@article {pmid40770083,
year = {2025},
author = {Wang, S and Kou, Z and Jiang, Y and Tomberlin, JK and Zhang, W and Wang, S and Huang, Y},
title = {Truncated Higiant enhances the bioconversion ability of Hermetia illucens.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1164},
pmid = {40770083},
issn = {2399-3642},
mesh = {Animals ; Gene Editing ; CRISPR-Cas Systems ; *Diptera/genetics/growth & development/metabolism/physiology ; *Insect Proteins/genetics/metabolism ; },
abstract = {The black soldier fly (BSF), Hermetia illucens, has garnered attention for its proficiency in converting organic waste into valuable biomass. In the context of rapid population growth and urbanization, BSF with higher bioconversion efficiency is in urgent need. One promising approach to improve the efficiency is generating new lines by molecular breeding, which has succeeded in plants and various livestock. Here, we developed a BSF strain with an enhanced bioconversion efficiency using CRISPR/Cas9-mediated genome editing. By knocking out the gene giant (gt) in BSF, we obtained individuals with larger size and higher reproductive capacity. Notably, the mutant strain increased the bioconversion efficiency of food waste by 13.10%, and exhibited consistent performance across diverse organic waste substrates. In addition, our study elucidates multifaceted roles of gt in larval growth and developmental plasticity. Moreover, mutants showed reduced reproductive competitiveness against WT in mixed populations. In conclusion, our study provides a superior and safe insect chassis for biomanufacturing and underscores the potential of molecular breeding to enhance efficiency in BSF farming for sustainable organic waste recycling.},
}

Animals
Gene Editing
CRISPR-Cas Systems
*Diptera/genetics/growth & development/metabolism/physiology
*Insect Proteins/genetics/metabolism

@article {pmid40769560,
year = {2025},
author = {Yang, Y and Zhang, Z and Liu, J and Tang, L and Wei, Y and Nong, W and Yin, L and Li, S and Duan, P and Wang, Y and Rao, Y},
title = {[Identification of rice htd1 allelic mutant and its regulatory role in grain size].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {7},
pages = {2789-2802},
doi = {10.13345/j.cjb.250179},
pmid = {40769560},
issn = {1872-2075},
mesh = {*Oryza/genetics/growth & development ; *Mutation ; *Edible Grain/genetics/growth & development ; Alleles ; *Plant Proteins/genetics ; *Dioxygenases/genetics ; Lactones/metabolism ; Gene Expression Regulation, Plant ; Genes, Plant ; Gene Editing ; CRISPR-Cas Systems ; Phenotype ; },
abstract = {Rice is the world's largest food crop, and its yield and quality are directly related to food security and human health. Grain size, as one of the important factors determining the rice yield, has been widely concerned by breeders and researchers for a long time. To decipher the regulatory mechanism of rice grain size, we obtained a multi-tiller, dwarf, and small-grain mutant htd1 by ethyl methanesulfonate (EMS) mutation from the Japonica rice cultivar 'Zhonghua 11' ('ZH11'). Genetic analysis indicated that the phenotype of htd1 was controlled by a single recessive gene. Using the mutation site map (Mutmap) method, we identified the candidate gene OsHTD1, which encoded a carotenoid cleavage dioxygenase involved in the biosynthesis of strigolactone (SL). The SL content in htd1 was significantly lower than that in 'ZH11'. Cytological analysis showed that the grain size of the mutant decreased due to the reductions in the length and width of glume cells. The function of htd1 was further verified by the CRISPR/cas9 gene editing technology. The plants with the gene knockout exhibited similar grain size to the mutant. In addition, gene expression analysis showed that the expression levels of multiple grain size-related genes in the mutant changed significantly, suggesting that HTD1 may interact with other genes regulating grain size. This study provides a new theoretical basis for research on the regulatory mechanism of rice grain size and potential genetic resources for breeding the rice cultivars with high yields.},
}

*Oryza/genetics/growth & development
*Mutation
*Edible Grain/genetics/growth & development
Alleles
*Plant Proteins/genetics
*Dioxygenases/genetics
Lactones/metabolism
Gene Expression Regulation, Plant
Genes, Plant
Gene Editing
CRISPR-Cas Systems
Phenotype

