way for more equitable and effective diagnostics on a global scale.},
}

*CRISPR-Cas Systems
Bibliometrics
Humans
*Viruses/isolation & purification/genetics
*Virus Diseases/diagnosis

@article {pmid40558442,
year = {2025},
author = {Du, K and Zeng, Q and Jiang, M and Hu, Z and Zhou, M and Xia, K},
title = {CRISPR/Cas12a-Based Biosensing: Advances in Mechanisms and Applications for Nucleic Acid Detection.},
journal = {Biosensors},
volume = {15},
number = {6},
pages = {},
doi = {10.3390/bios15060360},
pmid = {40558442},
issn = {2079-6374},
support = {No. 82330035//National Natural Science Foundation of China/ ; No. 82130043//National Natural Science Foundation of China/ ; No. 82361138573//National Natural Science Foundation of China/ ; No. 2021SK1010//National Natural Science Foundation of Hunan Province/ ; No. 2024JJ5340//Natural Science Foundation of Hunan Province/ ; No. 2024RC3207//Science and Technology innovation Program of Hunan Province/ ; No. 23B0437//Scientific Research Fund of Hunan Provincial Education Department/ ; },
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Humans ; *Nucleic Acids/analysis ; DNA/analysis ; },
abstract = {Nucleic acid detection technology is crucial for molecular diagnosis. The advent of CRISPR/Cas12a-based nucleic acid detection has considerably broadened its scope, from the identification of infectious disease-causing microorganisms to the detection of disease-associated biomarkers. This innovative system capitalizes on the non-specific single-strand cleavage activity of Cas12a upon target DNA recognition. By employing a fluorescent probe in the form of a single-stranded DNA/RNA, this technology enables the observation of fluorescence changes resulting from nonspecific cleavage, thereby facilitating detection. CRISPR/Cas12a-based detection systems can be regarded as a new type of biosensor, offering a practical and efficient approach for nucleic acid analysis in various diagnostic settings. CRISPR/Cas12a-based biosensors outperform conventional nucleic acid detection methods in terms of portability, simplicity, speed, and efficiency. In this review, we elucidate the detection principle of CRISPR/Cas12a-based biosensors and their application in disease diagnostics and discuss recent innovations and technological challenges, aiming to provide insights for the research and further development of CRISPR/Cas12a-based biosensors in personalized medicine. Our findings show that although CRISPR/Cas12a-based biosensors have considerable potential for various applications and theoretical research, certain challenges remain. These include simplifying the reaction process, enhancing precision, broadening the scope of disease detection, and facilitating the translation of research findings into clinical practice. We anticipate that ongoing advancements in CRISPR/Cas12a-based biosensors will address these challenges.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems
Humans
*Nucleic Acids/analysis
DNA/analysis

@article {pmid40558428,
year = {2025},
author = {De Silva, PIT and Hiniduma, K and Canete, R and Bhalerao, KS and Shawky, SM and Gunathilaka, H and Rouge, JL and Mosa, IM and Steffens, DC and Manning, K and Diniz, BS and Rusling, JF},
title = {Multiplexed CRISPR Assay for Amplification-Free Detection of miRNAs.},
journal = {Biosensors},
volume = {15},
number = {6},
pages = {},
doi = {10.3390/bios15060346},
pmid = {40558428},
issn = {2079-6374},
support = {2231490//U.S. National Science Foundation/ ; },
mesh = {*MicroRNAs/blood/analysis ; Humans ; *Biosensing Techniques ; *CRISPR-Cas Systems ; Limit of Detection ; },
abstract = {CRISPR-Cas proteins from bacteria are powerful tools for gene editing and molecular diagnostics. Expanding capacity of CRISPR to low cost, multiplexed assays of biomarkers is a key to future disease diagnostics, since multiple biomarker detection is essential for reliable diagnostics. Herein we describe a multiplexed assay in a 3D-printed 96-well plate with CRISPR-Cas13a immobilized in each well to target three circulating blood biomarker microRNAs (miRNAs 34c-5p, 200c-3p, and 30e-5p) for Alzheimer's disease (ALZ). Immobilized Cas13a is equipped with different crRNAs complementary to each miRNA target. MiRNA binding to crRNA complements activates the collateral RNase activity of Cas13a, cleaving a quenched fluorescent reporter (RNaseAlert) with fluorophore and quencher connected by an RNA oligonucleotide to enable fluorescence measurements. We achieved ultralow limits of detection (LOD) of 0.74 fg/mL for miRNA 34c-5p, 0.70 fg/mL for miRNA 30e-5p, and 7.4 fg/mL for miRNA 200c-3p, with dynamic ranges from LODs up to about 1800 pg/mL. The accuracy of the assay was validated by spike-recovery studies and good correlation of levels of patient plasma samples vs. a referee method. This new approach provides selective, sensitive multiplex miRNA biosensing, and simultaneously accommodates analysis of standards and controls.},
}

*MicroRNAs/blood/analysis
Humans
*Biosensing Techniques
*CRISPR-Cas Systems
Limit of Detection

@article {pmid40558037,
year = {2025},
author = {Taran, JA and Mintaev, RR and Glazkova, DV and Belugin, BV and Bogoslovskaya, EV and Shipulin, GA},
title = {[Influence of Homology Arm Length and Structure on the Efficiency of Long Transgene Integration into a Cleavage Site Induced by SpCas9 or AsCpf1].},
journal = {Molekuliarnaia biologiia},
volume = {59},
number = {2},
pages = {255-265},
pmid = {40558037},
issn = {0026-8984},
mesh = {Humans ; *Receptors, CCR5/genetics/metabolism ; Dependovirus/genetics ; *CRISPR-Cas Systems ; *Transgenes ; Green Fluorescent Proteins/genetics/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; Genetic Vectors/genetics ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {One of the promising new approaches to the treatment of HIV infection is CRISPR/Cas-mediated knockout of the CCR5 receptor gene followed by the integration of an anti-HIV gene into the break site. Numerous studies have focused on the knockout of the CCR5 gene; however, the efficiency of subsequent targeted integration of long fragments remains poorly studied. To evaluate the efficiency of this approach, we used HT1080 cells and investigated the integration of a cassette expressing the EGFP gene into the CCR5 locus using two different nucleases (SpCas9 and AsCpf1) and various donor DNA constructs delivered by recombinant adeno-associated viral vectors (rAAV). For each nuclease, we designed five variants of donor DNA differing in the length (ranging from 150 to 1000 bp) or structure of the homology arms. The efficiency of transgene integration with 150 bp homology arms was the lowest for both nucleases and differed significantly from constructs with longer homology arms. Furthermore, it was shown that the presence of nuclease cleavage sites in the donor DNA flanking the cassette with homology arms did not affect the efficiency of transgene integration during AAV delivery. We demonstrated that the AsCpf1 nuclease provided higher efficiency of EGFP transgene integration than SpCas9, despite the lower efficiency of CCR5 knockout. The maximum percentage of cells with the integrated transgene was achieved using the AsCpf1 nuclease and an expression cassette with 600 bp homology arms, reaching 59 ± 6%.},
}

Humans
*Receptors, CCR5/genetics/metabolism
Dependovirus/genetics
*CRISPR-Cas Systems
*Transgenes
Green Fluorescent Proteins/genetics/metabolism
*CRISPR-Associated Protein 9/genetics/metabolism
Genetic Vectors/genetics
*Bacterial Proteins/genetics/metabolism

@article {pmid40558035,
year = {2025},
author = {Mintaev, RR and Glazkova, DV and Taran, JA and Bogoslovskaya, EV and Shipulin, GA},
title = {[Improving the Efficiency and Safety of Human CCR5 Gene Editing by Selection of Optimal Guide RNAs for SpCAS9 and CAS12A].},
journal = {Molekuliarnaia biologiia},
volume = {59},
number = {2},
pages = {234-243},
pmid = {40558035},
issn = {0026-8984},
mesh = {*Receptors, CCR5/genetics/metabolism ; Humans ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; *HIV Infections/genetics/therapy ; *CRISPR-Associated Proteins/genetics/metabolism ; HEK293 Cells ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {Advances in CRISPR/Cas-mediated genome editing have opened up treatment alternatives for many human diseases, including HIV infection. Knockout of the CCR5 gene as a potential way to treat HIV infection has long been studied. Here we analyzed guide RNAs for SpCas9 and AsCas12a nucleases targeting CCR5 gene which had been previously studied and selected the most effective among them. We also designed novel guide RNAs for the same nucleases using bioinformatics resources. We compared the efficiency of target site cleavage for all selected gRNAs using three nucleases: wt SpCas9, SpCas9-HF1-plus, and AsCas12a, as well as their off- target activities. We demonstrated that among the tested guide RNAs two for SpCas9- HF1-plus and three for AsCas12a exhibited high cleavage activity, cutting CCR5 gene in 60-72% of cells, and had off-target activities below the limit of detection. Thus, these guide RNAs may be candidates for future development of gene therapies against HIV infection.},
}

*Receptors, CCR5/genetics/metabolism
Humans
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/genetics/metabolism
*HIV Infections/genetics/therapy
*CRISPR-Associated Proteins/genetics/metabolism
HEK293 Cells
*Endodeoxyribonucleases/genetics/metabolism
*Bacterial Proteins/genetics/metabolism

@article {pmid40557869,
year = {2025},
author = {Li, X and Zhu, Z and Wu, J and Li, C and Liu, Z and Wang, J and Li, P and Zhang, Z and Huang, Y and Hong, J and Wu, T},
title = {PAM-free hairpin target binding activates trans-cleavage activity of Cas12a.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf596},
pmid = {40557869},
issn = {1362-4962},
support = {22474045//National Natural Science Foundation of China/ ; 82172372//National Natural Science Foundation of China/ ; 82303589//National Natural Science Foundation of China/ ; 82000512//National Natural Science Foundation of China/ ; 2024JYCXJJ010//Central Universities, HUST/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *CRISPR-Cas Systems ; Molecular Docking Simulation ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; *Bacterial Proteins/metabolism/chemistry/genetics ; *DNA/chemistry/metabolism/genetics ; Calcium/analysis ; Nucleic Acid Conformation ; Protein Binding ; },
abstract = {CRISPR-Cas12a has been demonstrated to be activated for its trans-cleavage activity by single- and double-stranded DNA containing a protospacer adjacent motif (PAM), but other types of activators have remained undiscovered. In this work, we found that a hairpin-structured substrate can activate the trans-cleavage activity of Cas12a without a PAM, and the parameters of the hairpin loop obviously affect the activity. Cas12a exhibits sequence preference for proximal loops, preferring to recognize polyadenine hairpin loop activators. Molecular docking and dynamic calculations provide a theoretical basis for the activation of Cas12a by hairpin activators. Leveraging the efficient activation capability of the hairpin activator, we constructed an allosteric detection platform for non-nucleic acid targets, capable of sensitively and specifically detecting hypochlorous acid and calcium ions. This novel activator of Cas12a holds enormous potential for the development of multi-functional biological platforms.},
}

*CRISPR-Associated Proteins/metabolism/chemistry/genetics
*CRISPR-Cas Systems
Molecular Docking Simulation
*Endodeoxyribonucleases/metabolism/chemistry/genetics
*Bacterial Proteins/metabolism/chemistry/genetics
*DNA/chemistry/metabolism/genetics
Calcium/analysis
Nucleic Acid Conformation
Protein Binding

@article {pmid40482035,
year = {2025},
author = {Dai, P and Liu, T and Yang, D and Luo, Y and Liu, LD and Xie, X and Shang, Y and Liu, X and Meng, FL},
title = {Protocol for genetic dissection of class switch recombination using genome-editing tools.},
journal = {STAR protocols},
volume = {6},
number = {2},
pages = {103882},
pmid = {40482035},
issn = {2666-1667},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Immunoglobulin Class Switching/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Animals ; Electroporation/methods ; *Recombination, Genetic ; Mice ; },
abstract = {Antibody class switch recombination is achieved through programmed DNA damage, and the processing of programmed DNA lesions requires the coordinated action of many DNA metabolic factors. Here, we present a protocol for inducing class switch recombination using base editors or CRISPR-Cas9. We provide optimized guide RNA (gRNA) sequences and describe steps for cytokine activation, electroporation, surface immunoglobulin detection, and data analysis. This method allows researchers to investigate the involvement of specific factors in antibody diversification and elucidate their functional roles. For complete details on the use and execution of this protocol, please refer to Dai et al.[1].},
}

*Gene Editing/methods
CRISPR-Cas Systems/genetics
*Immunoglobulin Class Switching/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Humans
Animals
Electroporation/methods
*Recombination, Genetic
Mice

@article {pmid40436039,
year = {2025},
author = {Tang, S and Žedaveinytė, R and Burman, N and Pandey, S and Ramirez, JL and Kulber, LM and Wiegand, T and Wilkinson, RA and Ma, Y and Zhang, DJ and Lampe, GD and Berisa, M and Jovanovic, M and Wiedenheft, B and Sternberg, SH},
title = {Protein-primed homopolymer synthesis by an antiviral reverse transcriptase.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {40436039},
issn = {1476-4687},
abstract = {Bacteria defend themselves from viral predation using diverse immune systems, many of which target foreign DNA for degradation[1]. Defence-associated reverse transcriptase (DRT) systems provide an intriguing counterpoint to this strategy by using DNA synthesis instead[2,3]. We and others recently showed that DRT2 systems use an RNA template to assemble a de novo gene that encodes the antiviral effector protein Neo[4,5]. It remains unclear whether similar mechanisms of defence are used by other related DRT families. Here, we show that DRT9 systems defend against phage using DNA homopolymer synthesis. Viral infection triggers polydeoxyadenylate (poly-dA) accumulation in the cell, driving abortive infection and population-level immunity. Cryo-electron microscopy structures reveal how a non-coding RNA serves as both a structural scaffold and reverse transcription template to direct hexameric complex assembly and poly-dA synthesis. Notably, biochemical and functional experiments identify tyrosine residues within the reverse transcriptase itself that probably prime DNA synthesis, leading to the formation of protein-DNA covalent adducts. Synthesis of poly-dA by DRT9 in vivo is regulated by the competing activities of phage-encoded triggers and host-encoded silencers. Collectively, our study identifies a nucleic-acid-driven defence system that expands the paradigm of bacterial immunity and broadens the known functions of reverse transcriptases.},
}

@article {pmid40418630,
year = {2025},
author = {Biar, CG and Bodkin, N and Carvill, GL and Calhoun, JD},
title = {Protocol to perform multiplexed assays of variant effect using curated loci prime editing.},
journal = {STAR protocols},
volume = {6},
number = {2},
pages = {103851},
pmid = {40418630},
issn = {2666-1667},
mesh = {*Gene Editing/methods ; Humans ; *Genetic Variation/genetics ; CRISPR-Cas Systems/genetics ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {Multiplexed assays of variant effect (MAVEs) perform simultaneous characterization of many variants. Here, we present a protocol to perform MAVEs using curated loci prime editing (cliPE), an accessible experimental pipeline that enables prime editing of a target gene. We describe steps for designing prime editing reagents, screening for genome editing efficiency, selecting a pool of cells edited to harbor different genetic variants, and sequencing. Lastly, we detail procedures for performing enrichment analysis to identify variants with normal or aberrant activity.},
}

*Gene Editing/methods
Humans
*Genetic Variation/genetics
CRISPR-Cas Systems/genetics
High-Throughput Nucleotide Sequencing/methods

@article {pmid40397576,
year = {2025},
author = {Zhong, X and Wu, H and Wang, G and Sun, Z},
title = {Protocol for CRISPR-mediated deletion of cis-regulatory element in murine Th17 cells for in vivo assessment of effector function.},
journal = {STAR protocols},
volume = {6},
number = {2},
pages = {103831},
pmid = {40397576},
issn = {2666-1667},
support = {P30 CA033572/CA/NCI NIH HHS/United States ; R01 AI109644/AI/NIAID NIH HHS/United States ; R21 AI163256/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Th17 Cells/metabolism/immunology/cytology ; Encephalomyelitis, Autoimmune, Experimental/genetics/immunology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Cell Differentiation ; *Regulatory Sequences, Nucleic Acid/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mice, Inbred C57BL ; },
abstract = {Studying the cis-regulatory elements (CREs) of genes in Th17 cells during autoimmune disease progression, such as experimental autoimmune encephalomyelitis (EAE), is often limited by the availability of gene-edited mice. Here, we present a protocol for CRISPR-mediated deletion of a CRE in murine Th17 cells for in vivo assessment of effector function in EAE. We describe steps for dual U6gRNA construction, preparation of retroviruses, viral delivery, and Th17 differentiation. We then detail procedures for in vivo functionality analysis. For complete details on the use and execution of this protocol, please refer to Zhong et al.[1][,][2].},
}

Animals
Mice
*Th17 Cells/metabolism/immunology/cytology
Encephalomyelitis, Autoimmune, Experimental/genetics/immunology
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Cell Differentiation
*Regulatory Sequences, Nucleic Acid/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Mice, Inbred C57BL

@article {pmid40310725,
year = {2025},
author = {Hao, M and Lu, P and Guan, JL},
title = {Protocol for in vivo CRISPR knockout screening of autophagy genes to identify breast cancer metastasis suppressors.},
journal = {STAR protocols},
volume = {6},
number = {2},
pages = {103798},
pmid = {40310725},
issn = {2666-1667},
mesh = {Animals ; *Autophagy/genetics ; *Breast Neoplasms/genetics/pathology ; Mice ; Female ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; Humans ; Neoplasm Metastasis/genetics ; Lung Neoplasms/secondary/genetics ; },
abstract = {Metastasis represents the primary cause of mortality among patients with breast cancer. Here, we present a protocol for utilizing an in vivo custom CRISPR-Cas9 knockout library to systematically investigate autophagy regulatory genes implicated in breast cancer metastasis to the lung in mice. We describe steps for library synthesis, cloning, and virus packaging and transfection. We then detail procedures for genome DNA collection and transplantation, followed by analysis of screening data. This protocol enables efficient identification of potential suppressors of breast cancer metastasis in vivo. For complete details on the use and execution of this protocol, please refer to Hao et al.[1].},
}

Animals
*Autophagy/genetics
*Breast Neoplasms/genetics/pathology
Mice
Female
*CRISPR-Cas Systems/genetics
*Gene Knockout Techniques/methods
Humans
Neoplasm Metastasis/genetics
Lung Neoplasms/secondary/genetics

@article {pmid40215169,
year = {2025},
author = {Ortega, P and Sanchez, A and Seldin, M and Buisson, R},
title = {Oligo-seq protocol for mapping DNA motifs targeted by base editors.},
journal = {STAR protocols},
volume = {6},
number = {2},
pages = {103758},
pmid = {40215169},
issn = {2666-1667},
mesh = {*Gene Editing/methods ; *Nucleotide Motifs/genetics ; CRISPR-Cas Systems/genetics ; *Sequence Analysis, DNA/methods ; *DNA/genetics/chemistry ; Humans ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {Determining which DNA sequences are preferentially targeted by base editors is critical for understanding how APOBECs, AID, and other CRISPR-Cas9 base editors edit DNA in cells or improve their editing efficiency. We have developed Oligo-seq, an in vitro sequencing-based method to identify the preferred sequence motifs targeted by these enzymes. This assay monitors DNA deaminase activity on DNA oligonucleotides containing random nucleotides and/or DNA structures and determines by sequencing which sequences are preferentially deaminated. For complete details on the use and execution of this protocol, please refer to Sanchez et al.[1].},
}