@article {pmid40769554,
year = {2025},
author = {Lü, W and Yang, H and Xu, H and Zhang, Y},
title = {[Construction of a Sox17 activation vector based on the CRISPR/dCas9 system and its validation in sheep embryonic stem cells].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {7},
pages = {2707-2718},
doi = {10.13345/j.cjb.250136},
pmid = {40769554},
issn = {1872-2075},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Sheep ; *SOXF Transcription Factors/genetics ; *Embryonic Stem Cells/metabolism/cytology ; *Genetic Vectors/genetics ; Cell Differentiation/genetics ; Transfection ; Gene Editing/methods ; },
abstract = {The CRISPR/dCas9 system is a gene editing tool that has proven to be highly efficient and precise. By utilizing transcriptional activators, such as VP64, p65, and Rta, the system can effectively and stably activate target genes. Sox17, a transcription factor belonging to the SOX family, plays a crucial role in the differentiation of the germ layers and the determination of cell fates during the early stages of embryonic development. Sheep embryonic stem cells (sESCs) are characterized by their capacity for self-renewal and multidirectional differentiation, serving as a significant in vitro model for studying the mechanisms of cell differentiation during early embryonic development. However, the importing of exogenous genes into sESCs is challenging due to their unique growth characteristics. The objective of this study was to investigate the conditions necessary for successfully activating Sox17 in sESCs. To this end, we employed the CRISPR/dCas9 system along with liposome transfection, lentivirus invasion, and electroporation to activate Sox17 in sESCs. The expression of Sox17 was then determined by fluorescence quantitative PCR, on the basis of which the performance of different transfection methods was compared. The results indicated that the electroporation group had the best transfection effect and the highest Sox17 expression among the three transfection methods. The efficient and stable gene activation protocol will provide a reference for embryonic stem cell research in other species, especially livestock animals, and lay the foundation for the subsequent study of gene function and realization of precise cell fate regulation by regulating gene expression in sheep embryonic stem cells.},
}

Animals
*CRISPR-Cas Systems/genetics
Sheep
*SOXF Transcription Factors/genetics
*Embryonic Stem Cells/metabolism/cytology
*Genetic Vectors/genetics
Cell Differentiation/genetics
Transfection
Gene Editing/methods

@article {pmid40769177,
year = {2025},
author = {Liu, Y and Yu, L and Gan, Z and Feng, Y and Tong, J and Xiao, Y},
title = {Lanthanide Metal-Organic Framework-Integrated CRISPR-Cas Technology for Amplification-free Gene Mutation Assay.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c01949},
pmid = {40769177},
issn = {1520-6882},
abstract = {Nucleic acid amplification remains a major bottleneck in CRISPR-based molecular diagnostics, limiting assay speed, simplicity, and accuracy. Herein, we report a lanthanide metal-organic framework (Ln-MOF)-integrated CRISPR-Cas12a platform for amplification-free detection of gene mutations with high sensitivity and specificity. In this system, target recognition activates Cas12a trans-cleavage, degrading a single-stranded DNA linker and releasing alkaline phosphatase (ALP). The liberated ALP hydrolyzes p-nitrophenyl phosphate (pNPP) to generate phosphate ions, which interact with Eu-TPTC (TPTC: [1,1':4',1″] terphenyl-3,3″,5,5″-tetracarboxylic acid) MOF sensor, inducing a ratiometric fluorescence change by selectively quenching the Eu[3+] emission and enhancing the ligand emission. This mechanism enables quantitative, amplification-free detection of the oncogenic BRAF V600E mutation down to 0.1 pM, with excellent specificity and anti-interference ability. The assay demonstrates full agreement with conventional quantitative PCR when applied to clinical samples, accurately distinguishing mutant from wild-type genotypes. This strategy couples CRISPR-driven enzymatic signaling with Ln-MOF fluorescence modulation, offering an accurate and rapid readout. By eliminating amplification and streamlining workflow, this method holds significant potential for gene mutation analysis in clinical diagnostics.},
}

@article {pmid40768591,
year = {2025},
author = {Stundl, J and Desingu Rajan, AR and Urrutia, HA and Leyhr, J and Stundlova, J and Solovieva, T and Haitina, T and Sanchez, S and Musilova, Z and Martik, ML and Bronner, ME},
title = {Acquisition of neural crest promoted thyroid evolution from chordate endostyle.},
journal = {Science advances},
volume = {11},
number = {32},
pages = {eadv2657},
doi = {10.1126/sciadv.adv2657},
pmid = {40768591},
issn = {2375-2548},
mesh = {Animals ; *Neural Crest/embryology/metabolism ; *Thyroid Gland/embryology/metabolism ; *Biological Evolution ; *Chordata/embryology ; Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; },
abstract = {The endostyle is an endodermal organ unique to nonvertebrate chordates except for lamprey larvae, where it serves as forerunner to the adult thyroid. Here, we examine whether the acquisition of neural crest in the vertebrate lineage played a role in the elaboration of the endostyle. CM-DiI lineage tracing reveals a neural crest contribution to the endostyle, and CRISPR-Cas9 mutagenesis of key neural crest genes causes endostyle defects including formation of a single rather than bilobed structure. RNA sequencing reveals gene profiles characteristic of embryonic neural crest cells and Schwann cell precursors in the developing endostyle. Contrasting with the prevailing view that the endostyle is an endoderm-derived organ, we propose that the acquisition of the neural crest played a critical step in promoting thyroid evolution from chordate endostyle.},
}

Animals
*Neural Crest/embryology/metabolism
*Thyroid Gland/embryology/metabolism
*Biological Evolution
*Chordata/embryology
Gene Expression Regulation, Developmental
CRISPR-Cas Systems