*Gene Editing/methods
*Nucleotide Motifs/genetics
CRISPR-Cas Systems/genetics
*Sequence Analysis, DNA/methods
*DNA/genetics/chemistry
Humans
High-Throughput Nucleotide Sequencing/methods

@article {pmid40198219,
year = {2025},
author = {Obolenski, S and Olvera-León, R and Sun, D and Adams, DJ and Waters, AJ},
title = {Protocol for the functional evaluation of genetic variants using saturation genome editing.},
journal = {STAR protocols},
volume = {6},
number = {2},
pages = {103710},
pmid = {40198219},
issn = {2666-1667},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Humans ; *Genetic Variation/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; High-Throughput Nucleotide Sequencing/methods ; Gene Library ; },
abstract = {Saturation genome editing (SGE) employs CRISPR-Cas9 and homology-directed repair (HDR) to introduce exhaustive nucleotide modifications at specific genomic sites in multiplex, enabling the functional analysis of genetic variants while preserving their native genomic context. Here, we present a protocol for SGE-based variant evaluation in HAP1-A5 cells. We describe the steps for designing variant libraries, single-guide RNAs (sgRNAs), and oligonucleotide primers for PCR. We also detail the sample preparation before the SGE screen, the cellular screening process, and subsequent next-generation sequencing (NGS) library preparation. For complete details on the use and execution of this protocol, please refer to Radford et al.,[1] Waters et al.,[2] and Olvera-León et al.[3].},
}

*Gene Editing/methods
CRISPR-Cas Systems/genetics
Humans
*Genetic Variation/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
High-Throughput Nucleotide Sequencing/methods
Gene Library

@article {pmid40173037,
year = {2025},
author = {Della Volpe, L and Vacca, R and Di Micco, R},
title = {Protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human hematopoietic stem and progenitor cells.},
journal = {STAR protocols},
volume = {6},
number = {2},
pages = {103722},
pmid = {40173037},
issn = {2666-1667},
mesh = {Humans ; *Gene Editing/methods ; *Hematopoietic Stem Cells/cytology/metabolism ; *CRISPR-Cas Systems/genetics ; *Cell Culture Techniques/methods ; },
abstract = {Long-range correction strategies require ex vivo activation of hematopoietic stem and progenitor cells (HSPCs) to engage the homology-directed repair (HDR) mechanism, but prolonged culture causes harmful cellular responses, reducing the long-term functionality of gene-edited (GE) HSPCs. Here, we present a protocol for optimizing culture conditions for ex vivo activation during CRISPR-Cas9 gene editing in human HSPCs. We describe steps for HSPC thawing, ex vivo treatments, gene editing, and downstream in vitro and in vivo analyses to assess the functionality of GE-HSPCs. For complete details on the use and execution of this protocol, please refer to della Volpe et al.[1].},
}

Humans
*Gene Editing/methods
*Hematopoietic Stem Cells/cytology/metabolism
*CRISPR-Cas Systems/genetics
*Cell Culture Techniques/methods

@article {pmid40120111,
year = {2025},
author = {Liang, Z and Wu, X and Ye, Z and She, L and Ma, X and Huo, YX},
title = {Protocol for identification of sgRNA mutants using high-throughput screening technique and multiplex genome editing.},
journal = {STAR protocols},
volume = {6},
number = {2},
pages = {103690},
pmid = {40120111},
issn = {2666-1667},
mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; *High-Throughput Screening Assays/methods ; *Mutation ; Humans ; Gene Library ; },
abstract = {In the CRISPR-Cas9 system, tandem expression of multiple identical single-guide RNAs (sgRNAs) often triggers homologous sequences loss, which affects multiplex genome editing efficiencies. Here, we present a protocol for high-throughput screening of functional sgRNAs with nonrepetitive mutants. We describe steps for constructing the screening platform, designing and constructing sgRNA libraries, and screening sgRNA mutants. These mutants can interact with the Cas9 protein, enabling multiplex genome editing. For complete details on the use and execution of this protocol, please refer to Liang et al.[1].},
}

*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems/genetics
*High-Throughput Screening Assays/methods
*Mutation
Humans
Gene Library

@article {pmid40088451,
year = {2025},
author = {Kohabir, KAV and Linthorst, J and Wolthuis, RMF and Sistermans, EA},
title = {Protocol for high-precision CRISPR-Cas12a-based SNV detection on synthetic DNA, cell line cfDNA models, and liquid biopsies.},
journal = {STAR protocols},
volume = {6},
number = {2},
pages = {103696},
pmid = {40088451},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Liquid Biopsy/methods ; *Cell-Free Nucleic Acids/genetics ; *Polymorphism, Single Nucleotide/genetics ; *DNA/genetics ; Cell Line ; Gene Editing/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {CRISPR-based diagnostics (CRISPRdx) offer promising tools for rapid and cost-effective genetic testing, but achieving single-nucleotide fidelity remains a challenge. Here, we present a protocol for high-precision detection of single-nucleotide variants (SNVs) using a Cas12a-based approach. We describe how to apply our publicly available ARTEMIS algorithm to identify targetable SNVs, design optimized CRISPR RNAs (crRNAs), and perform fluorescence-based CRISPRdx assays on synthetic DNA, cell line-derived cell-free DNA (cfDNA), and liquid biopsy samples. For complete details on the use and execution of this protocol, please refer to Kohabir et al.[1].},
}

*CRISPR-Cas Systems/genetics
Humans
Liquid Biopsy/methods
*Cell-Free Nucleic Acids/genetics
*Polymorphism, Single Nucleotide/genetics
*DNA/genetics
Cell Line
Gene Editing/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40555885,
year = {2025},
author = {Asah-Asante, R and Tang, L and Gong, X and Fan, S and Yan, C and Asante, JO and Zeng, Q},
title = {Exploring pigment-producing Streptomyces as an alternative source to synthetic pigments: diversity, biosynthesis, and biotechnological applications. A review.},
journal = {World journal of microbiology & biotechnology},
volume = {41},
number = {7},
pages = {211},
pmid = {40555885},
issn = {1573-0972},
mesh = {*Streptomyces/metabolism/genetics/classification ; *Pigments, Biological/biosynthesis/chemistry ; Biosynthetic Pathways/genetics ; *Biotechnology/methods ; Multigene Family ; CRISPR-Cas Systems ; Melanins/biosynthesis ; Gene Editing ; Anthraquinones/metabolism ; Prodigiosin/analogs & derivatives/biosynthesis ; Synthetic Biology ; Fermentation ; Benzoisochromanequinones ; },
abstract = {The increasing health and environmental concerns associated with synthetic pigments have intensified the global search for natural, eco-friendly alternatives. Among microbial sources, Streptomyces, a genus within the class Actinomycetia, has emerged as a prolific source of bioactive pigments with wide-ranging industrial applications. The review provides a comprehensive synthesis of pigment-producing Streptomyces, focusing on their ecological diversity, biosynthetic pathways, and taxonomic relevance. This review has discussed key pigment classes, including melanin, prodiginine, quinone, and actinorhodin, with their bioactive properties, such as antioxidants, antimicrobials, and anticancer. The review further emphasizes recent advancements in synthetic biology, including clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas)-based gene editing, which has facilitated the activation of silent biosynthetic gene clusters (BGCs) that can enhance pigment yield. Additionally, this review discusses the optimization and fermentation protocols, industrial scalability, and the application of pigments in food, pharmaceutical, cosmetic, textile, and environmental sectors. Despite these advancements, critical research gaps persist, particularly in toxicological evaluation, pigment stability under industrial conditions, high-throughput screening of unexplored Streptomyces strains, and the integration of AI-based predictive tools for pathway optimization. Addressing these gaps is essential to unlocking the full potential of Streptomyces-derived pigments to replace harmful synthetic colorants.},
}

*Streptomyces/metabolism/genetics/classification
*Pigments, Biological/biosynthesis/chemistry
Biosynthetic Pathways/genetics
*Biotechnology/methods
Multigene Family
CRISPR-Cas Systems
Melanins/biosynthesis
Gene Editing
Anthraquinones/metabolism
Prodigiosin/analogs & derivatives/biosynthesis
Synthetic Biology
Fermentation
Benzoisochromanequinones

@article {pmid40554662,
year = {2025},
author = {Li, C and Liu, X and Li, Y and Peng, Q and Miao, J and Liu, X},
title = {The Tetracycline-Inducible/CRISPR-Cas9 System is an Efficient Tool for Studying Gene Function in Phytophthora sojae.},
journal = {Molecular plant pathology},
volume = {26},
number = {6},
pages = {e70114},
doi = {10.1111/mpp.70114},
pmid = {40554662},
issn = {1364-3703},
support = {32372602//National Natural Science Foundation of China/ ; 32272599//National Natural Science Foundation of China/ ; 2024ZC-KJXX-062//the Project of Shaanxi Young Stars in Science and Technology/ ; },
mesh = {*Phytophthora/genetics/drug effects ; *CRISPR-Cas Systems/genetics/drug effects ; *Tetracycline/pharmacology ; Promoter Regions, Genetic/genetics ; },
abstract = {The present study presents a novel approach combining a tetracycline-inducible system (Tet-On) and CRISPR-Cas9 techniques to investigate the function of two essential genes in Phytophthora sojae. We constructed a donor vector in which the reverse tetracycline transactivator (rtTA) is driven by an oomycete promoter. Additionally, it contains a fused TetR binding site and the minimum oomycete promoter, as well as 1000-bp homologous arms of the promoter upstream and downstream sequences. The promoter of the target gene was replaced with a tetracycline-responsive promoter (Ptet) using a CRISPR-Cas9 system. In the native transformants, the target gene was induced by the administration of tetracycline and repressed in its absence. Using the Tet-On/CRISPR-Cas9 system, we obtained inducible transformants of PsAF5 and PsCesA3. The phenotype of PsAF5 inducible transformants without doxycycline was consistent with that of ΔPsAF5 transformants, specifically characterised by an increase in oospore production and heightened sensitivity to H2O2. PsCesA3 inducible transformants could not grow in the absence of doxycycline, which means PsCesA3 is an essential protein for P. sojae. In conclusion, the Tet-On/CRISPR-Cas9 system represents an effective approach for investigating crucial genes in P. sojae.},
}

*Phytophthora/genetics/drug effects
*CRISPR-Cas Systems/genetics/drug effects
*Tetracycline/pharmacology
Promoter Regions, Genetic/genetics

@article {pmid40554553,
year = {2025},
author = {Wei, SC and Cantor, AJ and Walleshauser, J and Mepani, R and Melton, K and Bans, A and Khekare, P and Gupta, S and Wang, J and Soares, C and Kiwan, R and Lee, J and McCawley, S and Jani, V and Leong, WI and Shahi, PK and Chan, J and Boivin, P and Otoupal, P and Pattnaik, BR and Gamm, DM and Saha, K and Gowen, BG and Haak-Frendscho, M and Janatpour, MJ and Silverman, AP},
title = {Evaluation of subretinally delivered Cas9 ribonucleoproteins in murine and porcine animal models highlights key considerations for therapeutic translation of genetic medicines.},
journal = {PloS one},
volume = {20},
number = {6},
pages = {e0317387},
doi = {10.1371/journal.pone.0317387},
pmid = {40554553},
issn = {1932-6203},
mesh = {Animals ; Mice ; *Gene Editing/methods ; *Ribonucleoproteins/genetics/administration & dosage ; Swine ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/administration & dosage ; Retina/metabolism ; *Genetic Therapy/methods ; Retinal Pigment Epithelium/metabolism ; Disease Models, Animal ; },
abstract = {Genetic medicines, including CRISPR/Cas technologies, extend tremendous promise for addressing unmet medical need in inherited retinal disorders and other indications; however, there remain challenges for the development of therapeutics. Herein, we evaluate genome editing by engineered Cas9 ribonucleoproteins (eRNP) in vivo via subretinal administration using mouse and pig animal models. Subretinal administration of adenine base editor and double strand break-inducing Cas9 nuclease eRNPs mediate genome editing in both species. Editing occurs in retinal pigmented epithelium (RPE) and photoreceptor cells, with favorable tolerability in both species. Using transgenic reporter strains, we determine that editing primarily occurs close to the site of administration, within the bleb region associated with subretinal injection. Our results show that subretinal administration of BE-eRNPs in mice mediates base editing of up to 12% of the total neural retina, with an average rate of 7% observed at the highest dose tested. In contrast, a substantially lower editing efficiency was observed in minipigs; even with direct quantification of only the treated region, a maximum base editing rate of 1.5%, with an average rate of <1%, was observed. Our data highlight the importance of species consideration in preclinical studies for the development of genetic medicines targeting the eye and provide an example of a lack of translation between small and larger animal models in the context of subretinal administration of Cas9 eRNPs.},
}

Animals
Mice
*Gene Editing/methods
*Ribonucleoproteins/genetics/administration & dosage
Swine
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/genetics/administration & dosage
Retina/metabolism
*Genetic Therapy/methods
Retinal Pigment Epithelium/metabolism
Disease Models, Animal

@article {pmid40553877,
year = {2025},
author = {Saleem, S and Amin, W and Bhatti, F and Majid, M and Fazal, A},
title = {Nucleic acid-based strategies to mitigate stripe rust disease of wheat for achieving global food security - A review.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {145353},
doi = {10.1016/j.ijbiomac.2025.145353},
pmid = {40553877},
issn = {1879-0003},
abstract = {Wheat (Triticum aestivum), being a global staple crop, is critical in ensuring food security due to its significant nutritional value. However, it faces numerous challenges from both biotic and abiotic stresses, with fungal diseases being particularly detrimental to yield. Among these, wheat stripe rust, caused by the fungal pathogen Puccinia striiformis, poses a severe threat to wheat. Globally, 5.47 million tons of grains are lost due to the stripe rust pathogen, equivalent to a loss of USD 979 million annually; almost 88 % of the world's wheat production is susceptible to stripe rust. This review accentuates the global extensive distribution of stripe rust, detailing its causes and impact on crop productivity and mitigating approaches following traditional, genomic, and post-genomics. The mitigation approaches to wheat stripe rust have been mainly categorized into primitive (pre-genomic), modern (genomic), and next-generation (post-genomic) approaches. The primitive approaches include traditional breeding, phenotypic selection, and exotic germplasm to introduce resistance leads to early success in disease management. The advanced genomic era, with tools like QTL mapping, GWAS, marker-assisted selection, and high-throughput sequencing to deploy resistance genes, helps in precise mapping and developing high-throughput genotyping for large-scale screening and introgression of multiple resistant genes. The gene-editing approaches, including CRISPR/Cas9, RNAi, and epigenomics, now enable precise gene editing and regulation for durable resistance, together with multi-omics techniques, to identify resistant pathways and biomarkers with enhanced understanding of host-pathogen interactions and resistance mechanisms. Climate change events like shifts in rainfall patterns and rising temperatures expand the rust-prone area and pose more challenges in developing durable rust-resistant cultivars. Furthermore, the review explores using wheat's valuable genetic resources and integrating AI-based technologies to enhance stripe rust resistance by analyzing large datasets, including pathogen evolution and growth stages, allowing for timely interventions of the stripe rust epidemic. The role of multiomics approaches, particularly genomics and transcriptomics, in unraveling the genetic basis of stress tolerance is highlighted. A forward-looking framework is proposed, emphasizing the use of interdisciplinary methodologies, including big data, multi-omics, and AI-driven approaches, that hold immense promise to revolutionize wheat protection with the development of climate-resilient wheat genotypes and ensure real-time disease monitoring and precision-resistant strategies against the evolving rust pathogen.},
}

@article {pmid40553564,
year = {2025},
author = {Kratzmeier, C and Taheri, M and Mei, Z and Lim, I and Khalil, MA and Carter-Cooper, B and Fanaroff, RE and Ong, CS and Schneider, EB and Chang, S and Leyder, E and Li, D and Luzina, IG and Banerjee, A and Krupnick, AS},
title = {Lung adenocarcinoma-derived IFN-γ promotes growth by modulating CD8+ T cell production of CCR5 chemokines.},
journal = {The Journal of clinical investigation},
volume = {},
number = {},
pages = {},
doi = {10.1172/JCI191070},
pmid = {40553564},
issn = {1558-8238},
abstract = {Since the lung is a mucosal barrier organ with a unique immunologic environment, mechanisms of immunoregulation in lung cancer may differ from those of other malignancies. Consistent with this notion, we found that CD8+ T cells play a paradoxical role in facilitating, rather than ameliorating, the growth of multiple lung adenocarcinoma models. These include spontaneous, carcinogen-induced, and transplantable tumor cell line models. Specifically, we found that CD8+ T cells promote homing of CD4+Foxp3+ T regulatory cells to the tumor bed by increasing levels of CCR5 chemokines in the tumor microenvironment in an IFN-γ and TNF-α dependent manner. Contrary to their canonical role, these Th1 cytokines contributed to accelerated growth of murine lung adenocarcinomas while suppressing the growth of other malignancies. Surprisingly, lung cancer cells themselves can serve as a dominant source of IFN-γ, and deletion of this cytokine from cancer cells using CRISPR/Cas-9 decreases tumor growth. Importantly for translational applications, a high level of IFN-γ was also found in human lung cancer patients at both the mRNA and protein level. Our data outlines what we deem a novel and previously undefined lung cancer specific immunoregulatory pathway that may be harnessed to tailor immune based therapy specifically for this malignancy.},
}

@article {pmid40553349,
year = {2025},
author = {Mirchandani, I and Khandhediya, Y and Chauhan, K},
title = {Review on Advancement of AI in Synthetic Biology.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2952},
number = {},
pages = {483-490},
pmid = {40553349},
issn = {1940-6029},
mesh = {*Synthetic Biology/methods ; *Artificial Intelligence ; Gene Editing/methods ; CRISPR-Cas Systems ; Machine Learning ; Humans ; },
abstract = {The way biological systems are built and designed has been revolutionized by synthetic biology. Further enhancements like predictive modeling, optimization and systematic design of complex biological systems, is now possible due to integration of Artificial Intelligence into synthetic biology. This review shares insights on the role of AI in advancement of synthetic biology, including genome editing, metabolic pathway optimization and biological circuit design etc. AI-driven tools contribute to the increased efficiency and precision. Application of deep learning and machine learning has made it possible to make CRISPR-cas9, de novo protein design and gene circuit development more precise. However, there are still some persistent challenges, especially in curating high-quality biological datasets and bridging interdisciplinary gaps between computational and experimental scientists. Future perspectives focus on causal reasoning in AI models, integration of physics based algorithms, and promoting collaboration across disciplines to achieve breakthroughs in both synthetic biology and AI. By joining these fields, the transformative power of synthetic biology and AI can be unlocked and applied in the fields of medicine, biotechnology and environmental sustainability, pioneering a way for a new era of bioengineering.},
}