@article {pmid40768573,
year = {2025},
author = {Bell, AG and Dunkley, ORS and Modi, NH and Huang, Y and Tseng, S and Reiss, R and Daivaa, N and Davis, JL and Vargas, DA and Banada, P and Xie, YL and Myhrvold, C},
title = {A streamlined CRISPR-based test for tuberculosis detection directly from sputum.},
journal = {Science advances},
volume = {11},
number = {32},
pages = {eadx2067},
doi = {10.1126/sciadv.adx2067},
pmid = {40768573},
issn = {2375-2548},
mesh = {*Sputum/microbiology ; Humans ; *Mycobacterium tuberculosis/genetics/isolation & purification ; *Tuberculosis/diagnosis/microbiology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Nucleic Acid Amplification Techniques ; },
abstract = {Mycobacterium tuberculosis (Mtb) is a major threat to global health, and there is an urgent need for affordable, simple tuberculosis (TB) diagnosis in underresourced areas. Here, we combine recombinase polymerase amplification with Cas13a and Cas12a detection to create two parallelized one-pot assays that detect two conserved elements of Mtb (IS6110 and IS1081) and a human DNA internal control. These assays are compatible with lateral flow and can be readily lyophilized. Our final assay showed a limit of detection of 69.0 CFU per milliliter for Mtb H37Rv and 80.5 CFU per milliliter for Mycobacterium bovis BCG in spiked sputum, with no cross-reactivity to diverse bacterial or fungal isolates. Clinical tests on 13 blinded sputum samples revealed 100% (six of six) sensitivity and 100% (seven of seven) specificity compared to culture. SHINE-TB streamlines TB diagnosis from sample to answer by combining amplification and detection while being compatible with lateral flow and lyophilization.},
}

*Sputum/microbiology
Humans
*Mycobacterium tuberculosis/genetics/isolation & purification
*Tuberculosis/diagnosis/microbiology
*CRISPR-Cas Systems
Sensitivity and Specificity
*Clustered Regularly Interspaced Short Palindromic Repeats
Nucleic Acid Amplification Techniques

@article {pmid40768535,
year = {2025},
author = {de Roest, RH and Buijze, M and Veth, M and de Lint, K and Pai, G and Rooimans, MA and Wolthuis, RMF and Brink, A and Poell, JB and Brakenhoff, RH},
title = {An unexpected role of CLASP1 in radiation response and S-phase regulation of head and neck cancer cells.},
journal = {PloS one},
volume = {20},
number = {8},
pages = {e0329731},
doi = {10.1371/journal.pone.0329731},
pmid = {40768535},
issn = {1932-6203},
mesh = {Humans ; *Head and Neck Neoplasms/genetics/radiotherapy/pathology/metabolism ; Cell Line, Tumor ; *Microtubule-Associated Proteins/genetics/metabolism ; Squamous Cell Carcinoma of Head and Neck/genetics ; *S Phase/radiation effects/genetics ; *Radiation Tolerance/genetics ; CRISPR-Cas Systems ; DNA Damage/radiation effects ; Gene Expression Regulation, Neoplastic/radiation effects ; Gene Knockout Techniques ; },
abstract = {Radiotherapy is a mainstay of treatment for head and neck squamous cell carcinoma (HNSCC), either definitive or adjuvant to surgery. Biological factors known to affect radiation response are hypoxia and DNA repair capacity, but several lines of evidence indicate that other genes and pathways in the tumor cells might be involved that have not been elucidated. Here, we report the results of a genome-wide CRISPR-Cas9 functional genomics screen in HNSCC cells to identify radiosensitizing genes. Remarkably, microtubule organizing genes were identified with CLASP1 as most unexpected radiosensitizing hit. Clonogenic assay confirmed the radiosensitizing effect of CLASP1 knockout. Functional analysis showed that CLASP1 knockout has major impact during S-phase, and resulted in mitotic cells with broken chromosomes and cell death. CLASP1 and possibly the microtubule machinery in broader sense seem involved in protection of HNSCC cells against radiation-induced DNA damage. This newly identified mechanism provides an outlook for novel treatment approaches in HNSCC.},
}

Humans
*Head and Neck Neoplasms/genetics/radiotherapy/pathology/metabolism
Cell Line, Tumor
*Microtubule-Associated Proteins/genetics/metabolism
Squamous Cell Carcinoma of Head and Neck/genetics
*S Phase/radiation effects/genetics
*Radiation Tolerance/genetics
CRISPR-Cas Systems
DNA Damage/radiation effects
Gene Expression Regulation, Neoplastic/radiation effects
Gene Knockout Techniques