*Synthetic Biology/methods
*Artificial Intelligence
Gene Editing/methods
CRISPR-Cas Systems
Machine Learning
Humans

@article {pmid40553339,
year = {2025},
author = {Pandey, S and Choudhari, JK and Tripathi, A and Singh, A and Antony, A and Chouhan, U},
title = {Artificial Intelligence-Based Genome Editing in CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2952},
number = {},
pages = {273-282},
pmid = {40553339},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Artificial Intelligence ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Precision Medicine ; Genomics/methods ; },
abstract = {Artificial intelligence (AI) plays a critical role in predicting and improving genome editing methods, including CRISPR/Cas9. Recently, several AI models, such as DeepCRISPR, CRISTA, and Deep High Fidelity (DeepHF), have been utilized to design guide RNAs (gRNAs) for CRISPR-Cas systems. These models assess genomic context, desired mutation type, on-target and off-target scores, and potential off-target locations. AI models help improve various genome editing methods, such as base, prime, and epigenome editing, which allow for precise and intentional changes to DNA sequences without the need for donor DNA templates. Furthermore, integrating AI with genome editing and precision medicine enables the creation of personalized treatments tailored to each individual's unique genetic profile. Examining genomic data enables the identification of mutations, variations, and biomarkers linked to diseases like cancer, diabetes, and Alzheimer's disease. Integrating AI with genome editing can potentially enhance genetic modification techniques' precision, efficiency, and cost-effectiveness. Furthermore, it presents novel prospects for fields like genetics, biomedicine, and healthcare, which could significantly impact human health. However, several challenges still exist, including high costs, inaccurate edits, and effective delivery methods for CRISPR components, improved editing performance, and safety in clinical applications.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Artificial Intelligence
Humans
RNA, Guide, CRISPR-Cas Systems/genetics
Precision Medicine
Genomics/methods

@article {pmid40553337,
year = {2025},
author = {Khammampalli, S and Vindal, V},
title = {Artificial Intelligence in CRISPR-Cas Systems: A Review of Tool Applications.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2952},
number = {},
pages = {243-257},
pmid = {40553337},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Artificial Intelligence ; *Gene Editing/methods ; Humans ; Machine Learning ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA Editing ; },
abstract = {Genetic engineering is a method used to alter an organism's DNA, which could entail altering a base pair, removing a section of DNA, or introducing a new DNA segment. Over time, genetic engineering has progressed from basic cloning for research purposes to advanced synthetic biology, leading to new biomedical applications. Targeted genomic editing is one method of cellular reprogramming that aims to change the state of a cell. The invention of CRISPR Cas systems has greatly simplified gene editing. These systems use a unique RNA-guided DNA endonuclease, a protein that can cut DNA and be trained to target new places by changing the sequence of its guide RNA. Integrating CRISPR-Cas systems with artificial intelligence opens new insights into the study of genetic engineering and its applications. Extensive research utilizing deep learning and machine learning has been conducted to predict the outcomes of CRISPR-Cas9 editing. Artificial intelligence also predicts RNA editing events and CRISPR off-target cleavage sites. Scientists often struggle to identify the ideal perturbation for their specific application because of the ample search space and expensive genetic trials. The algorithmic method using artificial intelligence utilizes the cause-and-effect link between variables in a complicated system like genome regulation to determine which perturbation is most effective in each successive round of testing, thereby making artificial intelligence an effective technique in gene editing.},
}

*CRISPR-Cas Systems
*Artificial Intelligence
*Gene Editing/methods
Humans
Machine Learning
RNA, Guide, CRISPR-Cas Systems/genetics
RNA Editing

@article {pmid40553335,
year = {2025},
author = {Johnson, SJS},
title = {AI Revolutionizing Cell and Genetic Engineering: Innovations and Applications.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2952},
number = {},
pages = {219-232},
pmid = {40553335},
issn = {1940-6029},
mesh = {Humans ; *Artificial Intelligence ; *Genetic Engineering/methods ; Gene Editing/methods ; CRISPR-Cas Systems ; Precision Medicine/methods ; *Cell Engineering/methods ; Drug Discovery/methods ; Genomics/methods ; },
abstract = {The integration of Artificial Intelligence (AI) into the realms of cell and genetic engineering has ushered in a transformative era of innovation and discovery. This chapter explores the myriad ways in which AI technologies are revolutionizing these fields, from data analysis and drug discovery to genomic sequencing and gene editing. AI algorithms are adept at analyzing vast datasets, uncovering intricate patterns, and predicting biological phenomena with unparalleled accuracy. In drug discovery, AI-driven platforms expedite the identification of potential therapeutics by simulating molecular interactions and predicting their efficacy. Genomic sequencing efforts benefit from AI's ability to interpret genetic variations and their implications for health and disease. Furthermore, AI-guided gene editing techniques, such as CRISPR-Cas9, enable precise and targeted modifications of the genome. Beyond the laboratory, AI facilitates personalized medicine by analyzing genetic data to tailor treatments to individual patients. This chapter underscores the pivotal role of AI in advancing cell and genetic engineering, promising a future of unprecedented scientific breakthroughs and personalized healthcare solutions.},
}

Humans
*Artificial Intelligence
*Genetic Engineering/methods
Gene Editing/methods
CRISPR-Cas Systems
Precision Medicine/methods
*Cell Engineering/methods
Drug Discovery/methods
Genomics/methods

@article {pmid40553283,
year = {2025},
author = {Suk, Y and Apel, E and Custers, S and Miletic, P and Zhai, K and Chokshi, C and Venugopal, C and Moffat, J and Singh, SK},
title = {CRISPR-Cas9-Guided Genetic Manipulation of Patient-Derived Brain Tumor Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2944},
number = {},
pages = {173-184},
pmid = {40553283},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Humans ; *Brain Neoplasms/genetics/pathology ; *Gene Editing/methods ; Gene Knockout Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; },
abstract = {CRISPR-Cas9 technology has revolutionized scientific research and has provided scientists with the ability to change DNA bases specifically and precisely at predetermined sites. The CRISPR-Cas9 knockout (KO) and activation (a) platforms developed by Hart et al. and Sanson et al. allow for RNA-directed genome editing to both decrease or increase gene expression, respectively [1, 2]. In this chapter the two techniques, CRISPR KO and CRISPRa, will be discussed and explained in further detail for optimized application against patient derived brain tumor cells.},
}

*CRISPR-Cas Systems
Humans
*Brain Neoplasms/genetics/pathology
*Gene Editing/methods
Gene Knockout Techniques/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Cell Line, Tumor

@article {pmid40553057,
year = {2025},
author = {Long, W and Li, Q and Jin, T and Lu, Z and Hu, F and Zhang, H and Xu, Y},
title = {Dual detection of hypervirulent genes of Klebsiella pneumoniae using a single CRISPR-Cas12a system modulated using entropy-driven circuits.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5ay00563a},
pmid = {40553057},
issn = {1759-9679},
abstract = {A set of different CRISPR-based analyses have been extensively used to detect nucleic acids owing to their characteristics of being sensitive, specific, rapid and easy to operate. However, one of its major challenges is application to multiplex detection (https://pubmed.ncbi.nlm.nih.gov/31460243/) using a single CRISPR-Cas system because the detection relies on the indiscriminate trans-cleavage activity of the Cas protein. Here we developed a CRISPR-based dual gene assay system for hypervirulent Klebsiella pneumoniae (hvKp) by integrating two parallel entropy-driven circuits (EDCs) and the trans-cleavage activity of the Cas12a protein towards fluorescent substrate reporters. The EDC reaction released two different fluorescent reporters and the same trigger probe according to different targets. The trigger probe activated the trans-cleavage activity of Cas12a towards reporter probes to achieve the purpose of the dual detection of different genes with a single CRISPR-Cas12a system in one tube. Furthermore, the assay system was designed to detect different mRNA sequences (rmpA and peg-344) of hvKp, requiring no reverse transcription procedure and providing technical support for the identification of hvKp and classical Klebsiella pneumoniae (cKp). The detection limits for rmpA and peg-344 were as low as 0.10 fmol L[-1] and 0.17 fmol L[-1], respectively, within 25 minutes under isothermal conditions. It enabled rapid dual detection of hvKp in a single tube using only a CRISPR-Cas12a system, demonstrating great potential for point-of-care testing due to its high sensitivity, low cost, and robust ability to detect various mRNA biomarkers.},
}

@article {pmid40551276,
year = {2025},
author = {Zhang, Q and Yu, Y and Yin, B and Xu, L and Chen, H and Qiao, R and Chen, A and Zhu, N and Wu, X},
title = {An ultrasensitive and specific CRISPR-Cas13a-mediated point-of-care assay for monkeypox detection and PCR-based clade detection.},
journal = {Infectious diseases of poverty},
volume = {14},
number = {1},
pages = {56},
pmid = {40551276},
issn = {2049-9957},
support = {202308003//Nanjing Science and technology plan project/ ; BE2022669//Social Development Project of Jiangsu Provincial Science and Technology Department/ ; SJCX23_0855//Graduate Research and Innovation Projects of Jiangsu Province/ ; RCMS24008//Talent Lift Program of The Second Hospital of Nanjing/ ; },
mesh = {Sensitivity and Specificity ; Humans ; *CRISPR-Cas Systems ; *Point-of-Care Systems ; *Mpox, Monkeypox/diagnosis/virology ; *Monkeypox virus/isolation & purification/genetics/classification ; *Polymerase Chain Reaction/methods ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; },
abstract = {BACKGROUND: The rapid increase in the number of monkeypox cases poses a considerable threat to the international community, necessitating sensitive, fast, and available diagnostic methods. Therefore, the objective of this study was to develop a rapid, sensitive and simple method with high clinical applicability.

METHODS: We developed a simple, rapid point-of-care assay to detect monkeypox virus (MPXV) using multienzyme isothermal rapid amplification (MIRA) coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a system. The detection system was optimized by synthesizing plasmids, and the detection sensitivity was explored by the continuous dilution of the plasmid. We validated the accuracy of this assay on 202 clinical MPXV samples and 104 interference samples through the kappa test. The visual interpretation of the results was realized by combining the assay with lateral flow strips. In addition, we developed a PCR-based method to identify MPXV Clades I and II, and the accuracy was tested through a kappa test on 202 clinical monkeypox samples and 104 interference samples.

RESULTS: Our assay achieved an analytical sensitivity of 14.4 copies/ml and high selectivity, as it differentiated MPXV from three other Orthopoxvirus species. The clinical testing results for 202 monkeypox samples and 104 interference samples demonstrated 100% sensitivity and specificity. Compared with quantitative PCR (qPCR), three samples tested as positive using our assay, which showed that the performance of this assay was superior to that of the qPCR assay. Combined with lateral flow strips, its availability and simplicity provide an alternative point-of-care diagnostic method for MPXV testing in remote settings and resource-poor areas. The results of 32 clinical samples showed that lateral flow strips had a high detection sensitivity and could identify samples with Ct value of 39 as positive. The clade identification assay detected as few as 200 copies/ml within 40 min and no cross-reaction was observed between Clades I and II. The clinical samples tested were all Clade II, which was consistent with the circulating clade in the Chinese mainland.

CONCLUSIONS: The MIRA-CRISPR-Cas13a-MPXV system offers a rapid, sensitive and specific approach for monkeypox diagnosis, with significance for monitoring monkeypox epidemics. The clade identification assay based on PCR could accurately distinguish Clade I from Clade II within 40 min and can be implemented for high-throughput operation.},
}

Sensitivity and Specificity
Humans
*CRISPR-Cas Systems
*Point-of-Care Systems
*Mpox, Monkeypox/diagnosis/virology
*Monkeypox virus/isolation & purification/genetics/classification
*Polymerase Chain Reaction/methods
*Nucleic Acid Amplification Techniques/methods
*Molecular Diagnostic Techniques/methods

@article {pmid40551141,
year = {2025},
author = {Kim, MS and Jeong, DE and Choi, SK},
title = {Harnessing an anti-CRISPR protein for powering CRISPR/Cas9-mediated genome editing in undomesticated Bacillus strains.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {143},
pmid = {40551141},
issn = {1475-2859},
support = {KGM1192511//Korea Research Institute of Bioscience and Biotechnology/ ; IGM042411//HLB Genex/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Bacillus/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Plasmids/genetics ; CRISPR-Associated Protein 9/antagonists & inhibitors ; RNA, Guide, CRISPR-Cas Systems/genetics ; Bacillus subtilis/genetics ; },
abstract = {BACKGROUND: Wild-type Bacillus strains have significant industrial and medical value, but their effective utilization often requires strain improvement. The CRISPR/Cas9 system has become the primary tool for genome editing, allowing precise introduction of desired mutations at specific chromosomal locations. However, the practical application of CRISPR/Cas9 in most wild-type Bacillus strains remains challenging due to cellular toxicity resulting from Cas9/sgRNA activity. Therefore, controlling Cas9 toxicity is essential for the widespread application of the CRISPR/Cas9 system in wild-type Bacillus strains.

RESULTS: We employed AcrIIA4, an anti-CRISPR protein that inhibits the Cas9/sgRNA ribonucleoprotein complex from interacting with DNA, to mitigate Cas9/sgRNA-mediated toxicity, thereby enabling CRISPR/Cas9-based genome editing in wild-type strains. The newly constructed CRISPR/anti-CRISPR (CAC) plasmids harbor both cas9 and acrIIA4 genes controlled by the Pspac and Pxyl promoters, respectively, along with the repressor genes lacI and xylR. This design allows precise control of Cas9 activity through inducers. Xylose, which induces AcrIIA4 expression, effectively alleviated Cas9/sgRNA-mediated toxicity during transformation. Under xylose induction, the CAC plasmid led to a remarkable 139-fold increase in the transformation efficiency of wild-type Bacillus subtilis compared to a plasmid lacking anti-CRISPR. Meanwhile, IPTG induction promoted Cas9 expression, facilitating efficient genome editing. Upon IPTG induction, the genome editing efficiency in wild-type B. subtilis increased from 0 to 95.8% in transformants carrying the CAC plasmid. Importantly, our findings extend beyond B. subtilis, revealing that the anti-CRISPR protein dramatically enhanced transformation and genome editing efficiencies in Bacillus pumilus. Moreover, we demonstrated that the CAC system successfully enabled the generation of spo0A mutants in Bacillus mojavensis, Bacillus tequilensis, and Paenibacillus polymyxa.

CONCLUSIONS: In this study, we developed a CAC system that utilizes the anti-CRISPR protein AcrIIA4 to reduce Cas9/sgRNA-mediated toxicity in Bacillus strains. This system enables precise control of AcrIIA4 and Cas9 expression through inducers, significantly enhancing the efficiency of transformation and genome editing in wild-type Bacillus strains. Therefore, the CAC system stands as a powerful tool to facilitate genome editing in diverse wild-type Bacillus species.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Bacillus/genetics/metabolism
*Bacterial Proteins/genetics/metabolism
Plasmids/genetics
CRISPR-Associated Protein 9/antagonists & inhibitors
RNA, Guide, CRISPR-Cas Systems/genetics
Bacillus subtilis/genetics

@article {pmid40551124,
year = {2025},
author = {Shu, J and Tan, Q and Huang, Z and Zhang, T and Ye, L and Fu, S and Mao, Z},
title = {One-pot one-step detection platform for severe fever with thrombocytopenia syndrome virus via the CRISPR/Cas12a detection system.},
journal = {Virology journal},
volume = {22},
number = {1},
pages = {203},
pmid = {40551124},
issn = {1743-422X},
support = {2025KY435//Medical and Health Research Project of Zhejiang Province/ ; 2022CZBJ088//the Top Talent of Changzhou "The 14th Five-Year Plan" High Level Health Talents Training Project/ ; },
mesh = {*Phlebovirus/genetics/isolation & purification ; *Severe Fever with Thrombocytopenia Syndrome/diagnosis/virology ; Humans ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Limit of Detection ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne virus that primarily causes SFTS. Although a common testing route is available, a timely, conventional and accurate method for SFTSV detection is urgently needed. In the present study, we established a platform that combines the recombinase polymerase amplification (RPA) assay with the clustered regularly interspaced short palindromic repeats-CRISPR associated proteins (CRISPR/Cas) 12a technique in one step in one pot. The procedure can be completed within 45 min at a constant temperature without a sophisticated instrument. This method targets the S gene of SFTSV, with a detection limit (LoD) of 11.7 copies per reaction and high specificity, without cross reactivity with other pathogens. Furthermore, across 46 test samples, this method achieved 89.13% consistency with the PCR method (41/46). Together, the reaction system developed in the present study provides not only a novel method for SFTSV detection but also an alternative method for detecting RNA viruses.},
}

*Phlebovirus/genetics/isolation & purification
*Severe Fever with Thrombocytopenia Syndrome/diagnosis/virology
Humans
Sensitivity and Specificity
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*Molecular Diagnostic Techniques/methods
Limit of Detection
CRISPR-Associated Proteins/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40550676,
year = {2025},
author = {Zhan, Y and Zheng, L and Shen, J and Hu, Y and Luo, X and Dai, L},
title = {[Development of a miniaturized CRISPR/Cas gene editing tool for human gut Bacteroides].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {6},
pages = {2360-2372},
doi = {10.13345/j.cjb.240959},
pmid = {40550676},
issn = {1872-2075},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Bacteroides/genetics ; Humans ; *Gastrointestinal Microbiome/genetics ; Bacteroides fragilis/genetics ; },
abstract = {: Bacteroides, as one of the most abundant and diverse genera in the human gut, is regarded as a window into the study of gut microbiota-host interactions. Currently, CRISPR/Cas-based gene editing systems targeting Bacteroides have been widely applied, while the large size of Cas nucleases limits their potential application scenarios (such as in situ gut Bacteroides editing based on phage delivery). Therefore, this study aims to develop a compact and highly efficient genetic editing tool in Bacteroides., We developed a miniaturized CRISPR/Cas gene editing system for human gut Bacteroides. First, the editing capabilities of different miniaturized CRISPR/Cas systems, including AsCas12f, CasΦ2, and ISDge10, were evaluated in Bacteroides fragilis. Subsequently, the editing capability of AsCas12f was assessed across various Bacteroides species, and the size of this system was further optimized. The results demonstrated that the CRISPR/AsCas12f genome editing system exhibited the highest editing efficiency in B. fragilis. The CRISPR/AsCas12f system achieved efficient genome editing in B. fragilis, Bacteroides thetaiotaomicron, and Phocaeicola vulgatus. Furthermore, with a repair template of 500 bp homologous arms, the editing efficiency remained as high as 94.7%. In conclusion, CRISPR/AsCas12f can serve as a chassis tool enzyme for the development of Bacteroides-based miniature gene editors and derivative technologies, laying a foundation for the further development of gene editing technology for Bacteroides.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Bacteroides/genetics
Humans
*Gastrointestinal Microbiome/genetics
Bacteroides fragilis/genetics