@article {pmid40768438,
year = {2025},
author = {Wang, Y and Zhao, W and He, Y and Li, J and Mao, R},
title = {Advanced Heterogeneous Network-Based Graph Neural Network Framework for Predicting Anti-CRISPR Protein Sequences.},
journal = {IEEE journal of biomedical and health informatics},
volume = {29},
number = {8},
pages = {6092-6104},
doi = {10.1109/JBHI.2025.3548463},
pmid = {40768438},
issn = {2168-2208},
mesh = {*Neural Networks, Computer ; *Computational Biology/methods ; *CRISPR-Cas Systems/genetics ; *Sequence Analysis, Protein/methods ; Algorithms ; Databases, Protein ; Bacteriophages/genetics ; Graph Neural Networks ; },
abstract = {Anti-CRISPR proteins play a crucial role in bacterial-phage interactions by inhibiting the CRISPR/Cas system and thus enhancing phage survival. Accurately predicting these proteins is essential for understanding phage-host immune interactions and progressing CRISPR/ Cas-based technologies. Current approaches primarily analyze proteins individually, which may overlook the intrinsic similarities and potential connections among protein sequences. This study introduces PACRGNN, a graph neural network framework that creates a heterogeneous protein network by integrating sequence and structural similarities, wherein nodes represent proteins and edges signify their relationships. By combining Graph Attention (GAT) and Graph Sample and Aggregation (GraphSAGE) layers, PACRGNN captures both local and global topological dependencies, while incorporating six protein feature categories to enrich node representations. PACRGNN achieves an accuracy of 0.9577, an F1-Score of 0.9572, and a PRAUC of 0.9876 on the validation set. The model demonstrated superior performance to existing methods on the independent test set derived from NCBI database (Jan.-Oct. 2024).},
}

*Neural Networks, Computer
*Computational Biology/methods
*CRISPR-Cas Systems/genetics
*Sequence Analysis, Protein/methods
Algorithms
Databases, Protein
Bacteriophages/genetics
Graph Neural Networks

@article {pmid40766843,
year = {2025},
author = {Zhang, Y and Chen, J and Feng, H and Yang, L and Hu, L and Gao, Y and Wang, Z and Feng, F and Zhao, J and Zhang, P and Zhang, R},
title = {Rapid generation of HCoV-229E and HCoV-OC43 reporter viruses and replicons for antiviral research.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1614369},
pmid = {40766843},
issn = {2235-2988},
mesh = {*Antiviral Agents/pharmacology ; *Genes, Reporter ; *Replicon/genetics ; Humans ; *Coronavirus 229E, Human/genetics/drug effects ; *Coronavirus OC43, Human/genetics/drug effects ; *Reverse Genetics/methods ; CRISPR-Cas Systems ; Luciferases/genetics ; Recombination, Genetic ; Cloning, Molecular ; Saccharomyces cerevisiae/genetics ; Animals ; Adenosine Monophosphate/analogs & derivatives ; Alanine/analogs & derivatives ; },
abstract = {INTRODUCTION: The large size of coronavirus genome, along with the instability of certain genomic sequences, makes the construction of reverse genetics for coronaviruses particularly challenging. The rapid development and application of reverse genetics systems for coronaviruses require further exploration.

METHODS: Using transformation-associated recombination (TAR) cloning in yeast and the in vitro CRISPR-Cas9 system, reverse genetics systems of two mild coronaviruses HCoV-OC43 and HCoV-229E were rapidly established. Antiviral assays, high-content imaging, and NanoLuc luciferase assays were used to characterize reporter viruses and replicon systems.

RESULTS: We rapidly assembled infectious clones for two mild coronaviruses, HCoV-OC43 and HCoV-229E, using transformation-associated recombination (TAR) cloning in yeast. The infected clones could stably express the mGreenLantern reporter gene. We further generated T7 promoter-driven RNA replicon of HCoV-229E and CMV promoter-driven DNA replicon of HCoV-OC43, with the readout of NanoLuc luciferase activity. The effectiveness of these tools for antiviral study was evaluated using the broad-spectrum RNA-dependent RNA polymerase inhibitor remdesivir, exhibiting high sensitivity, efficiency, and convenience.

DISCUSSION: The application of yeast-based TAR cloning significantly facilitates the rapid assembly of large viral genome, and the establishment of HCoV-OC43 and HCoV-229E reverse genetics systems provides valuable platforms for studying the biology and developing antivirals against coronaviruses.},
}

*Antiviral Agents/pharmacology
*Genes, Reporter
*Replicon/genetics
Humans
*Coronavirus 229E, Human/genetics/drug effects
*Coronavirus OC43, Human/genetics/drug effects
*Reverse Genetics/methods
CRISPR-Cas Systems
Luciferases/genetics
Recombination, Genetic
Cloning, Molecular
Saccharomyces cerevisiae/genetics
Animals
Adenosine Monophosphate/analogs & derivatives
Alanine/analogs & derivatives