@article {pmid40549893,
year = {2025},
author = {Wei, Y and Yue, T and Wang, Y and Yang, Y},
title = {Fertile androgenetic mice generated by targeted epigenetic editing of imprinting control regions.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {27},
pages = {e2425307122},
doi = {10.1073/pnas.2425307122},
pmid = {40549893},
issn = {1091-6490},
support = {2018YFC1004500//National Key Research and Developmental Program of China/ ; 2017YFC1001300//National Key Research and Developmental Program of China/ ; },
mesh = {Animals ; *Genomic Imprinting/genetics ; Male ; Mice ; DNA Methylation/genetics ; *Gene Editing/methods ; Female ; *Epigenesis, Genetic ; *Fertility/genetics ; Spermatozoa/metabolism ; CRISPR-Cas Systems ; Alleles ; Epigenome Editing ; },
abstract = {Each new mammalian life begins with the fusion of an oocyte and a sperm to produce a fertilized egg containing two sets of genomes, one from the mother and one from the father. Androgenesis, a way for producing offspring solely from male genetic material, is limited in mammals, presumably due to barriers arising from genomic imprinting, an epigenetic mechanism leading to monoallelic gene expression. Here, we report adult mammalian offspring derived from the genetic material of two sperm cells. These mice, which we refer to as androgenetic mice, were produced via targeted DNA methylation editing of seven imprinting control regions (ICRs) through CRISPR-based epigenome engineering. Two sperm cells were injected into an enucleated oocyte to form putatively diploid embryos. Allele-specific epigenetic editing was achieved by injecting guide RNAs with protospacer adjacent motif (PAM) sequences designed to match one allele but not the other. The birth of androgenetic mice that were able to develop to adulthood demonstrates that mammalian androgenesis is achievable by targeted epigenetic remodeling of a few defined ICRs.},
}

Animals
*Genomic Imprinting/genetics
Male
Mice
DNA Methylation/genetics
*Gene Editing/methods
Female
*Epigenesis, Genetic
*Fertility/genetics
Spermatozoa/metabolism
CRISPR-Cas Systems
Alleles
Epigenome Editing

@article {pmid40549156,
year = {2025},
author = {Sui, M and Zhou, M and Cui, M and Liu, H and Zhang, X and Hu, N and Li, Y and Wang, B and Yang, G and Gui, P and Zhu, L and Wan, F and Zhang, B},
title = {Novel drug-inducible CRISPRa/i systems for rapid and reversible manipulation of gene transcription.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {82},
number = {1},
pages = {249},
pmid = {40549156},
issn = {1420-9071},
support = {82371443//National Natural Science Foundation of China grant/ ; 2023YFE0117600//National Key R&D Program of China grant/ ; },
mesh = {Humans ; Tamoxifen/pharmacology/analogs & derivatives ; *CRISPR-Cas Systems/genetics ; *Transcription, Genetic/drug effects ; Animals ; Gene Expression Regulation/drug effects ; Mice ; HEK293 Cells ; Receptors, Estrogen/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR activation and interference (CRISPRa/i) are highly effective tools to regulate transcription by fusing dead Cas9 (dCas9) with transcriptional regulatory factors guided by small guide RNA (sgRNA) in mammalian cells and mice. Still, a controllable gene regulation system is desired to investigate and manipulate dynamic biological processes. Here, we reported flexible drug-responsive CRISPRa/i systems by fusing mutated human estrogen receptor (ERT2) domains, which responded to estrogen analogue tamoxifen or its active metabolite 4-hydroxy-tamoxifen (4OHT), to CRISPRa/i components for transcriptional regulation. Upon 4OHT treatment, the optimal variants, ERT2-ERT2-CRISPRa/i-ERT2 (iCRISPRa/i), showed rapid protein translocation of iCRISPRa/i from cytoplasm to nucleus and subsequent transcriptional response. The inducible transcriptional manipulation could be restored to its original level when 4OHT was withdrawn. Moreover, the efficiencies of gene expression regulation of iCRISPRa/i were comparable to those of non-inducible and doxycycline-inducible counterparts, with a lower leakage and a faster drug response activity. The iCRISPRa/i systems successfully induced phenotypic changes in various cell lines. These results highlight that iCRISPRa/i systems could achieve fast and flexible drug-responsive transcriptional modulation and phenotypic changes, and thus provide better options for gain- and loss-of-function model construction and gene therapy.},
}

Humans
Tamoxifen/pharmacology/analogs & derivatives
*CRISPR-Cas Systems/genetics
*Transcription, Genetic/drug effects
Animals
Gene Expression Regulation/drug effects
Mice
HEK293 Cells
Receptors, Estrogen/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40548936,
year = {2025},
author = {Li, Y and Zhao, C and Cao, Y and Chen, X and Tang, Y and Zhou, X and Ingmer, H and Jiao, X and Li, Q},
title = {Oxidative stress elicited by phage infection induces Staphylococcal type III-A CRISPR-Cas system.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf541},
pmid = {40548936},
issn = {1362-4962},
support = {32430103//National Natural Science Foundation of China/ ; B2302024//National Natural Science Foundation of China/ ; 2023YFD1800503//National Key Research and Development Program of China/ ; 2022YFC2604200//National Key Research and Development Program of China/ ; RZZ202302//Jiangsu Key Laboratory of Zoonosis Major Independent Research/ ; 2024ZB284//Jiangsu Funding Program for Excellent Postdoctoral Talent/ ; 2023B1515120016//Guangdong Basic and Applied Basic Research Foundation/ ; JCYJ20220818100616034//Shenzhen Science and Technology Innovation Commission/ ; KYCX24_3850//Yangzhou University/ ; 32430103//Priority Academic Program Development of Jiangsu Higher Education Institution (PAPD)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Oxidative Stress/genetics ; *Staphylococcus aureus/virology/genetics/metabolism/immunology ; Promoter Regions, Genetic ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; *Bacteriophages ; *Staphylococcus Phages ; CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {In prokaryotes, the CRISPR-Cas system provides immunity to invading mobile genetic elements, but its expression is commonly repressed in the absence of phage infection to prevent autoimmunity. How bacteria senses phage infection and activates CRISPR-Cas system are poorly understood. Here, we demonstrate that an essential promoter Pcas, located within the cas1 gene, is the primary promoter driving expression of cas genes encoding the Cas10-Csm interference complex in Staphylococcus aureus type III-A CRISPR-Cas system during phage infection. As a conserved promoter in Staphylococci type III-A CRISPR-Cas system, the Pcas loses its ability to activate cas genes expression when mutated at the C186 site. Importantly, we find that the transcriptional regulator MgrA directly represses type III-A CRISPR-Cas system by interacting with Pcas to prevent autoimmunity. Upon phage infection, MgrA senses oxidative stress and dissociates from the Pcas, alleviating the transcriptional repression and subsequently triggering a robust immunity against phages. Our work provides evidence for the requirement of Pcas within cas1 during type III-A CRISPR-Cas interference stage, and reveals that MgrA-mediated regulation provides an effective mechanism for bacteria to balance avoiding autoimmunityand defending against phages.},
}

*CRISPR-Cas Systems/genetics
*Oxidative Stress/genetics
*Staphylococcus aureus/virology/genetics/metabolism/immunology
Promoter Regions, Genetic
Bacterial Proteins/genetics/metabolism
Gene Expression Regulation, Bacterial
*Bacteriophages
*Staphylococcus Phages
CRISPR-Associated Proteins/genetics/metabolism

@article {pmid40548934,
year = {2025},
author = {Flores-Fernández, CN and Lin, D and Robins, K and O'Callaghan, CA},
title = {UniClo: scarless hierarchical DNA assembly without sequence constraint.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf548},
pmid = {40548934},
issn = {1362-4962},
support = {BB/X511122/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; //University of Oxford/ ; },
mesh = {DNA Methylation ; *DNA/genetics/metabolism/chemistry ; Plasmids/genetics ; CRISPR-Cas Systems ; *Deoxyribonucleases, Type II Site-Specific/metabolism/genetics ; Genetic Vectors/genetics ; },
abstract = {Type IIS restriction enzyme-mediated DNA assembly is efficient but has sequence constraints and can result in unwanted sequence scars. To overcome these drawbacks, we developed UniClo, a type IIS restriction enzyme-mediated method for universal and flexible DNA assembly. This is achieved through a combination of vector engineering, DNA methylation using recombinant methylases, and steric blockade using CRISPR-dCas9 technology to regulate this methylation. Type IIS restriction enzyme sites within fragments to be assembled are methylated using recombinant methylases, while the fragment-flanking outer sites are protected from methylation by a recombinant dCas9-sgRNA complex. During the subsequent assembly reaction, only the protected flanking sites are cut as only they are unmethylated. Fragments are correctly assembled, despite containing internal sites for the single type IIS restriction enzyme used for the one-pot assembly. The assembled plasmid can be used as a donor plasmid in a subsequent assembly round with the same type IIS restriction enzyme and the assembly vector engineering ensures removal of potential scars by a trimming process. This simplifies assembly design and only three vectors are required for any multi-round assembly. These vectors all use the same pair of overhangs. UniClo provides a simple scarless approach for hierarchical assembly of any sequence and has wide potential application.},
}

DNA Methylation
*DNA/genetics/metabolism/chemistry
Plasmids/genetics
CRISPR-Cas Systems
*Deoxyribonucleases, Type II Site-Specific/metabolism/genetics
Genetic Vectors/genetics

@article {pmid40548648,
year = {2025},
author = {Alariqi, M and Ramadan, M and Yu, L and Hui, F and Hussain, A and Zhou, X and Yu, Y and Zhang, X and Jin, S},
title = {Enhancing Specificity, Precision, Accessibility, Flexibility, and Safety to Overcome Traditional CRISPR/Cas Editing Challenges and Shape Future Innovations.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e2416331},
doi = {10.1002/advs.202416331},
pmid = {40548648},
issn = {2198-3844},
support = {2023ZD04074//Biological Breeding-Major Projects/ ; 32325039//National Natural Science Fund of China for Distinguished Young Scholars/ ; 2021hszd013//Hubei Hongshan Laboratory/ ; W2433067//National Natural Science Foundation of China for International Young Scientists/ ; 32272128//National Natural Science Foundation of China/ ; },
abstract = {Derived from the bacterial immune system, CRISPR/Cas9 induces DSBs at specific DNA sequences, which are repaired by the cell's endogenous mechanisms, leading to gene insertions, deletions, or substitutions. Despite its transformative potential, several challenges remain in optimizing of CRISPR/Cas systems, including off-target effects, delivery methods, PAM restrictions, and the limitations of traditional editing approaches. This review focuses on the interplay between these challenges and their contributions to gene editing precision, specificity, accessibility, flexibility, and safety. How reducing off-target effects enhances specificity and safety is explored, while discussing the role of HDR-based editing in achieving precise gene modifications, alongside alternative methods such as base editing and prime editing. Improved delivery mechanisms are examined for their impact on accessibility and efficiency, while the reduction of PAM restrictions is highlighted for its contributions to flexibility. Lastly, emerging cleavage-free editing technologies are evaluated as they relate to safety and accessibility. This focused review aims to clarify the connections among these aspects and outline future research directions for advancing CRISPR-based applications.},
}

@article {pmid40548119,
year = {2025},
author = {Kumbhakar, DV and Thakkar, L and Akhand, C and Sharaf, S and Vemuganti, GK},
title = {Nanomaterials targeting cancer stem cells to overcome drug resistance and tumor recurrence.},
journal = {Frontiers in oncology},
volume = {15},
number = {},
pages = {1499283},
pmid = {40548119},
issn = {2234-943X},
abstract = {A cancer stem cell (CSC) is an immortal cell that is capable of self-renewal, continuous proliferation, differentiation into various cancer cell lineages, metastatic dissemination, tumorigenesis, maintaining tumor heterogeneity, and resistance to conventional treatments. Targeted therapies have made huge advances in the past few years, but resistance is still a major roadblock to their success, in addition to their life-threatening side effects. Progressive treatments are now available, including immunotherapies, CRISPR-Cas 9, sonodynamic therapy, chemodynamic therapy, antibody-drug nanoconjugates, cell-based therapies, gene therapy, and ferroptosis-based therapy, which have replaced surgery, chemotherapy, and radiotherapy for cancer treatment. The challenge is to develop targeted treatment strategies that are effective in eradicating CSCs, as they are resistant to anticancer drugs, causing treatment failure, relapse, and recurrence of cancer. An overview of the fundamental characteristics of CSCs, drug resistance, tumor recurrence, and signaling pathways as well as biomarkers associated with their metastatic potential of CSC is elucidated in this review. The regulatory frameworks for manufacturing and conducting clinical trials on cancer therapy are explicated. Furthermore, we summarize a variety of promising nanocarriers (NCs) that have been used directly and/or synergistic therapies coupled with the therapeutic drug of choice for the detection, targeting, and imaging of CSCs to surmount therapeutic resistance and stemness-related signaling pathways and eradicate CSCs, hence alleviating the limitation of conventional therapies. Nanoparticle-mediated ablation therapies (NMATs) are also being argued as a method for burning or freezing cancer cells without undergoing open surgery. Additionally, we discuss the recent clinical trials testing exosomes, CRISPR/Cas9, and nanodrugs, which have already received approval for several new technologies, while others are still in the early stages of testing. The objective of this review is to elucidate the advantages of nanocarriers in conquering cancer drug resistance and to discuss the most recent developments in this field.},
}

@article {pmid40544217,
year = {2025},
author = {Ossio, A and Merino-Mascorro, A and Leon, JS and Heredia, N and Garcia, S},
title = {Detection of Murine Norovirus on Fresh Produce Through a CRISPR/Cas13a RNase-Based Capsid Integrity Assay.},
journal = {Food and environmental virology},
volume = {17},
number = {3},
pages = {35},
pmid = {40544217},
issn = {1867-0342},
support = {2019-67017-29642//National Institute of Food and Agriculture/ ; PROVERICYT//Universidad Autonoma de Nuevo Leon/ ; PROVERICYT//Universidad Autonoma de Nuevo Leon/ ; },
mesh = {*Norovirus/genetics/isolation & purification ; CRISPR-Cas Systems ; *Ribonucleases/genetics/metabolism ; Animals ; *Capsid/chemistry/metabolism ; *Food Contamination/analysis ; Mice ; *Capsid Proteins/genetics/metabolism ; Food Microbiology/methods ; Fruit/virology ; Humans ; },
abstract = {Standard food detection methods do not distinguish between infectious and non-infectious human norovirus leading to uncertainty in the management of a norovirus positive food sample. These methods also require expensive RT-qPCR-based equipment and reagents. In contrast, CRISPR-based, compared to RT-qPCR-based, detection methods are generally less expensive and yield similar sensitivity and specificity. Our goal was to detect norovirus with an intact capsid, a proxy for infectivity, through a CRISPR-Cas13a-based detection method together with an RNase-capsid integrity assay. We termed this assay: Foodborne RNA-virus Enzymatic Sensing for High-throughput on fresh produce (CRISPR FRESH) reflecting its potential to detect infectious or potentially infectious virus particles. Our CRISPR FRESH method detected murine norovirus (MNV-1), with an intact capsid, at a limit of detection of 2.59 log10 gc/25 g (5 gc/rx). This method did not cross-react with other targets (synthetic DNA targets for hepatitis A virus; human norovirus GI, GII; rotavirus). Compared with RT-qPCR, CRISPR FRESH showed an increased sensitivity when detecting low copy numbers of RNase-pre-treated MNV-1 in lettuce and blueberries samples. Viral detection with the RT-qPCR assay is quantifiable while the CRISPR assay is present/absent. This report describes a CRISPR-based detection of potentially infectious viruses in food samples.},
}

*Norovirus/genetics/isolation & purification
CRISPR-Cas Systems
*Ribonucleases/genetics/metabolism
Animals
*Capsid/chemistry/metabolism
*Food Contamination/analysis
Mice
*Capsid Proteins/genetics/metabolism
Food Microbiology/methods
Fruit/virology
Humans

@article {pmid40540504,
year = {2025},
author = {Yamashita, K and Muramoto, T},
title = {Efficient endogenous protein labelling in Dictyostelium using CRISPR/Cas9 knock-in and split fluorescent proteins.},
journal = {PloS one},
volume = {20},
number = {6},
pages = {e0326577},
pmid = {40540504},
issn = {1932-6203},
mesh = {*Dictyostelium/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *Luminescent Proteins/genetics/metabolism ; *Protozoan Proteins/genetics/metabolism ; Green Fluorescent Proteins/genetics/metabolism ; Histones/genetics/metabolism ; },
abstract = {Fluorescent protein tagging is a powerful technique for visualising protein dynamics; however, full-length fluorescent protein knock-in can be inefficient at certain genomic loci, making it challenging to achieve stable and uniform expression. To address this issue, we used CRISPR/Cas9-mediated knock-in strategies with split fluorescent proteins in Dictyostelium discoideum. This approach enabled efficient integration of the short mNeonGreen2 (mNG2) fragment, mNG211, particularly at functionally critical loci such as major histone h2bv3, where full-length tagging was unsuccessful. Our analysis revealed that inserting tandem repeats of mNG211 at the h2bv3 locus progressively impaired cell proliferation, indicating that functional disruption depends on insert size. These findings suggest that using short tags like mNG211 minimises functional interference and facilitates knock-in at sensitive loci. We further optimised the fluorescence intensity by fine-tuning the expression of the long fragment, mNG21-10, and introducing tandem repeats of mNG211. This approach provides a reliable method for precise and stable endogenous protein labelling, facilitating live-cell imaging and functional studies in D. discoideum.},
}

*Dictyostelium/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Gene Knock-In Techniques/methods
*Luminescent Proteins/genetics/metabolism
*Protozoan Proteins/genetics/metabolism
Green Fluorescent Proteins/genetics/metabolism
Histones/genetics/metabolism