@article {pmid40764301,
year = {2025},
author = {Aussel, C and Cathomen, T and Fuster-García, C},
title = {The hidden risks of CRISPR/Cas: structural variations and genome integrity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7208},
pmid = {40764301},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; DNA Repair/genetics ; Translocation, Genetic ; Animals ; },
abstract = {CRISPR/Cas technology has revolutionized genome engineering, unlocking unprecedented therapeutic potential. However, beyond well-documented concerns of off-target (OT) mutagenesis, recent studies reveal a more pressing challenge: large structural variations (SVs), including chromosomal translocations and megabase-scale deletions, particularly in cells treated with DNA-PKcs inhibitors. These undervalued genomic alterations raise substantial safety concerns for clinical translation. As more CRISPR-based therapies progress toward the clinic, understanding and mitigating these risks is paramount. Here, we review emerging evidence on on-target aberrations and chromosomal translocations, identify key gaps in our understanding of the DNA repair pathways underlying these adverse effects, and discuss strategies to improve the safety of genome editing.},
}

*CRISPR-Cas Systems/genetics
Humans
*Gene Editing/methods
DNA Repair/genetics
Translocation, Genetic
Animals

@article {pmid40716589,
year = {2025},
author = {Du, H and Huang, X and Ren, R and Sun, Y and Wang, Y},
title = {TSPEAR S475TfsX79 mutation does not affect auditory function, tooth morphology or hair development in mice.},
journal = {Gene},
volume = {965},
number = {},
pages = {149687},
doi = {10.1016/j.gene.2025.149687},
pmid = {40716589},
issn = {1879-0038},
mesh = {Animals ; Mice ; *Tooth/growth & development/metabolism ; Mutation ; Wnt Signaling Pathway/genetics ; Mice, Knockout ; *Hair/growth & development/metabolism ; CRISPR-Cas Systems ; *Hearing/genetics ; },
abstract = {TSPEAR is a member of the EAR (epilepsy-associated repeat) protein family with poorly characterized function. Several lines of evidence suggest that mutations in the human TSPEAR gene are associated with hearing loss or ectodermal dysplasia. Although tooth abnormalities and a reduced capacity for caudal fin regeneration were observed in the Tspeara[-/-];Tspearb[-/-] knockout zebrafish model, there have been no reports of the Tspear knockout mouse model to date, which hampers further investigation of its physiological role. Here, we inactivated the Tspear gene in mice using CRISPR/Cas9-mediated genome editing. Intriguingly, stereociliary morphology and auditory function remain unaffected in TSPEAR S475TfsX79 mutant mice. Similarly, tooth morphology and hair development are unaltered in these mutants. Nevertheless, the S475TfsX79 mutation appears to perturb both Notch and Wnt signaling pathways. Specifically, Notch1 and several downstream target genes are downregulated, whereas Heyl expression is upregulated in the skin. Additionally, Wnt4 expression is elevated in both the skin and inner ear. In conclusion, our data demonstrate that TSPEAR S475TfsX79 mutation does not compromise auditory function, tooth morphology, or hair development in mice, but TSPEAR may modulate both Notch and Wnt signaling pathways in the mouse.},
}

Animals
Mice
*Tooth/growth & development/metabolism
Mutation
Wnt Signaling Pathway/genetics
Mice, Knockout
*Hair/growth & development/metabolism
CRISPR-Cas Systems
*Hearing/genetics

@article {pmid40673518,
year = {2025},
author = {Wan, W and Cui, C and Zhou, Y and Wang, J and Zhao, X and Cui, X and Sun, J and Yu, P and Feng, J and Wang, T and Wang, L and Xu, J},
title = {Targeting Mettl14 Using an RNA-Targeting Clustered Regularly Interspaced Short Palindromic Repeat-High-Fidelity Cas13x System Attenuates Doxorubicin-Induced Cardiotoxicity.},
journal = {Journal of the American Heart Association},
volume = {14},
number = {15},
pages = {e040700},
doi = {10.1161/JAHA.124.040700},
pmid = {40673518},
issn = {2047-9980},
mesh = {Animals ; *Doxorubicin/toxicity/adverse effects ; *Methyltransferases/genetics/metabolism ; Male ; Cardiotoxicity ; *CRISPR-Cas Systems ; Mice ; Disease Models, Animal ; Mice, Inbred C57BL ; *Cardiomegaly/chemically induced/genetics/prevention & control ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Physical Conditioning, Animal ; Myocytes, Cardiac/pathology/drug effects ; },
abstract = {BACKGROUND: Doxorubicin is an effective chemotherapy drug used to treat various types of cancer. However, doxorubicin treatment is associated with cardiotoxicity, which limits its clinical use. Exercise can benefit both cancer and cardiovascular disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas13 (CRISPR-associated protein 13) platforms have emerged as effective technologies for targeting the expression of RNA in transcript levels. To develop exercise mimetics that can mimic the beneficial effects of exercise training to attenuate doxorubicin-induced cardiotoxicity, we are using the CRISPR-hf (high-fidelity)Cas13x system.

METHODS: Adult male mice were swim-trained twice a day for 4 weeks to induce exercise-induced physiological cardiac hypertrophy. Adeno-associated virus 9-mediated METTL14 (methyltransferase-like 14) overexpression under the cardiac-specific ctnt promoter was used to overexpression METTL14 in vivo. RNA N[6]-methyladenosine inhibitor STM2457 was used to modulate global total RNA m[6]A levels in vivo. CRISPR-cr3-4/hfCas13x system was generated by hfCas13x guided crRNA3 and crRNA4 targeting the Mettl14 expressed under ctnt promoter and packaged in an adeno-associated virus 9.

RESULTS: Swimming exercise alleviated doxorubicin-induced cardiotoxicity. METTL14 was increased in doxorubicin-treated hearts but decreased in exercised hearts. METTL14 overexpression inhibited exercise-induced physiological cardiac hypertrophy. Conversely, STM2457 treatment reversed the suppressive effects of METTL14 overexpression on the physiological cardiac hypertrophy induced by exercise. Treatment with CRISPR-cr3-4/hfCas13x effectively inhibiting the expression of METTL14 in the heart, alleviating doxorubicin treatment-induced cardiac dysfunction and cardiac fibrosis.

CONCLUSIONS: Our results suggest that the CRISPR-hfCas13x system has the potential for generating exercise mimetics. Mimicking exercise by RNA-targeting Mettl14 suppression could be a therapeutic strategy for doxorubicin-induced cardiotoxicity.},
}

Animals
*Doxorubicin/toxicity/adverse effects
*Methyltransferases/genetics/metabolism
Male
Cardiotoxicity
*CRISPR-Cas Systems
Mice
Disease Models, Animal
Mice, Inbred C57BL
*Cardiomegaly/chemically induced/genetics/prevention & control
*Clustered Regularly Interspaced Short Palindromic Repeats
Physical Conditioning, Animal
Myocytes, Cardiac/pathology/drug effects

@article {pmid40631538,
year = {2025},
author = {Jahani, R and Munusamy, S and Zheng, H and Kong, J and Chen, J and Zhou, S and Guan, X},
title = {Ultrasensitive Detection of Attomolar Neurofilament Light Chain Using Gold Nanoparticle-Assisted CRISPR-Cas12a Enhanced Fluorescent Assay.},
journal = {ACS chemical neuroscience},
volume = {16},
number = {15},
pages = {2844-2853},
doi = {10.1021/acschemneuro.5c00137},
pmid = {40631538},
issn = {1948-7193},
mesh = {*Gold/chemistry ; *Neurofilament Proteins/analysis/blood ; *Metal Nanoparticles/chemistry ; Humans ; *CRISPR-Cas Systems ; Biosensing Techniques/methods ; Limit of Detection ; },
abstract = {Neurofilament light chain (NfL) is a nonspecific biomarker that can indicate neuronal damage, and elevated NfL levels have been reported in several neurological disorders and traumatic brain injury and are correlated with disease severity and progression. However, quantitative detection of NfL in bodily fluids, especially in blood, is challenging due to its ultralow levels and potential interference from matrix components. Herein, we report a DNA-assisted CRISPR-Cas12a-enhanced fluorescent assay for ultrasensitive and specific detection of NfL. By utilizing antibody-functionalized magnetic beads for capturing the target NfL, dual-functionalized gold nanoparticles for DNA introduction and controlled release as well as signal amplification, and the CRISPR-Cas12a system for further enhancement in the sensor sensitivity, our method was able to detect NfL with a limit of detection reaching as low as 0.012 pg/mL. Furthermore, the high selectivity of the sensor was verified by examining its response to a number of nontarget proteins such as C-reactive protein, interleukin-6, interleukin-2β, bovine serum albumin, and human serum albumin. To validate its clinical utility, simulated serum samples were analyzed, with the percent recoveries determined by the use of our sensor ranging from 85% to 95%. Given the high sensitivity and selectivity as well its ability to accurately detect trace amounts of NfL in human biospecimens, the CRISPR-based florescence sensor constructed in this work may find useful application in neurological disease diagnosis.},
}

*Gold/chemistry
*Neurofilament Proteins/analysis/blood
*Metal Nanoparticles/chemistry
Humans
*CRISPR-Cas Systems
Biosensing Techniques/methods
Limit of Detection