@article {pmid40539512,
year = {2025},
author = {Sung, K and Jung, Y and Kim, N and Kim, YW and Kim, HH and Kim, SK and Bae, S},
title = {A rational engineering strategy for structural dynamics modulation enables target specificity enhancement of the Cas9 nuclease.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf535},
pmid = {40539512},
issn = {1362-4962},
support = {2018R1A2B2001422//National Research Foundation of Korea/ ; 2021M3A9H3015389//National Research Foundation of Korea/ ; RS-2024-00451880//National Research Foundation of Korea/ ; RS-2024-00455559//National Research Foundation of Korea/ ; SRC-NRF2022R1A5A102641311//National Research Foundation of Korea/ ; RS-2022-NR070713//MSIT/ ; RS-2023-00260968//MSIT/ ; RS-2024-00357556//MSIT/ ; RS-2024-00338871//MSIT/ ; RS-2024-00332601//Korean Fund for Regenerative Medicine (KFRM)/ ; },
mesh = {Substrate Specificity ; *Protein Engineering/methods ; Humans ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; DNA/metabolism/chemistry ; Mutation ; Gene Editing ; Models, Molecular ; *Endonucleases/metabolism/genetics/chemistry ; },
abstract = {Structural dynamics of an enzyme plays a crucial role in enzymatic activity and substrate specificity, yet rational engineering of the dynamics for improved enzymatic properties remains a challenge. Here, we present a new biochemical strategy of intermediate state stabilization that modulates the multistep dynamic mechanisms of enzyme reactions to improve substrate specificity. We employ this strategy to enhance CRISPR-Cas9 nuclease specificity. By incorporating positively charged residues into the noncatalytic REC2 domain of Cas9, we stabilize the REC2-DNA interaction that forms exclusively in a catalytically inactive intermediate conformation of the Cas9 complex. This enables off-target trapping in the inactive conformation and thus reduces off-target cleavage in human cells. Furthermore, we combine the REC2 modification with mutations in previous rational variants, leading to the development of a combinational variant named Correct-Cas9, which connotes "combined with rationally engineered REC-Two" Cas9. Assessed by high-throughput analysis at thousands of target sequences, Correct-Cas9 exhibits increased target specificity compared to its parental variants, demonstrating a synergy between our strategy and previous rational approaches. Our method of intermediate state stabilization, either alone or combined with conventional approaches, could be applied to various nucleic acid-processing enzymes that undergo conformational changes upon target binding, to enhance their target specificity effectively.},
}

Substrate Specificity
*Protein Engineering/methods
Humans
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/chemistry/genetics/metabolism
DNA/metabolism/chemistry
Mutation
Gene Editing
Models, Molecular
*Endonucleases/metabolism/genetics/chemistry

@article {pmid40539281,
year = {2025},
author = {Glenthøj, A and Carlsen, SBIS and Hoffmann, M and Haastrup, EK and Andersen, LP and Toft, N and Kornblit, BT and Petersen, JB and Hasle, H and Ifversen, MRS},
title = {[CRISPR as a functional cure for hemoglobinopathies].},
journal = {Ugeskrift for laeger},
volume = {187},
number = {22},
pages = {},
doi = {10.61409/V12240888},
pmid = {40539281},
issn = {1603-6824},
mesh = {Humans ; *Gene Editing/methods ; *Hemoglobinopathies/therapy/genetics ; *Genetic Therapy/methods ; Anemia, Sickle Cell/therapy/genetics ; *CRISPR-Cas Systems ; beta-Thalassemia/therapy/genetics ; Fetal Hemoglobin/genetics/biosynthesis ; },
abstract = {Severe haemoglobinopathies, including sickle cell disease and β-thalassaemia, represent significant global health burdens. CRISPR technology enables precise genetic editing of haematopoietic stem cells, with current therapies focused on boosting fetal haemoglobin production for a functional cure. This review finds that, while promising, ex vivo approaches require advanced facilities and substantial resources, limiting accessibility where the need is highest. Future development of in vivo methods may expand global access, addressing the urgent need for scalable and affordable treatments for these debilitating diseases.},
}

Humans
*Gene Editing/methods
*Hemoglobinopathies/therapy/genetics
*Genetic Therapy/methods
Anemia, Sickle Cell/therapy/genetics
*CRISPR-Cas Systems
beta-Thalassemia/therapy/genetics
Fetal Hemoglobin/genetics/biosynthesis

@article {pmid40538650,
year = {2025},
author = {Song, Y and Hu, Q and Han, Y and Liu, H and Huang, Z and Niu, M and Dong, X and Yan, K and Jin, L and Li, H and Sun, Y},
title = {Detection assay of polymyxin resistance coding mcr-1 gene based on CRISPR/Cas13a system.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1553681},
pmid = {40538650},
issn = {2235-2988},
mesh = {*CRISPR-Cas Systems ; *Escherichia coli Proteins/genetics ; *Polymyxins/pharmacology ; *Escherichia coli/genetics/drug effects/isolation & purification ; *Anti-Bacterial Agents/pharmacology ; *Drug Resistance, Bacterial/genetics ; Humans ; Sensitivity and Specificity ; Real-Time Polymerase Chain Reaction/methods ; Microbial Sensitivity Tests ; Escherichia coli Infections/microbiology ; },
abstract = {INTRODUCTION: Polymyxins are reserved as an ultimate defense against multidrug-resistant bacteria. The emergence of the polymyxin resistance gene mcr-1 poses a potential risk for the treatment of severe infections caused by Gram-negative bacteria. Timely detection and monitoring the mcr-1 gene are essential for guiding anti-infective therapy and controlling the spread of polymyxin resistance. Quantitative real-time PCR (qPCR) is one of the common methods for detecting resistance genes. However, qPCR has equipment dependency, and is not feasible in primary healthcare settings. Currently, there remains a lack of a highly sensitive and portable method for detecting the mcr-1 gene.

METHODS: We established and optimized detection assays of the mcr-1 gene based on CRISPR/Cas13a system and lateral flow strips. The detection method was preliminarily evaluated using clinical isolates from Escherichia coli, compared with qPCR.

RESULTS: The method for detecting the mcr-1 gene based on the CRISPR/Cas13a system and lateral flow strips was established, with a detection limit of 100 copies/mL. This method demonstrated high analytical specificity, with no cross-reactivity detected in non-mcr-1 and non-resistant strains. Among 36 clinical isolates, the method identified 31 strains as positive for the mcr-1 gene, and had a 100% concordance rate with the results of qPCR.

CONCLUSIONS: We established a detection method for the polymyxin resistance mcr-1 gene based on the CRISPR/Cas13a system. This method enables visual readouts without instruments, making it potentially applicable to primary healthcare settings and field surveillance.},
}

*CRISPR-Cas Systems
*Escherichia coli Proteins/genetics
*Polymyxins/pharmacology
*Escherichia coli/genetics/drug effects/isolation & purification
*Anti-Bacterial Agents/pharmacology
*Drug Resistance, Bacterial/genetics
Humans
Sensitivity and Specificity
Real-Time Polymerase Chain Reaction/methods
Microbial Sensitivity Tests
Escherichia coli Infections/microbiology

@article {pmid40537891,
year = {2025},
author = {Gao, Q and Zhang, T and Yuan, Y and Li, G and Li, B and Xiong, C},
title = {Detection of KPC-Producing Carbapenem-Resistant Klebsiella pneumoniae Based on CRISPR Cas12a.},
journal = {Journal of microbiology and biotechnology},
volume = {35},
number = {},
pages = {e2502042},
doi = {10.4014/jmb.2502.02042},
pmid = {40537891},
issn = {1738-8872},
mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation & purification/enzymology ; *Bacterial Proteins/genetics/metabolism ; *beta-Lactamases/genetics/metabolism ; *Carbapenems/pharmacology ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; Humans ; Klebsiella Infections/microbiology ; Anti-Bacterial Agents/pharmacology ; *Carbapenem-Resistant Enterobacteriaceae/genetics/isolation & purification/enzymology ; *CRISPR-Associated Proteins/genetics ; *Endodeoxyribonucleases/genetics ; Polymerase Chain Reaction/methods ; ROC Curve ; },
abstract = {To develop a detection system for Klebsiella pneumoniae carbapenemase (KPC) and provide a reference for clinical prevention and control of nosocomial infections caused by multidrug-resistant K. pneumoniae. The KPC resistance gene was amplified by PCR. Guided by crRNA, Cas12a specifically identified the resistance gene and activated its trans-cleavage activity. In the detection system, a fluorescence probe was cleaved by activated Cas12a, and the fluorescence signal was measured using a microplate reader. Under optimized conditions, the fluorescence signal appeared within 12 min, peaked at 40 min and completed detection within 60 min. sensitivity: 91.2%, specificity: 84.1%, detection limit: 0.01 ng/μl. The samples were examined by fluorescence-CRISPR Cas12a and PCR. The coincidence rate was 85.9%, Kappa value was 0.8. The ROC curve analysis revealed an AUC of 0.916, with an optimal cutoff value of 1.55, sensitivity of 91.2%, and specificity of 84.1%. The CRISPR Cas12a detection of carbapenem-resistant K. pneumoniae (CRKP) demonstrates high sensitivity, specificity, and broad applicability. This method requires standard molecular biology equipment but does not rely on sequencing-based platforms.},
}

*Klebsiella pneumoniae/genetics/drug effects/isolation & purification/enzymology
*Bacterial Proteins/genetics/metabolism
*beta-Lactamases/genetics/metabolism
*Carbapenems/pharmacology
Sensitivity and Specificity
*CRISPR-Cas Systems
Humans
Klebsiella Infections/microbiology
Anti-Bacterial Agents/pharmacology
*Carbapenem-Resistant Enterobacteriaceae/genetics/isolation & purification/enzymology
*CRISPR-Associated Proteins/genetics
*Endodeoxyribonucleases/genetics
Polymerase Chain Reaction/methods
ROC Curve

@article {pmid40537846,
year = {2025},
author = {Sarno, F and Jacob, JJ and Eilers, RE and Nebbioso, A and Altucci, L and Rots, MG},
title = {Epigenetic editing and epi-drugs: a combination strategy to simultaneously target KDM4 as a novel anticancer approach.},
journal = {Clinical epigenetics},
volume = {17},
number = {1},
pages = {105},
pmid = {40537846},
issn = {1868-7083},
mesh = {Humans ; *Jumonji Domain-Containing Histone Demethylases/genetics/antagonists & inhibitors ; *Epigenesis, Genetic/drug effects ; DNA Methylation/drug effects ; *Gene Editing/methods ; Cell Line, Tumor ; *Antineoplastic Agents/pharmacology ; Gene Expression Regulation, Neoplastic/drug effects ; *Neoplasms/genetics/drug therapy ; CRISPR-Cas Systems ; Epigenome Editing ; },
abstract = {KDM4-A/B/C, preferentially demethylating di- and tri-methylated lysine 9 on histone H3, are overexpressed in cancers and considered interesting therapeutic targets. Consequently, KDM4 inhibitors have been developed to block their enzymatic activity. However, the potential lack of specificity of such small molecules (epi-drugs) may contribute to dose-limiting toxicities. In the pursuit of more specific interventions, epigenetic editing (epi-editing) has emerged as a powerful tool to modulate gene expression by modifying the epigenetic profile of specific genomic locations. The recently developed CRISPRoff (dCas9 fused to DNMT3A/3L and KRAB), guided by sgRNAs, is successfully used for gene repression by introducing methylation of DNA and (indirectly) of histones at the targeted genomic region. We propose that combining epi-editing (here to prevent the expression of KDM4) with epi-drugs (to inhibit the KDM4 protein activity) may represent a novel path for synergistic anticancer effects through simultaneous inhibition of gene expression and protein activity. Upon validating the downregulation of KDM4A in HEK293T cells through epi-editing, we demonstrated its repression in colon, breast and hepatocellular carcinomas which was effective in preventing (breast, MCF7) or inhibiting (colon, HCT116) cancer cell growth. Anticancer effect was also confirmed for these cell lines using the KDM4 inhibitor QC6352. In parallel, our studies demonstrate a previously unnoticed increase in the expression of KDM4-A/B/C genes following the inhibition of protein activity using the pan-KDM4 inhibitors QC6352 and JIB-04. Importantly, this induction of gene expression was fully prevented or even further inhibited by epi-editing. Then, we assessed the efficacy of our dual-targeted silencing approach in cancer cells and demonstrated that the inhibition in cancer cell growth by epi-drug or epigenetic editing could be further improved by combining the treatments. Building upon these findings, we introduce a novel, potentially synergistic, therapeutic strategy that combines epi-drug administration with epi-editing. This innovative approach aims to reduce drug toxicity and the potential development of resistance by preventing drug-induced upregulation of target enzyme expression, thereby further increasing anticancer effects.},
}

Humans
*Jumonji Domain-Containing Histone Demethylases/genetics/antagonists & inhibitors
*Epigenesis, Genetic/drug effects
DNA Methylation/drug effects
*Gene Editing/methods
Cell Line, Tumor
*Antineoplastic Agents/pharmacology
Gene Expression Regulation, Neoplastic/drug effects
*Neoplasms/genetics/drug therapy
CRISPR-Cas Systems
Epigenome Editing

@article {pmid40537532,
year = {2025},
author = {Norota, K and Ishizuka, S and Hirose, M and Sato, Y and Maeki, M and Tokeshi, M and Ibrahim, SM and Harashima, H and Yamada, Y},
title = {Lipid nanoparticle delivery of the CRISPR/Cas9 system directly into the mitochondria of cells carrying m.7778G>T mutation in MtDNA (mt-Atp8).},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {18717},
pmid = {40537532},
issn = {2045-2322},
support = {HI1813/7-1//Deutsche Forschungsgemeinschaft/ ; 23H00541//Ministry of Education, Culture, Sports, Science and Technology/ ; JPMJFR203X//Japan Science and Technology Corporation/ ; },
mesh = {*CRISPR-Cas Systems ; *DNA, Mitochondrial/genetics ; Humans ; Animals ; *Nanoparticles/chemistry/administration & dosage ; HeLa Cells ; Mice ; *Point Mutation ; *Mitochondria/genetics/metabolism ; *Lipids/chemistry ; Mitochondrial Diseases/genetics/therapy ; DNA Breaks, Double-Stranded ; Ribonucleoproteins/genetics ; Liposomes ; },
abstract = {Mitochondrial genome mutations are associated with various diseases and gene therapy targeted to mitochondria has the potential to effectively treat such diseases. Here, we targeted a point mutation in mitochondrial DNA (mtDNA) that can cause mitochondrial diseases via delivery of the clustered, regularly interspaced, short palindromic repeats/Cas9 (CRISPR/Cas9) system to mitochondria using an innovative lipid nanoparticle (LNP) delivery system. To overcome the major barrier of the mitochondrial membrane structure, we investigated a strategy to deliver ribonucleoprotein (RNP) directly to mitochondria via membrane fusion using MITO-Porter, a mitochondria-targeting lipid nanoparticle. First, we constructed RNP-MITO-Porter, in which an RNP was loaded into MITO-Porter using a microfluidic device. Sequence-specific double-strand breaks were confirmed when the constructed RNP-MITO-Porter was applied to isolated mitochondria. Next, the RNP-MITO-Porter was applied to HeLa cells, and a portion of the RNP-MITO-Porter was colocalized with mitochondria and caused sequence-specific double-strand breaks in mtDNA. Finally, RNP-MITO-Porter was successfully delivered to mitochondria of cells derived from a mouse carrying a point mutation (m.7778G > T) in mtDNA (mt-Atp8) (LMSF-N-MTFVB cells), and created double-strand breaks at the target sequence. RNP-MITO-Porter is expected to contribute significantly to the clinical application of mitochondrion-targeted gene therapy.},
}

*CRISPR-Cas Systems
*DNA, Mitochondrial/genetics
Humans
Animals
*Nanoparticles/chemistry/administration & dosage
HeLa Cells
Mice
*Point Mutation
*Mitochondria/genetics/metabolism
*Lipids/chemistry
Mitochondrial Diseases/genetics/therapy
DNA Breaks, Double-Stranded
Ribonucleoproteins/genetics
Liposomes

@article {pmid40480225,
year = {2025},
author = {Zou, S and Sun, Y and Tang, W},
title = {Charting the development and engineering of CRISPR base editors: lessons and inspirations.},
journal = {Cell chemical biology},
volume = {32},
number = {6},
pages = {789-808},
doi = {10.1016/j.chembiol.2025.05.003},
pmid = {40480225},
issn = {2451-9448},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cytosine/metabolism/chemistry ; Adenine/chemistry/metabolism ; },
abstract = {CRISPR base editors (BEs) have introduced a new chapter in precise genome editing. The brief but fruitful history of BE development documents many case studies that not only lay the foundation of base-editing technology but are also instrumental to future protein engineering efforts. In this review, we summarize the development and engineering of various BEs with a focus on recent progress. These include traditional cytosine and adenine base editors (CBEs and ABEs), novel TadA-derived CBEs, transversion BEs, dual BEs, and CRISPR-free BEs. We discuss each aspect of the workflow and highlight the successes and challenges encountered in the engineering process.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Cytosine/metabolism/chemistry
Adenine/chemistry/metabolism

@article {pmid40456614,
year = {2025},
author = {Zetterdahl, OG and Crowe, JA and Reyhani, S and Güra, MA and Labastida-Botey, O and Girard, AS and Froese, DS and Ahlenius, H and Canals, I},
title = {Generation of iPSC Lines with Tagged α-Synuclein for Visualization of Endogenous Protein in Human Cellular Models of Neurodegenerative Disorders.},
journal = {eNeuro},
volume = {12},
number = {6},
pages = {},
doi = {10.1523/ENEURO.0093-25.2025},
pmid = {40456614},
issn = {2373-2822},
mesh = {Humans ; *alpha-Synuclein/metabolism/genetics ; *Induced Pluripotent Stem Cells/metabolism ; *Neurodegenerative Diseases/metabolism/pathology ; Neurons/metabolism ; Cell Line ; CRISPR-Cas Systems ; Gene Editing ; Cell Differentiation ; },
abstract = {α-Synuclein is a synaptic protein that accumulates primarily in synucleinopathies and secondarily in certain lysosomal storage disorders. However, its physiological roles in health and disease are not fully understood. In part, this has been hampered by the inability to visualize α-synuclein and its cellular localization, due to the lack of specific antibodies and faithful reporters. Here, we used CRISPR/Cas9-based genome editing to generate human-induced pluripotent stem cell (iPSC) lines in which the α-synuclein (SNCA) gene has been tagged with the short HA peptide either at the N-terminus or C-terminus or with the fluorescent protein mCherry at the C-terminus of the protein. These diverse strategies revealed the C-terminus HA-tag as the best option. C-Terminus HA-tagged α-synuclein had unchanged protein expression and did not generate degradation by-products. Importantly, we show that following differentiation to neurons, the C-terminus HA-tagged iPSC line had unaffected electrophysiological properties and could be used to visualize accumulation of α-synuclein upon inhibition of lysosomal function and under physiological protein levels. It is our expectation that this line and tagging approach will be very useful in further studies examining α-synuclein aggregation and its role in cellular dysfunction and neurodegeneration.},
}

Humans
*alpha-Synuclein/metabolism/genetics
*Induced Pluripotent Stem Cells/metabolism
*Neurodegenerative Diseases/metabolism/pathology
Neurons/metabolism
Cell Line
CRISPR-Cas Systems
Gene Editing
Cell Differentiation

@article {pmid40349174,
year = {2025},
author = {Cui, Z and Huang, F and Fang, K and Yan, J and Zhang, Y and Kang, DD and Zhou, Y and Zhao, Y and Everitt, JI and Hankey, W and Armstrong, AJ and Huang, J and Wang, H and Jin, VX and Dong, Y and Wang, Q},
title = {SCORT-Cas13d Nanotherapy Precisely Targets the 'Undruggable' Transcription Factor HoxB13 in Metastatic Prostate Cancer In Vivo.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {23},
pages = {e2417605},
doi = {10.1002/advs.202417605},
pmid = {40349174},
issn = {2198-3844},
support = {R01CA287510/CA/NCI NIH HHS/United States ; R35GM144117/GM/NIGMS NIH HHS/United States ; //Department of Pathology/ ; //Duke University School of Medicine/ ; 2023-TRG-0006//North Carolina Biotechnology Center/ ; R01CA287510/CA/NCI NIH HHS/United States ; R35GM144117/GM/NIGMS NIH HHS/United States ; },
mesh = {Male ; *Homeodomain Proteins/genetics/metabolism ; *Prostatic Neoplasms/genetics/pathology/metabolism/therapy ; Animals ; Mice ; Humans ; Cell Line, Tumor ; *Nanoparticles ; CRISPR-Cas Systems/genetics ; Neoplasm Metastasis ; Cell Proliferation ; },
abstract = {Metastatic cancer, the primary cause of cancer mortality, frequently exhibits heightened dependence on certain transcription factors (TFs), which serve as master regulators of oncogenic signaling yet are often untargetable by small molecules. Selective Cell in ORgan Targeting (SCORT) nanoparticles are developed for precise CRISPR/Cas13d mRNA and gRNA delivery to metastatic cancer cells in vivo, aiming to knock down the undruggable oncogenic TF HoxB13. In prostate cancer liver metastasis models driven by HoxB13, repeated systemic SCORT-Cas13d-gHoxB13 treatment significantly decreases HoxB13 expression, reduces metastasis, and extends mouse survival. Prolonged treatment shows no significant impact on major organ function, histology or immune markers. Mechanistically, SCORT-Cas13d-gHoxB13 treatment suppresses metastatic tumor proliferation and angiogenesis while promoting apoptosis by regulating multiple gene pathways. Unexpectedly, it inhibits the non-canonical, EMT-independent oncogenic function of Snail. These findings suggest that SCORT-Cas13d-gHoxB13 can effectively and safely target the undruggable HoxB13 in metastatic prostate cancer, positioning CRISPR/Cas13d as a potential treatment.},
}

Male
*Homeodomain Proteins/genetics/metabolism
*Prostatic Neoplasms/genetics/pathology/metabolism/therapy
Animals
Mice
Humans
Cell Line, Tumor
*Nanoparticles
CRISPR-Cas Systems/genetics
Neoplasm Metastasis
Cell Proliferation

@article {pmid40325913,
year = {2025},
author = {Sugita, K and Kurata, M},
title = {Identification of Target Genes Using Innovative Screening Systems.},
journal = {Pathology international},
volume = {75},
number = {6},
pages = {257-266},
pmid = {40325913},
issn = {1440-1827},
mesh = {Humans ; *Neoplasms/genetics ; *Genetic Testing/methods ; Oncogenes/genetics ; High-Throughput Nucleotide Sequencing ; Single-Cell Analysis/methods ; CRISPR-Cas Systems ; Mutation ; Animals ; },
abstract = {Advances in cancer biology have been achieved by the identification of oncogenes and tumor suppressor genes through the remarkable progression of next-generation sequencing. New techniques, such as single-cell analysis, help uncover cancer progression and heterogeneity. Reverse genetic screenings, including methods like random mutagenesis via retroviruses, transposons, RNA interference, and CRISPR, are useful for exploring gene functions and their roles in cancer. Especially in random mutagenesis, CRISPR screening and its modifications have recently emerged as powerful tools due to their comprehensiveness and simplicity in inducing genetic mutations. Initially, CRISPR screening focused on analyzing biological phenotypes in a cell population. It has since evolved to incorporate advanced techniques, such as combining single-cell and spatial analyses. These developments enable the investigation of cell-cell and spatial interactions, which more closely mimic In Vivo microenvironments, offering deeper insights into complex biological processes. These approaches allow for the identification of essential genes involved in cancer survival, drug resistance, and tumorigenesis. Together, these technologies are advancing cancer research and therapeutic development.},
}

Humans
*Neoplasms/genetics
*Genetic Testing/methods
Oncogenes/genetics
High-Throughput Nucleotide Sequencing
Single-Cell Analysis/methods
CRISPR-Cas Systems
Mutation
Animals

@article {pmid40301572,
year = {2025},
author = {Zamora-Dorta, M and Laine-Menéndez, S and Abia, D and González-García, P and López, LC and Fernández-Montes, P and Calvo, E and Vázquez, J and Enríquez, JA and Balsa, E},
title = {Time-resolved mitochondrial screen identifies regulatory components of oxidative metabolism.},
journal = {EMBO reports},
volume = {26},
number = {12},
pages = {3045-3074},
pmid = {40301572},
issn = {1469-3178},
support = {PID2019-110766GA-I00//Ministerio de Ciencia, Innovación y Universidades (MCIU)/ ; PID2022-137404OB-I00//Ministerio de Ciencia, Innovación y Universidades (MCIU)/ ; PID2021-1279880B//Ministerio de Ciencia, Innovación y Universidades (MCIU)/ ; FPU21/06416//Ministerio de Ciencia, Innovación y Universidades (MCIU)/ ; CEX2021-001154-S//Ministerio de Ciencia, Innovación y Universidades (MCIU)/ ; CEX2020-001041-S//Ministerio de Ciencia, Innovación y Universidades (MCIU)/ ; Margarita Salas-UAM postdoctoral fellowship CA4/RSUE/2022-00037//Ministerio de Ciencia, Innovación y Universidades (MCIU)/ ; FPI-UAM PhD fellowship//Ministerio de Ciencia, Innovación y Universidades (MCIU)/ ; ERC-2020-STG grant agreement n{degree sign}
948478//EC | ERC | HORIZON EUROPE European Research Council (ERC)/ ; PR_EX_2022-01//CRIS Cancer Foundation (CRIS Foundation)/ ; LCF/PR/H23/52430010//'la Caixa' Foundation ('la Caixa')/ ; PRTR//EC | NextGenerationEU (NGEU)/ ; CB16/10/00282//Centro de Investigación Biomédica en Red Fragilidad y Envejecimiento Saludable (CIBERFES)/ ; },
mesh = {Humans ; *Mitochondria/metabolism/genetics ; *Oxidative Phosphorylation ; CRISPR-Cas Systems ; Mitochondrial Proteins/metabolism/genetics ; Oxidation-Reduction ; Amino Acids, Branched-Chain/metabolism ; Mitochondrial Diseases/genetics/metabolism ; },
abstract = {Defects in mitochondrial oxidative metabolism underlie many genetic disorders with limited treatment options. The incomplete annotation of mitochondrial proteins highlights the need for a comprehensive gene inventory, particularly for Oxidative Phosphorylation (OXPHOS). To address this, we developed a CRISPR/Cas9 loss-of-function library targeting nuclear-encoded mitochondrial genes and conducted galactose-based screenings to identify novel regulators of mitochondrial function. Our study generates a gene catalog essential for mitochondrial metabolism and maps a dynamic network of mitochondrial pathways, focusing on OXPHOS complexes. Computational analysis identifies RTN4IP1 and ECHS1 as key OXPHOS genes linked to mitochondrial diseases in humans. RTN4IP1 is found to be crucial for mitochondrial respiration, with complexome profiling revealing its role as an assembly factor required for the complete assembly of complex I. Furthermore, we discovered that ECHS1 controls oxidative metabolism independently of its canonical function in fatty acid oxidation. Its deletion impairs branched-chain amino acids (BCAA) catabolism, disrupting lipoic acid-dependent enzymes such as pyruvate dehydrogenase (PDH). This deleterious phenotype can be rescued by restricting valine intake or catabolism in ECHS1-deficient cells.},
}

Humans
*Mitochondria/metabolism/genetics
*Oxidative Phosphorylation
CRISPR-Cas Systems
Mitochondrial Proteins/metabolism/genetics
Oxidation-Reduction
Amino Acids, Branched-Chain/metabolism
Mitochondrial Diseases/genetics/metabolism

@article {pmid40268165,
year = {2025},
author = {Jerke, U and Eulenberg-Gustavus, C and Wagner, DL and Schreiber, A and Kettritz, R},
title = {CRISPR-Cas9 mediated proteinase 3 autoantigen deletion as a treatment strategy for anti-neutrophil cytoplasmic autoantibody-associated vasculitis.},
journal = {Kidney international},
volume = {108},
number = {1},
pages = {145-149},
doi = {10.1016/j.kint.2025.03.020},
pmid = {40268165},
issn = {1523-1755},
mesh = {Humans ; *Myeloblastin/genetics/immunology/metabolism ; *CRISPR-Cas Systems ; *Autoantigens/genetics/immunology ; Neutrophils/immunology ; *Gene Editing/methods ; *Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/therapy/immunology/genetics ; Hematopoietic Stem Cells/immunology ; Cell Differentiation ; Proof of Concept Study ; Antibodies, Antineutrophil Cytoplasmic/immunology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cells, Cultured ; *Genetic Therapy/methods ; Leukocyte Elastase ; },
abstract = {INTRODUCTION: Proteinase 3 (PR3) is a major autoantigen in patients with anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV). Here, we performed a proof-of-principle study using ex vivo CRISPR-Cas9 guided gene editing to eliminate the PR3 autoantigen as an alternative to suppressing the autoimmune response to PR3.

METHODS: A ribonucleoprotein (RNP) complex of Cas9 protein and a PR3-specific single guide-RNA was transfected into human CD34[+] hematopoietic stem and progenitor cells (HSPC) by electroporation. Effects on PR3 protein abundance, neutrophil differentiation, and ANCA-dependent and -independent neutrophil responses were assessed.

RESULTS: Gene editing introduced a frame shift in exon 2 of PRTN3. Consequently, PR3 protein was efficiently reduced in neutrophil-differentiated HSPCs as demonstrated by immunoblotting, ELISA, microscopy, and the complete absence of PR3-specific proteolytic activity. Human neutrophil elastase served as control and was not affected. PR3-deleted (PR3[KO])- and PR3 wild-type (PR3[WT])-HSPCs showed similar neutrophil differentiation. Importantly, general neutrophil defense functions to non-ANCA receptor-independent and -dependent stimuli were similar in PR3[KO]- and PR3[WT]-neutrophils as was constitutive apoptosis. Flow cytometry showed that cell membrane-PR3 was significantly reduced on PR3[KO]-neutrophils and consequent neutrophil activation to either monoclonal antibodies to PR3 or human PR3-ANCA was attenuated. In contrast, myeloperoxidase-ANCA stimulation was not affected.

CONCLUSIONS: We show the feasibility and efficacy of depleting the PR3 autoantigen in human CD34[+] HSPCs using CRISPR-Cas9. Depleting the PR3 autoantigen instead of suppressing the autoimmune response to PR3 could potentially lead to drug-free remission, particularly in patients with refractory or relapsing disease.},
}

Humans
*Myeloblastin/genetics/immunology/metabolism
*CRISPR-Cas Systems
*Autoantigens/genetics/immunology
Neutrophils/immunology
*Gene Editing/methods
*Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis/therapy/immunology/genetics
Hematopoietic Stem Cells/immunology
Cell Differentiation
Proof of Concept Study
Antibodies, Antineutrophil Cytoplasmic/immunology
RNA, Guide, CRISPR-Cas Systems/genetics
Cells, Cultured
*Genetic Therapy/methods
Leukocyte Elastase

@article {pmid40172759,
year = {2025},
author = {Yang, H and Ji, X and Zhong, H and Yang, X and Hu, D and Cai, G and Wu, Z},
title = {CRISPR screening identifies protein methylation and ubiquitination modifications that modulate aflatoxin B1 cytotoxicity.},
journal = {Science China. Life sciences},
volume = {68},
number = {7},
pages = {2121-2136},
pmid = {40172759},
issn = {1869-1889},
mesh = {*Aflatoxin B1/toxicity ; *Ubiquitination/drug effects ; Humans ; Protein Processing, Post-Translational/drug effects ; Methylation/drug effects ; *Cystathionine beta-Synthase/metabolism/genetics ; S-Adenosylmethionine/metabolism ; CRISPR-Cas Systems ; Mitochondria/metabolism/drug effects ; Cell Survival/drug effects ; Gene Knockout Techniques ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Death/drug effects ; },
abstract = {Aflatoxin B1 (AFB1) is one of the most potent mycotoxins affecting human health and animal production. To deeply understand the host-toxin interaction, we performed CRISPR screening and identified cystathionine β-synthase (CBS) as a critical host gene affecting AFB1 cytotoxicity. Mechanistic studies revealed that CBS affects AFB1-induced cell death by regulating the abundance of protein post-translational modifications (PTMs) in host cells. First, AFB1 disrupted the transfer of S-adenosylmethionine (SAM) from the cytoplasm to the mitochondria, thereby reducing the intra-mitochondrial protein methylation level. Deficient intra-mitochondrial protein methylation impaired mitochondrial function and caused cell death. CBS knockout (KO) can enhance SAM generation and mobilization to restore intra-mitochondrial SAM levels by rescuing the perturbed methionine cycle after AFB1 exposure, thereby alleviating AFB1-induced cell death. Second, AFB1 decreased global protein ubiquitination levels by affecting gene expression of ubiquitin-modified enzymes. CBS-KO and pharmaceutical treatment correcting gene expression of ubiquitin-modified enzymes can rescue AFB1-induced cell death. We also investigated two PTM-regulating small molecules, SAM and PR-619, which can increase cell viability in AFB1-exposed cells.},
}

*Aflatoxin B1/toxicity
*Ubiquitination/drug effects
Humans
Protein Processing, Post-Translational/drug effects
Methylation/drug effects
*Cystathionine beta-Synthase/metabolism/genetics
S-Adenosylmethionine/metabolism
CRISPR-Cas Systems
Mitochondria/metabolism/drug effects
Cell Survival/drug effects
Gene Knockout Techniques
*Clustered Regularly Interspaced Short Palindromic Repeats
Cell Death/drug effects

@article {pmid40100189,
year = {2025},
author = {Džafo, E and Hafezi, M and Attianese, GMPG and Reichenbach, P and Grillet, S and Garcia, H and Cribioli, E and Voize, C and Tissot, S and de Silly, RV and Coukos, G and Scholten, K and Irving, M and Gentner, B},
title = {DNA-dependent protein kinase inhibitors PI-103 and samotolisib augment CRISPR/Cas9 knock-in efficiency in human T cells.},
journal = {Cytotherapy},
volume = {27},
number = {6},
pages = {766-773},
doi = {10.1016/j.jcyt.2025.02.001},
pmid = {40100189},
issn = {1477-2566},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *T-Lymphocytes/drug effects/metabolism/immunology ; *DNA-Activated Protein Kinase/antagonists & inhibitors ; *Gene Knock-In Techniques/methods ; *Protein Kinase Inhibitors/pharmacology ; *Pyrimidines/pharmacology ; },
abstract = {The adoptive transfer of autologous peripheral blood T cells gene-modified to express preselected, tumor antigen-specific T-cell receptors (TCRs) is a promising treatment for solid cancers. While gene-transfer by viral transduction is highly efficient, the insertional site is not targeted and persistence of the T cells is oftentimes limited. In contrast, site-specific integration of the TCR into the TCR α chain (TRAC) locus by CRISPR/Cas9 has been shown to enable more consistent and physiologic levels of exogenous TCR expression coupled with superior persistence and tumor control in preclinical studies. Here, we sought to improve the efficiency of CRISPR/Cas9 mediated TCR knock-in (KI) into the TRAC locus of primary human T cells. In addition to the previously reported DNA-dependent protein kinase (DNA-PK) inhibitor M3814, we demonstrated that PI-103 and samotolisib markedly increase KI efficiency in a process that is good manufacturing process (GMP)-compatible. Importantly, samotolisib enabled the generation of a potent T-cell product, having no negative impact on T-cell viability, phenotype, expansion, effector function, and tumor control. Overall, we conclude that our GMP-compatible CRISPR/Cas9 protocol comprising samotolisib to augment TCR KI efficiency is suitable for the generation of genetically modified T cells for clinical use.},
}

Humans
*CRISPR-Cas Systems/genetics
*T-Lymphocytes/drug effects/metabolism/immunology
*DNA-Activated Protein Kinase/antagonists & inhibitors
*Gene Knock-In Techniques/methods
*Protein Kinase Inhibitors/pharmacology
*Pyrimidines/pharmacology

@article {pmid40035969,
year = {2025},
author = {Feng, Y and Liu, G and Li, H and Cheng, L},
title = {The landscape of cell lineage tracing.},
journal = {Science China. Life sciences},
volume = {68},
number = {7},
pages = {1941-1963},
pmid = {40035969},
issn = {1869-1889},
mesh = {*Cell Lineage/genetics ; Humans ; Animals ; *Cell Tracking/methods ; CRISPR-Cas Systems ; Cell Differentiation ; },
abstract = {Cell fate changes play a crucial role in the processes of natural development, disease progression, and the efficacy of therapeutic interventions. The definition of the various types of cell fate changes, including cell expansion, differentiation, transdifferentiation, dedifferentiation, reprogramming, and state transitions, represents a complex and evolving field of research known as cell lineage tracing. This review will systematically introduce the research history and progress in this field, which can be broadly divided into two parts: prospective tracing and retrospective tracing. The initial section encompasses an array of methodologies pertaining to isotope labeling, transient fluorescent tracers, non-fluorescent transient tracers, non-fluorescent genetic markers, fluorescent protein, genetic marker delivery, genetic recombination, exogenous DNA barcodes, CRISPR-Cas9 mediated DNA barcodes, and base editor-mediated DNA barcodes. The second part of the review covers genetic mosaicism, genomic DNA alteration, TCR/BCR, DNA methylation, and mitochondrial DNA mutation. In the final section, we will address the principal challenges and prospective avenues of enquiry in the field of cell lineage tracing, with a particular focus on the sequencing techniques and mathematical models pertinent to single-cell genetic lineage tracing, and the value of pursuing a more comprehensive investigation at both the spatial and temporal levels in the study of cell lineage tracing.},
}

*Cell Lineage/genetics
Humans
Animals
*Cell Tracking/methods
CRISPR-Cas Systems
Cell Differentiation