@article {pmid40550305,
year = {2025},
author = {León, E and Maya-Hoyos, M and Ramírez-Hernández, MH and Téllez, J and Contreras R, LE},
title = {Upregulation of nicotinamide/nicotinate mononucleotide adenylyl transferase increases resistance to oxidative stress and antimony in promastigotes of Leishmania braziliensis.},
journal = {Experimental parasitology},
volume = {275},
number = {},
pages = {108979},
doi = {10.1016/j.exppara.2025.108979},
pmid = {40550305},
issn = {1090-2449},
mesh = {*Oxidative Stress/drug effects ; Up-Regulation ; *Nicotinamide-Nucleotide Adenylyltransferase/genetics/metabolism ; *Leishmania braziliensis/drug effects/enzymology/genetics ; *Antimony/pharmacology ; Hydrogen Peroxide/pharmacology ; NAD/biosynthesis/metabolism ; CRISPR-Cas Systems ; Gene Editing ; *Antiprotozoal Agents/pharmacology ; Animals ; NADP/metabolism ; RNA, Messenger/metabolism ; Drug Resistance ; },
abstract = {Leishmaniasis is a prevalent parasitic neglected disease caused by protozoans of the genus Leishmania. Currently, no vaccines are available for humans, and existing treatments are ineffective because of their toxicity and the emergence of drug-resistant strains. Consequently, it is crucial to identify new potential therapeutic targets, thereby facilitating the development of effective therapies. This study indicates a correlation between the upregulated biosynthesis of nicotinamide adenine dinucleotide (NAD) and the resistance to hydrogen peroxide (H2O2) and trivalent antimony exposure (Sb[3+]) in L. braziliensis promastigotes. CRISPR/Cas9 gene-editing of the nicotinamide/nicotinate mononucleotide adenylyl transferase in L. braziliensis (Lbnmnat), the key gene in the NAD biosynthetic pathway, allowed upregulation at both the mRNA and protein levels, as well as elevated NAD and NADP contents in the Lbnmnat-edited parasites. A single guide RNA template, directed against the 5' end of the Lbnmnat and a donor DNA consisting of the mCherry reporter gene preceded by the 5' UTR from the Crithidia fasciculata phosphoglycerate kinase (CfPGKB) were electroporated into L. braziliensis promastigotes that constitutively express the Streptococcus pyogenes Cas9 and T7 RNA polymerase. PCR analysis and DNA sequencing demonstrated the successful editing of the Lbnmnat gene. Importantly, dose-dependent oxidative stress susceptibility assays to H2O2 and Sb[3+] revealed higher IC50 values in the Lbnmnat-edited parasites. These findings denote a correlation between the upregulated biosynthesis of NAD in L. braziliensis and its enhanced resistance to oxidative stress, indicating that the Lbnmnat gene may play a significant role in the survival and resistance mechanisms of the parasite against antimony compounds.},
}

*Oxidative Stress/drug effects
Up-Regulation
*Nicotinamide-Nucleotide Adenylyltransferase/genetics/metabolism
*Leishmania braziliensis/drug effects/enzymology/genetics
*Antimony/pharmacology
Hydrogen Peroxide/pharmacology
NAD/biosynthesis/metabolism
CRISPR-Cas Systems
Gene Editing
*Antiprotozoal Agents/pharmacology
Animals
NADP/metabolism
RNA, Messenger/metabolism
Drug Resistance

@article {pmid40359132,
year = {2025},
author = {Park, SY and Feng, Z and Choi, SH and Zhang, X and Tang, Y and Gasser, GN and Richart, D and Yuan, F and Qiu, J and Engelhardt, JF and Yan, Z},
title = {Recombinant Adeno-Associated Virus Vector Mediated Gene Editing in Proliferating and Polarized Cultures of Human Airway Epithelial Cells.},
journal = {Human gene therapy},
volume = {36},
number = {15-16},
pages = {1067-1082},
doi = {10.1089/hum.2024.260},
pmid = {40359132},
issn = {1557-7422},
mesh = {Humans ; *Gene Editing/methods ; *Dependovirus/genetics ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; *Genetic Vectors/genetics ; *Epithelial Cells/metabolism/cytology ; *Cystic Fibrosis/genetics/therapy/pathology ; Cell Proliferation ; CRISPR-Cas Systems ; Cell Line ; Genetic Therapy ; },
abstract = {Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. While CRISPR-based CFTR editing approaches have shown proof-of-concept for functional rescue in primary airway basal cells, induced pluripotent stem cells, and organoid cultures derived from patients with CF, their efficacy remains suboptimal. Here, we developed the CuFi[Cas9(Y66S)eGFP] reporter system by integrating spCas9 and a non-fluorescent Y66S eGFP mutant into CuFi-8 cells, an immortalized human airway epithelial cell line derived from a patient with CF with homozygous F508del mutations. These cells retain the basal cell phenotype in proliferating cultures and can differentiate into polarized airway epithelium at an air-liquid interface (ALI), enabling both visualized detection of gene editing and electrophysiological assessment of CFTR functional restoration. Using this system, recombinant adeno-associated virus (rAAV)-mediated homology-directed repair (HDR) was evaluated in proliferating cultures. A correction rate of 13.5 ± 0.8% was achieved in a population where 82.3 ± 5.6% of cells were productively transduced by AAV.eGFP630g2-CMVmCh, an rAAV editing vector with an mCherry reporter. Dual-editing of F508del CFTR and Y66S eGFP was explored using AAV.HR-eGFP630-F508(g03) to deliver two templates and single guide RNAs. eGFP[+] (Y66S-corrected) cells and eGFP[-] (non-corrected) cells were sorted via fluorescence-activated cell sorting and differentiated at an ALI to assess the recovery of CFTR function. Despite a low F508 correction rate of 2.8%, ALI cultures derived from the eGFP[-] population exhibited 25.2% of the CFTR-specific transepithelial Cl[-] transport observed in CuFi-ALI cultures treated with CFTR modulators. Next-generation sequencing revealed frequent co-editing at both genomic loci, with sixfold higher F508 correction rate in the eGFP[+] cells than eGFP[-] cells. In both populations, non-homology end joining predominated over HDR. This reporter system provides a valuable platform for optimizing editing efficiencies in proliferating airway basal cells, particularly for development of strategies to enhance HDR through modulation of DNA repair pathways.},
}