@article {pmid39828625,
year = {2025},
author = {Yang, J and Xu, X and Yang, L and Tian, Y and Wang, J and Han, D},
title = {Dynamic Genomic Imaging and Tracking in Living Cells by a DNA Origami-Based CRISPR‒dCas9 System.},
journal = {Small methods},
volume = {9},
number = {6},
pages = {e2401559},
doi = {10.1002/smtd.202401559},
pmid = {39828625},
issn = {2366-9608},
support = {2021YFA0909400//National Key Research and Development Program of China/ ; 2020YFA0211100//National Key Research and Development Program of China/ ; 22225402//National Natural Science Foundation of China/ ; 22204099//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *DNA/chemistry/genetics ; *Genomics/methods ; Nanostructures/chemistry ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Green Fluorescent Proteins/genetics/metabolism ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)-associated system has displayed promise in visualizing the dynamics of target loci in living cells, which is important for studying genome regulation. However, developing a cell-friendly and rapid transfection method for achieving dynamic and long-term genomic imaging in living cells with high specificity and accuracy is still challenging. Herein, a robust and versatile method is presented that employs a barrel-shaped DNA nanostructure (TUBE) modified with aptamers for loading, protecting, and delivering CRISPR-Cas9 to visualize specific genomic loci in living cells. This approach enables dynamic tracking of target genomic regions (Chr3q29, a repetitive region of chromosome 3) throughout the mitotic process and captures variations in their spatial distribution and quantity accurately. Distinct dynamic behaviors between the Chr3q29 and telomeres are observed, which are linked to their unique chromosomal positions and levels of mobility. High-resolution multicolor labeling of the target genes is achieved, with a high degree of colocalization between the enhanced green fluorescent protein and cyanine-5 channels, facilitating precise imaging of target loci. This method not only supports dynamic genomic imaging but also enables multiplexed tracking, providing a powerful visualization tool for studying cellular processes and genetic interactions in real time within living cells.},
}

*CRISPR-Cas Systems/genetics
Humans
*DNA/chemistry/genetics
*Genomics/methods
Nanostructures/chemistry
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Green Fluorescent Proteins/genetics/metabolism

@article {pmid39718905,
year = {2025},
author = {Yao, ZY and Yu, MJ and Li, QQ and Gong, JS and Zhang, P and Jiang, JY and Su, C and Xu, G and Jia, BY and Xu, ZH and Shi, JS},
title = {Unlocking Green Biomanufacturing Potential: Superior Heterologous Gene Expression with a T7 Integration Overexpression System in Bacillus subtilis.},
journal = {ACS synthetic biology},
volume = {14},
number = {6},
pages = {1977-1987},
doi = {10.1021/acssynbio.4c00694},
pmid = {39718905},
issn = {2161-5063},
mesh = {*Bacillus subtilis/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *DNA-Directed RNA Polymerases/genetics/metabolism ; *Viral Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Gene Expression ; },
abstract = {Industrial biotechnology employs cells for producing valuable products and serving as biocatalysts sustainably, addressing resource, energy, and environmental issues. Bacillus subtilis is a preferred host for creating microbial chassis cells and producing industrial enzymes and functional nutritional products. In this study, a dual-module T7 integration expression system in B. subtilis was established. The first module, driven by the T7 RNA polymerase, was integrated into the genome via the CRISPR/Cas9 system. Another module responsible for expression control was systematically integrated into 28 discrete chromosomal loci and the impact of different genomic positions on gene expression was explored, resulting in a high-intensity integrated expression system. Furthermore, by modifying the LacI repressor factor for biological regulation, we achieved a strong expression intensity without the inducer addition. This system was successfully used to express phospholipase D and hyaluronic acid lyase, resulting in extracellular enzyme activities of 339.12 U/mL and 2.60 × 10[4] U/mL, respectively. Additionally, by exclusively targeting the HA gene cluster for expression, a production yield of 6.86 g/L was achieved on a 5 L fermentation scale. The system eliminates the use of antibiotics and inducers, offering a controllable, efficient, and promising gene expression regulation tool in B. subtilis, enhancing its potential for biomanufacturing applications.},
}

*Bacillus subtilis/genetics/metabolism
CRISPR-Cas Systems/genetics
*DNA-Directed RNA Polymerases/genetics/metabolism
*Viral Proteins/genetics/metabolism
Gene Expression Regulation, Bacterial
Gene Expression

@article {pmid39236976,
year = {2025},
author = {Hou, ZH and Gao, Y and Zheng, JC and Zhao, MJ and Liu, Y and Cui, XY and Li, ZY and Wei, JT and Yu, TF and Zheng, L and Jiao, YC and Yang, SH and Hao, JM and Chen, J and Zhou, YB and Chen, M and Qiu, L and Ma, YZ and Xu, ZS},
title = {GmBSK1-GmGSK1-GmBES1.5 regulatory module controls heat tolerance in soybean.},
journal = {Journal of advanced research},
volume = {73},
number = {},
pages = {187-198},
doi = {10.1016/j.jare.2024.09.004},
pmid = {39236976},
issn = {2090-1224},
mesh = {*Glycine max/genetics/physiology/metabolism ; Plants, Genetically Modified/genetics ; Gene Expression Regulation, Plant ; *Thermotolerance/genetics ; CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; *Heat-Shock Response/genetics ; Reactive Oxygen Species/metabolism ; Signal Transduction/genetics ; Brassinosteroids/metabolism ; },
abstract = {INTRODUCTION: Heat stress poses a severe threat to the growth and production of soybean (Glycine max). Brassinosteroids (BRs) actively participate in plant responses to abiotic stresses, however, the role of BR signaling pathway genes in response to heat stress in soybean remains poorly understood.

OBJECTIVES: In this study, we investigate the regulatory mechanisms of GmBSK1 and GmBES1.5 in response to heat stress and the physiological characteristics and yield performance under heat stress conditions.

METHODS: Transgenic technology and CRISPR/Cas9 technology were used to generated GmBSK1-OE, GmBES1.5-OE and gmbsk1 transgenic soybean plants, and transcriptome analysis, LUC activity assay and EMSA assay were carried out to elucidate the potential molecular mechanism underlying GmBSK1-GmBES1.5-mediated heat stress tolerance in soybean.

RESULTS: CRISPR/Cas9-generated gmbsk1 knockout mutants exhibited increased sensitivity to heat stress due to a reduction in their ability to scavenge reactive oxygen species (ROS). The expression of GmBES1.5 was up-regulated in GmBSK1-OE plants under heat stress conditions, and it directly binds to the E-box motif present in the promoters of abiotic stress-related genes, thereby enhancing heat stress tolerance in soybean plants. Furthermore, we identified an interaction between GmGSK1 and GmBES1.5, while GmGSK1 inhibits the transcriptional activity of GmBES1.5. Interestingly, the interaction between GmBSK1 and GmGSK1 promotes the localization of GmGSK1 to the plasma membrane and releases the transcriptional activity of GmBES1.5.

CONCLUSION: Our findings suggest that both GmBSK1 and GmBES1.5 play crucial roles in conferring heat stress tolerance, highlighting a potential strategy for breeding heat-tolerant soybean crops involving the regulatory module consisting of GmBSK1-GmGSK1-GmBES1.5.},
}

*Glycine max/genetics/physiology/metabolism
Plants, Genetically Modified/genetics
Gene Expression Regulation, Plant
*Thermotolerance/genetics
CRISPR-Cas Systems
*Plant Proteins/genetics/metabolism
*Heat-Shock Response/genetics
Reactive Oxygen Species/metabolism
Signal Transduction/genetics
Brassinosteroids/metabolism

@article {pmid39097089,
year = {2025},
author = {Tan, S and Yuan, C and Zhu, Y and Chang, S and Li, Q and Ding, J and Gao, X and Tian, R and Han, Z and Hu, Z},
title = {Glutathione hybrid poly (beta-amino ester)-plasmid nanoparticles for enhancing gene delivery and biosafety.},
journal = {Journal of advanced research},
volume = {73},
number = {},
pages = {697-711},
doi = {10.1016/j.jare.2024.07.038},
pmid = {39097089},
issn = {2090-1224},
mesh = {*Glutathione/chemistry ; *Plasmids/chemistry/genetics ; Animals ; *Nanoparticles/chemistry ; Humans ; Mice ; Reactive Oxygen Species/metabolism ; *Gene Transfer Techniques ; *Polymers/chemistry ; Gene Editing/methods ; Genetic Therapy/methods ; Transfection/methods ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: CRISPR/Cas9 gene editing technology has significantly advanced gene therapy, with gene vectors being one of the key factors for its success. Poly (beta-amino ester) (PBAE), a distinguished non-viral cationic gene vector, is known to elevate intracellular reactive oxygen species (ROS) levels, which may cause cytotoxicity and, consequently, impact gene transfection efficacy (T.E.).

OBJECTIVES: To develop a simple but efficient strategy to improve the gene delivery ability and biosafety of PBAE both in vivo and in vitro.

METHODS: We used glutathione (GSH), a clinically utilized drug with capability to modulating intracellular ROS level, to prepare a hybrid system with PBAE-plasmid nanoparticles (NPs). This system was characterized by flow cytometry, RNA-seq, Polymerase Chain Reaction (PCR) and Sanger sequencing in vitro, and its safety and efficacy in vivo was evaluated by imaging, PCR, Sanger sequencing and histology analysis.

RESULTS: The particle size of GSH-PBAE-plasmid NPs were 168.31 nm with a ζ-potential of 15.21 mV. An enhancement in T.E. and gene editing efficiency, ranging from 10 % to 100 %, was observed compared to GSH-free PBAE-plasmid NPs in various cell lines. In vitro results proved that GSH-PBAE-plasmid NPs reduced intracellular ROS levels by 25 %-40 %, decreased the total number of upregulated/downregulated genes from 4,952 to 789, and significantly avoided the disturbance in gene expression related to cellular oxidative stress-response and cell growth regulation signaling pathway compared to PBAE-plasmid NPs. They also demonstrated lower impact on the cell cycle, slighter hemolysis, and higher cell viability after gene transfection. Furthermore, GSH hybrid PBAE-plasmid NPs exhibited superior safety and improved tumor suppression ability in an Epstein-Barr virus (EBV)-infected murine tumor model, via targeting cleavage the EBV related oncogene by delivering CRISPR/Cas9 gene editing system and down-regulating the expression levels. This simple but effective strategy is expected to promote clinical applications of non-viral vector gene delivery.},
}

*Glutathione/chemistry
*Plasmids/chemistry/genetics
Animals
*Nanoparticles/chemistry
Humans
Mice
Reactive Oxygen Species/metabolism
*Gene Transfer Techniques
*Polymers/chemistry
Gene Editing/methods
Genetic Therapy/methods
Transfection/methods
CRISPR-Cas Systems

@article {pmid38084666,
year = {2025},
author = {Ogawa, Y and Lu, Y and Kiyozumi, D and Chang, HY and Ikawa, M},
title = {CRISPR/Cas9-mediated genome editing reveals seven testis-enriched transmembrane glycoproteins dispensable for male fertility in mice.},
journal = {Andrology},
volume = {13},
number = {5},
pages = {1251-1260},
pmid = {38084666},
issn = {2047-2927},
support = {R01 HD088412/HD/NICHD NIH HHS/United States ; JP22K15103//Japan Society for the Promotion of Science/ ; JP21H02487//Japan Society for the Promotion of Science/ ; JP21H00231//Japan Society for the Promotion of Science/ ; JP21K19263//Japan Society for the Promotion of Science/ ; JP21H05033//Japan Society for the Promotion of Science/ ; JPMJPR2143//Japan Science and Technology Agency/ ; R01HD088412//Eunice Kennedy Shriver National Institute of Child Health and Human Development/ ; INV-001902/GATES/Gates Foundation/United States ; },
mesh = {Animals ; Male ; *Testis/metabolism ; *Fertility/genetics ; Mice ; *CRISPR-Cas Systems ; Mice, Knockout ; *Gene Editing ; *Membrane Glycoproteins/genetics/metabolism ; Female ; Sperm Motility/genetics ; Spermatozoa/metabolism ; Spermatogenesis/genetics ; },
abstract = {BACKGROUND: Mammalian fertilization is mediated by multiple sperm acrosomal proteins, many of which are testis-enriched transmembrane glycoproteins expressed during spermiogenesis (e.g., Izumo sperm-egg fusion 1, Sperm acrosome associated 6, and Transmembrane protein 95).

METHODS: We hypothesized that proteins with these features might have a role in sperm-egg interaction and thus carried out an in-silico screen based on multiple public databases. We generated knockout mouse lines lacking seven candidate proteins by the CRISPR/Cas9 system and conducted detailed analyses on the fecundity of the knockout males, as well as their testis appearance and weight, testis and epididymis histology, and sperm motility and morphology.

RESULTS: Through the in-silico screen, we identified 4932438H23Rik, A disintegrin and metalloproteinase domain-containing protein 29, SAYSvFN domain-containing protein 1, Sel-1 suppressor of lin-12-like 2 (C. elegans), Testis-expressed protein 2, Transmembrane and immunoglobulin domain-containing 3, and Zinc and ring finger 4. Phenotypic analyses unveiled that the knockout males showed normal testis gross appearance, normal testis and epididymis histology, and normal sperm morphology and motility. Fertility tests further indicated that the knockout male mice could sire pups with normal litter sizes when paired with wild-type females.

DISCUSSION AND CONCLUSION: These findings suggest that these seven proteins are individually dispensable for male reproduction and fertilization. Future studies are warranted to devise advanced in-silico screening approaches that permit effective identification of gamete fusion-required sperm proteins.},
}

Animals
Male
*Testis/metabolism
*Fertility/genetics
Mice
*CRISPR-Cas Systems
Mice, Knockout
*Gene Editing
*Membrane Glycoproteins/genetics/metabolism
Female
Sperm Motility/genetics
Spermatozoa/metabolism
Spermatogenesis/genetics

@article {pmid40545778,
year = {2025},
author = {Li, Y and Jin, L and Li, W and Wang, K and Su, H and Mao, H and Chen, W and Lan, C and Li, Q and Kaufmann, K and Yan, W},
title = {Modulation of an evolutionarily conserved epigenetic regulon controlling abscisic acid catabolism enhances drought tolerance in wheat.},
journal = {The New phytologist},
volume = {},
number = {},
pages = {},
doi = {10.1111/nph.70302},
pmid = {40545778},
issn = {1469-8137},
support = {32272127//National Natural Science Foundation of China/ ; },
abstract = {Drought stress significantly reduces crop yield by triggering abscisic acid (ABA) accumulation in plants. It involves the suppression of CYP707A genes, which encode enzymes that catalyze ABA. However, little is known about epigenetic control in the CYP707A gene-mediated drought stress response in wheat. In this study, we reported that TaCYP707A-6A/6B/6D but not TaCYP707A-5A/5B/5D participates in drought response in common wheat. Disruption of TaCYP707A-6B showed enhanced drought tolerance but also decreased fertility. Expression of TaCYP707A-6B is negatively associated with H3K27me3 level. An evolutionarily conserved CTCTGYTY motif cluster (binding site for a Jumonji H3K27me3 demethylase) was found in the intron of TaCYP707A-6B as well as the intron of CYP707A homologs in other plant species. Blocking the CTCTGYTY motif by dead Cas9 (dCas9) maintained a high level of H3K27me3 on the CYP707A gene, while decreasing its expression level leading to enhanced drought tolerance in both wheat and Arabidopsis. In particular, the mutant in which the intron bound by H3K27me3 demethylase was cut out without change of splicing pattern showed enhanced drought tolerance. Therefore, our study provides a novel approach to improve plant drought tolerance by manipulating an evolutionarily conserved cis-element bound by histone demethylases in the intron of CYP707A genes.},
}

@article {pmid40545632,
year = {2025},
author = {Swinnen, G and Lizé, E and Loera Sánchez, M and Stolz, S and Soyk, S},
title = {Application of a GRF-GIF chimera enhances plant regeneration for genome editing in tomato.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70212},
pmid = {40545632},
issn = {1467-7652},
support = {802008//European Research Council/International ; 310030_212218//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung/ ; },
abstract = {Genome editing has become a routine tool for functionally characterizing plant and animal genomes. However, stable genome editing in plants remains limited by the time- and labour-intensive process of generating transgenic plants, as well as by the efficient isolation of desired heritable edits. In this study, we evaluated the impact of the morphogenic regulator GRF-GIF on plant regeneration and genome editing outcomes in tomato. We demonstrate that expressing a tomato GRF-GIF chimera reliably accelerates the onset of shoot regeneration from callus tissue culture by approximately one month and nearly doubles the number of recovered transgenic plants. Consequently, the GRF-GIF chimera enables the recovery of a broader range of edited haplotypes and simplifies the isolation of mutants harbouring heritable edits, but without markedly interfering with plant growth and development. Based on these findings, we outline strategies that employ basic or advanced diagnostic pipelines for efficient isolation of single- and higher-order mutants in tomato. Our work represents a technical advantage for tomato transformation and genome editing, with potential applications across other Solanaceae species.},
}

@article {pmid40545185,
year = {2025},
author = {Mondal, AR and Misra, A},
title = {'Emerging cell-specific therapies in cardiovascular disease'.},
journal = {Vascular pharmacology},
volume = {},
number = {},
pages = {107516},
doi = {10.1016/j.vph.2025.107516},
pmid = {40545185},
issn = {1879-3649},
abstract = {Atherosclerosis is a leading cause of cardiovascular morbidity and mortality worldwide, driven by complex interactions among various plaque cell types, including endothelial cells, macrophages, and smooth muscle cells. Traditional therapies targeting systemic risk factors such as cholesterol and blood pressure fail to directly address the underlying mechanisms governing plaque formation and progression. Recent advances in cell-specific therapies offer new avenues for targeting the cellular and molecular processes driving atherosclerosis. This Review explores innovative strategies including nanoparticles, viral vectors and CRISPR-Cas9 technology, which have the potential to modulate gene expression and behaviour within plaques cells to alleviate disease. By focusing on the specific roles of key cell types in atherosclerosis, these emerging therapies promise to provide more precise, effective, and personalised treatment options without inducing off-target effects. Moreover, insights gained from successful applications of these technologies in oncology are considered for potential repurposing in atherosclerosis-related disease. As these cell-specific approaches advance through preclinical and clinical development, they may significantly enhance our ability to treat atherosclerosis at its cellular roots, offering new hope for reducing the burden of cardiovascular disease.},
}

@article {pmid40544537,
year = {2025},
author = {Sinha, S and Upadhyay, LSB},
title = {Understanding antimicrobial resistance (AMR) mechanisms and advancements in AMR diagnostics.},
journal = {Diagnostic microbiology and infectious disease},
volume = {113},
number = {2},
pages = {116949},
doi = {10.1016/j.diagmicrobio.2025.116949},
pmid = {40544537},
issn = {1879-0070},
abstract = {The overuse and abuse of antibiotics, which results in the evolution of resistant microorganisms, is the primary cause of the global health catastrophe known as antimicrobial resistance (AMR). The enzymatic breakdown of antibiotics, target site modification, efflux pump overexpression, and the formation of biofilm are some of the mechanisms responsible for acquiring antimicrobial resistance (AMR). These mechanisms enable bacteria to evade or neutralize the effects of antimicrobial agents, complicating treatment options and increasing mortality rates. The rapid dissemination of resistance genes via horizontal gene transfer further exacerbates the problem, necessitating urgent intervention. Advanced AMR diagnostics are transforming the fight against antimicrobial resistance. Biosensors enable rapid, point-of-care detection; Cluster regularly interspaced short palindromic repeat (CRISPR) technologies offer precise identification of resistance genes; and mass spectrometry provides fast, accurate profiling. Automated systems streamline workflows and boost throughput, while flow cytometry delivers real-time, single-cell analysis of phenotypic resistance. Together, these innovations accelerate detection and support targeted antimicrobial stewardship, essential for combating the global AMR threat. This review covers the mechanisms underlying antimicrobial resistance (AMR) and recent advancements in AMR diagnostic technologies.},
}

@article {pmid40544408,
year = {2025},
author = {Yu, D and Xie, Z and Zhang, Y and Xie, Z and Fan, Q and Luo, S and Xie, L and Li, M and Zeng, T and Zhang, M and Li, X and Wei, Y and Wu, A and Wan, L},
title = {A dual fluorescence channel RAA-based CRISPR-Cas12a/Cas13a system for highly sensitive detection of Gyrovirus galga1 and Gyrovirus homsa1.},
journal = {Virulence},
volume = {16},
number = {1},
pages = {2521012},
doi = {10.1080/21505594.2025.2521012},
pmid = {40544408},
issn = {2150-5608},
abstract = {Gyrovirus galga1 (GyG1) and Gyrovirus homsa1 (GyH1) are the second and third most common gyroviruses identified, respectively, after chicken anaemia virus. They were first reported in 2011 and are currently prevalent worldwide. However, limited research on these pathogens and a lack of prevention and control strategies have necessitated the establishment of a rapid diagnostic technique to address new challenges in infectious diseases. Recombinase acid amplification (RAA) combined with CRISPR - Cas12a or CRISPR - Cas13a technology has major advantages for highly sensitive and rapid diagnosis. Specific targets can activate CRISPR-Cas trans-cleavage activity, resulting in non-specific cleavage of single-stranded DNA by the CRISPR - Cas12a complex and RNA cleavage by the CRISPR - Cas13a complex. In this study, for the first time, we combined RAA-based CRISPR - Cas12a and CRISPR - Cas13a systems for simultaneous differential diagnosis of GyG1 and GyH1 infection. The results showed that dual fluorescence channel RAA-based CRISPR - Cas12a/Cas13a technology could detect GyG1 and GyH1 within one hour, with a minimum detection limit of 1.5 copies of the target DNA standard/µL and no cross-reactivity with other avian pathogens. In addition, this method could be used for clinical detection, with the results exhibiting high consistency with those obtained by qPCR. These findings demonstrate that our RAA-based CRISPR - Cas12a/Cas13a dual-channel detection system can detect two different subtypes of gyrovirus in a sample with good specificity and high sensitivity, improving the detection efficiency and providing a new technique for the study of viral infection dynamics.},
}

@article {pmid40544221,
year = {2025},
author = {Ajibode, ET and Bender, AR and Yehl, K},
title = {Profiling crRNA architectures for enhanced Cas12 biosensing.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {947},
pmid = {40544221},
issn = {2399-3642},
abstract = {CRISPR-Cas diagnostics are revolutionizing point-of-care molecular testing due to the programmability, simplicity, and sensitivity of Cas systems with trans-cleavage activity. CRISPR-Cas12 assays are promising for detecting single nucleotide polymorphisms (SNPs). However, reports vary widely describing Cas12 SNP sensitivity, and an underlying mechanism is lacking. We systematically varied crRNA length and valency to investigate the role of crRNA architectures on Cas12 biosensing in the context of speed-of-detection, sensitivity, and selectivity. Our results demonstrate that crRNAs complementary to 20 base pairs of the target DNA is optimal for rapid and sensitive detection, while a complementary length of 15 base pairs is ideal for robust SNP detection. Additionally, we uncovered a unique periodicity in SNP sensitivity based on nucleotide position and developed a structural model explaining what drives Cas12 SNP sensitivity. Lastly, we showed that bivalent CRISPR-Cas sensors have synergistic and enhanced activity that is distance dependent.},
}

@article {pmid40542569,
year = {2025},
author = {Han, R and Gao, X and Qi, Y and Lu, X and Wang, X and Tang, X},
title = {Spacer-Complementary Single-Stranded DNA Oligonucleotides Can Serve as Target-Specific Inhibitors in CRISPR/Cas9 Systems.},
journal = {Cell biochemistry and function},
volume = {43},
number = {6},
pages = {e70088},
doi = {10.1002/cbf.70088},
pmid = {40542569},
issn = {1099-0844},
support = {//This research was supported by Guangxi Natural Science Foundation Project (2025GXNSFAA069363, 2023GXNSFAA026260)./ ; },
abstract = {The continuous expression of the CRISPR/Cas system in organisms can lead to various potential issues. Some anti-CRISPR strategies have been developed to achieve precise control over CRISPR/Cas, yet these strategies are predominantly protein-based, with the most commonly used anti-CRISPR (Acr) proteins lacking sufficient target specificity. However, in this study, we designed a single-stranded DNA (ssDNA) inhibitor that was complementary to the spacer region on the guide RNA, operating at the nucleic acid level. We demonstrated that this method effectively inhibits the cleavage activity of Cas9-sgRNA ribonucleoprotein (RNP) in a target-specific manner through in vitro cleavage experiments. Furthermore, we explored the binding position and effective length of this inhibitory ssDNA, finding that its inhibitory effect was significantly reduced when the length of continuous complementarity with the 5' end of the spacer was less than 7nt. The truncated ssDNA also showed potential in reducing off-target effects. Moreover, we applied nucleic acid inhibitors to embryos via microinjection, and gene editing activity was significantly reduced, as evidenced by a decrease in the mosaicism rate of mouse embryos undergoing normal gene editing from (84.4 ± 4.4) % to 0%. Our study introduces a convenient and target-specific nucleic acid inhibitor capable of achieving precise regulation of gene editing.},
}

@article {pmid40540289,
year = {2025},
author = {Han, JH and Lee, SY and Park, HH},
title = {AcrVIA6 Is a Monomeric DNA-Binding Protein That Does Not Directly Inhibit Cas13a.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {39},
number = {12},
pages = {e70753},
pmid = {40540289},
issn = {1530-6860},
support = {RS-(2025-02316334)//National Research Foundation of Korea (NRF)/ ; },
abstract = {The CRISPR-Cas system is a crucial adaptive immune mechanism in prokaryotes, providing defense against invading genetic elements. Among various CRISPR-Cas systems, the type VI-A system, employing RNA-guided RNase Cas13a, has been extensively studied for its ability to target and degrade single-stranded RNA. Anti-CRISPR (Acr) proteins have evolved as natural inhibitors of these systems, with AcrVIA proteins specifically targeting the Cas13a enzyme. However, there is currently conflicting debate regarding the anti-CRISPR function of AcrVIA6. This study reveals that AcrVIA6 functions as a DNA-binding protein rather than a Cas13a inhibitor, as it does not block RNA cleavage. These findings challenge its role in CRISPR-Cas regulation.},
}

@article {pmid40540136,
year = {2025},
author = {Ponnurangan, V and Namachivayam, R and Pradeep, RKM and Jesudoss, D and Eswaran, K and Loganathan, A and Kumar, KK and Vaikuntavasan, P and Maduraimuthu, D and Shanmugam, V},
title = {Biotechnological breakthroughs in rice disease management: an overview from transgenics to CRISPR.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {616},
pmid = {40540136},
issn = {1573-4978},
support = {BT/PR40456/ AGIII/103/1248/2020//Department of Biotechnology, New Delhi, India/ ; },
abstract = {Rice production faces persistent challenges due to a plethora of infectious agents, which cause substantial economic losses and pose significant threats to food security. Effective management of rice diseases is hindered by the lack of resistant sources and the slow pace of conventional breeding, which often fails to keep up with the rapid evolution of pathogens. Although transgenic approaches have contributed to disease resistance, they are limited by regulatory hurdles and public concern over the use of genetically modified organisms. Emerging genome-editing technologies, particularly CRISPR/Cas systems, offer promising alternatives by enabling the precise modification of native genes involved in plant susceptibility. CRISPR/Cas9-mediated inactivation of negative defense regulators, such as SWEET and ERF family genes, conferred broad-spectrum disease resistance. This review discusses the use of CRISPR/Cas technologies in developing disease-resistant rice varieties, along with emerging approaches and databases relevant to genome editing in plant research. Furthermore, it also highlights recent developments in CRISPR-based diagnostics for pathogen detection and the regulatory frameworks for the commercialization of edited crops. Overall, it underscores the transformative potential of CRISPR technologies in developing elite, climate-resilient rice cultivars, thereby supporting sustainable disease management and global food security.},
}

@article {pmid40539514,
year = {2025},
author = {Li, Y and Li, J and Pei, X and Wei, J and Gan, J and Lin, J},
title = {Catalytic-state structure of Candidatus Hydrogenedentes Cas12b revealed by cryo-EM studies.},
journal = {Nucleic acids research},
volume = {53},
number = {12},
pages = {},
doi = {10.1093/nar/gkaf519},
pmid = {40539514},
issn = {1362-4962},
support = {2017YFA0504602//National Key Research and Development Program of China/ ; 32130063//National Key Research and Development Program of China/ ; 31770784//National Natural Science Foundation of China/ ; 32371252//National Natural Science Foundation of China/ ; 2021-01-07-00-07-E00074//Shanghai Municipal Education Commission/ ; SN-ZJU-SIAS009//Zhejiang University/ ; 31770784//National Natural Science Foundation of China/ ; 32371252//National Natural Science Foundation of China/ ; },
abstract = {The CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) systems are adaptive immune mechanisms that play critical roles in defending archaea and bacteria against invading entities. These systems can be divided into two classes, with class 2 comprising three types (II, V, and VI). Because of their ability to cleave double-stranded DNA, many class 2 CRISPR-Cas proteins have been harnessed as genome editing tools. Unlike the well-studied type II Cas9 proteins, the structural studies of the type V-B Cas12b proteins are limited, hindering their engineering and broader application. Here, we report four complex structures of ChCas12b, which reveal many unique structural features. The folding of the single guide RNA (sgRNA) of ChCas12b is distinct from that of AacCas12b and BthCas12b. Notably, many of these unique features are involved in ChCas12b-sgRNA interaction, suggesting that they are co-evolved. While ChCas12b shares a conserved two-cation-assisted catalytic mechanism with its homologs, it recognizes a longer guide:target heteroduplex, potentially offering higher fidelity in DNA editing. Altogether, our studies suggested that Cas12b family proteins exhibit significant diversity in their folding, sgRNA and target DNA binding. In the future, it is worth characterizing more representative proteins to identify CRISPR-Cas proteins with higher gene editing ability and fidelity.},
}

@article {pmid40539280,
year = {2025},
author = {Bak, RO and Holm, M and Møller, B and Mikkelsen, JG and Mogensen, TH},
title = {[CRISPR/Cas gene editing of haematopoietic stem cells for curing primary immunodeficiency].},
journal = {Ugeskrift for laeger},
volume = {187},
number = {22},
pages = {},
doi = {10.61409/V02250083},
pmid = {40539280},
issn = {1603-6824},
abstract = {Primary immunodeficiencies are rare monogenic inborn errors of immunity and can involve any combination of infection, autoimmunity, inflammation, and malignancy. While increased use of whole genome sequencing has vastly improved diagnosis, curative treatment options beyond haematopoietic stem cell transplantation are still lacking behind. In this review, we present and discuss the promising avenues of CRISPR/Cas gene editing of patient stem cells for curing these diseases through homology-directed repair, base- or prime editing and delivery by nanoparticles or viral derivatives. However, technological, regulatory, and economic challenges exist on the road to safe and broad implementation of this technology for personalized medicine in the clinic.},
}

@article {pmid40538711,
year = {2025},
author = {Lathakumari, RH and Vajravelu, LK and Gopinathan, A and Vimala, PB and Panneerselvam, V and Ravi, SSS and Thulukanam, J},
title = {The gut virome and human health: From diversity to personalized medicine.},
journal = {Engineering microbiology},
volume = {5},
number = {1},
pages = {100191},
pmid = {40538711},
issn = {2667-3703},
abstract = {The human gut virome plays a crucial role in the gut and overall health; its diversity and regulatory functions influence bacterial populations, metabolism, and immune responses. Bacteriophages (phages) and eukaryotic viruses within the gut microbiome contribute to these processes, and recent advancements in sequencing technologies and bioinformatics have greatly expanded our understanding of the gut virome. These advances have led to the development of phage-based therapeutics, diagnostics, and artificial intelligence-driven precision medicine. The emerging field of phageomics shows promise for delivering personalized phage therapies that combat antimicrobial resistance by specifically targeting pathogenic bacteria while preserving beneficial microbes. Moreover, CRISPR-Cas systems delivered via phages have shown success in selectively targeting antibiotic resistance genes and enhancing treatment effectiveness. Phage-based diagnostics are highly sensitive in detecting bacterial pathogens, offering significant benefits for human health and zoonotic disease surveillance. This synthesis of the current knowledge highlights the pivotal role of the gut virome in regulating microbial communities and its transformative potential in personalized medicine, emphasizing its importance in advancing therapeutic and diagnostic strategies for improving health outcomes.},
}

@article {pmid40536889,
year = {2025},
author = {Wall, LA and Wall, D},
title = {Acquired CRISPR spacers and rhamnose-glucose polysaccharide defects confer resistance to Streptococcus mutans phage ɸAPCM01.},
journal = {Microbiology (Reading, England)},
volume = {171},
number = {6},
pages = {},
pmid = {40536889},
issn = {1465-2080},
mesh = {*Streptococcus mutans/virology/genetics/metabolism ; *Rhamnose/metabolism/genetics ; Biofilms/growth & development ; *Streptococcus Phages/genetics/physiology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Glucose/metabolism ; Mutation ; *Polysaccharides/metabolism ; Bacterial Proteins/genetics/metabolism ; *Polysaccharides, Bacterial/genetics ; },
abstract = {Streptococcus mutans is commonly associated with the development of dental caries worldwide. Due to their specificity for S. mutans, phage represents a promising avenue for future targeted therapeutic strategies. In this study, we investigated how phage resistance develops in S. mutans. As a model phage, we used ɸAPCM01, which is known to infect a serotype e strain. We isolated and sequenced the genomes of 15 spontaneous resistant mutants and found that 10 had acquired novel clustered regularly interspaced short palindromic repeats (CRIPSR) spacers targeting the phage, with a total of 18 new spacers identified. Additionally, eight strains contained mutations in rhamnose-glucose polysaccharide biosynthetic genes, three of which also acquired spacers. Only the rgp mutants exhibited defects in phage adsorption, supporting the role of these cell surface glycans as the phage receptor. Mutations in rgpF and the newly identified gene rgpX led to severe cell division defects and impaired biofilm formation, the latter of which was also shared by an rgpD mutant. Thus, rgp mutations confer phage resistance but impose severe fitness costs, limiting pathogenic potential. Surprisingly, we found that ɸAPCM01 was capable of binding to and injecting its genome into UA159, a model serotype c strain. However, UA159 was resistant to infection due to an unknown post-entry defence mechanism. Consequently, ɸAPCM01 has the potential to infect both major serotypes associated with dental caries.},
}

*Streptococcus mutans/virology/genetics/metabolism
*Rhamnose/metabolism/genetics
Biofilms/growth & development
*Streptococcus Phages/genetics/physiology
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Glucose/metabolism
Mutation
*Polysaccharides/metabolism
Bacterial Proteins/genetics/metabolism
*Polysaccharides, Bacterial/genetics

@article {pmid40536816,
year = {2025},
author = {Liao, X and Liu, Q and Chuai, G},
title = {PrimeNet: rational design of Prime editing pegRNAs by deep learning.},
journal = {Briefings in bioinformatics},
volume = {26},
number = {3},
pages = {},
doi = {10.1093/bib/bbaf293},
pmid = {40536816},
issn = {1477-4054},
support = {T24250193//National Natural Science Foundation of China/ ; 32341008//National Natural Science Foundation of China/ ; 62002265//National Natural Science Foundation of China/ ; 2021YFF1201200//National Key Research and Development Program of China/ ; 2021YFF1200900//National Key Research and Development Program of China/ ; //Shanghai Pilot Program for Basic Research/ ; //Shanghai Science and Technology Innovation Action Plan-Key Specialization in Computational Biology/ ; //Shanghai Shuguang Scholars Project/ ; //Shanghai Excellent Academic Leader Project/ ; 2021SHZDZX0100//Shanghai Municipal Science and Technology Major Project/ ; //Fundamental Research Funds for the Central Universities/ ; 2025080107//Tongji University "Medicine + X" Cross Research Program/ ; },
mesh = {Humans ; *Gene Editing/methods ; *Deep Learning ; HEK293 Cells ; K562 Cells ; CRISPR-Cas Systems ; DNA Methylation ; Epigenesis, Genetic ; },
abstract = {The rapid development of gene editing technology has revolutionized life science research and biotechnology applications. Prime editing, a precise gene editing tool, has shown promise in various applications, including disease research and therapeutic interventions. However, its suboptimal editing efficiency for extensive fragments and lack of predictive models have hindered its widespread adoption. Existing models exhibit low prediction accuracy and limitations, such as neglecting epigenetic factors that impact gene editing effects. To address these challenges, we developed PrimeNet, a novel prediction model that integrates significant epigenetic factors, including chromatin accessibility and DNA methylation. By incorporating data from multiple cell lines and introducing multiscale convolution and attention mechanisms, PrimeNet enhances the accuracy of predictions and generalization performance. Our results show that PrimeNet achieves a Spearman correlation coefficient of 0.94 and 0.82 on two datasets originated from HEK293T and K562 cell lines, respectively, outperforming existing models. This novel model has the potential to guide experimental design, enhance the success rate of gene editing, and reduce unnecessary experimental costs, thereby advancing the application of gene editing technology in genetic disease treatment and related fields.},
}

Humans
*Gene Editing/methods
*Deep Learning
HEK293 Cells
K562 Cells
CRISPR-Cas Systems
DNA Methylation
Epigenesis, Genetic

@article {pmid40536049,
year = {2025},
author = {Ullah, M and Naeem, M and Andoh, V and Khan, MN and Han, J and Rizwan, M and Hussain, N and Saeed, M and Chen, Y and Chen, H},
title = {Lactic Acid Bacteria as Biofactories: Mechanistic Insights, Engineering Strategies, and Future Horizons for Heterologous Enzyme Expression.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c02311},
pmid = {40536049},
issn = {1520-5118},
abstract = {Lactic acid bacteria (LAB) constitute a genetically heterogeneous group that is uniquely capable of converting soluble carbohydrates into lactic acid. Such LAB, with a long history of safe consumption in fermented foods, are considered food-grade microorganisms and are highly sought after for a variety of biotechnological applications. Due to their unique properties, LAB can be genetically engineered to produce industrially significant enzymes. LAB act as an expression host for these enzymes by combining already existing engineering systems with techniques such as CRISPR-Cas. This review outlines the progress achieved to date on genome manipulation methods for LAB engineering and future perspectives of genetic tools. These strategies contribute greatly to fully unleashing the potential of LAB, and we further elaborate on how genome editing tools can enhance the capacity of heterologous expression in LAB.},
}