Humans
*Gene Editing/methods
*Dependovirus/genetics
*Cystic Fibrosis Transmembrane Conductance Regulator/genetics
*Genetic Vectors/genetics
*Epithelial Cells/metabolism/cytology
*Cystic Fibrosis/genetics/therapy/pathology
Cell Proliferation
CRISPR-Cas Systems
Cell Line
Genetic Therapy

@article {pmid40763977,
year = {2025},
author = {Li, Q and Wu, Y and Yuan, J and Liu, F and Cheng, W},
title = {[Application of base editing techniques in the identification of functional sites of genes].},
journal = {Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics},
volume = {42},
number = {6},
pages = {762-768},
doi = {10.3760/cma.j.cn511374-20241218-00666},
pmid = {40763977},
issn = {1003-9406},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Genome, Human ; Polymorphism, Single Nucleotide ; },
abstract = {The exploration of pathogenic single nucleotide polymorphisms in the genome plays a pivotal role in the study of human disease-associated genetic mutations. However, there remains a lack of suitable high-throughput screening platforms to investigate the impact of point mutations on genomic structure and function. CRISPR/Cas9-mediated base editors has enabled large-scale annotation of the human genome and phenotypic characterization of monogenic disorders. Base editors, a precise gene-editing technique capable of achieving targeted base substitutions, can be employed to induce mutations at specific functional sites, thereby observing their effects on gene expression, protein function, and cellular phenotypes. Furthermore, integrating base editors with high-throughput screening technologies allows for large-scale evaluation of multiple candidate sites, accelerating the identification of functional loci and providing a powerful tool for disease research and therapeutic target discovery. This article aims to introduce the working principles of various base editors, including cytosine base editors, adenine base editors, and prime editors, and summarize recent advances in high-throughput screening of functional genomic sites using base-editing techniques.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Genome, Human
Polymorphism, Single Nucleotide

@article {pmid40763823,
year = {2025},
author = {Xu, L and Wu, X},
title = {Viral detection using Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein and Argonaute nucleases.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {},
number = {},
pages = {120526},
doi = {10.1016/j.cca.2025.120526},
pmid = {40763823},
issn = {1873-3492},
abstract = {Viral pandemics pose severe threats to human health and societal stability, exemplified by the COVID-19 outbreak in 2019. Conventional viral detection methods such as Polymerase chain reaction (PCR) typically require trained personnel, expensive equipment, and 2-4 h for processing. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) and Argonaute (Ago) system-based detection methods achieve attomolar sensitivity or single-copy detection limits with single-base specificity within 1 h, without requiring complex or costly instruments. This review firstly introduces the mechanisms and functions of CRISPR/Cas systems (Cas9, Cas12, Cas13) and Ago systems. It also introduces viruses with significant social impact, and continued with reviewing applications of these systems in single and multiplex virus detection. Single viral detection includes recently developed DNA/RNA-activated Cas9 detection (DACD/RACD) using Cas9 trans-cleavage activity, Cas12-based DNA Endonuclease-targeted CRISPR Trans Reporter (DETECTR) with attomolar sensitivity, CRISPR/Cas13a-based Fluorescent Nanoparticle SARS-CoV-2 (CFNS) achieving 1 copy/mL sensitivity with quantum dot reporters, and amplification-free mobile phone detection detecting 31 copies/μL without amplification. Multiplex viral detection includes Microfluidic Device Integrated with CRISPR/Cas12a and Multiplex Recombinase Polymerase Amplification (MiCaR) enabling 30-plex detection through microfluidic chips with spatial discrimination, PfAgo-mediated Nucleic acid Detection (PAND) utilizing Ago-produced guide sequences for 5-plex detection, Specific High-Sensitivity Enzymatic Reporter UnLOCKing v2 (SHERLOCKv2) achieving 4-plex detection with multi-enzyme single-reaction systems, and Multiplexed Evaluation of Nucleic acids (CARMEN) supporting over 100 target assays. Finally, this review discusses challenges in CRISPR/Cas and Ago-based detection methods, including Protospacer Adjacent Motif (PAM) sequence requirements for Cas9/12, prolonged reaction times due to nucleic acid extraction/amplification, and instability of core components like nucleases and crRNAs. Detection specificity and multiplex capabilities could be further improved. Future directions are outlined for improving detection specificity, developing multiplex capabilities and advancing POCT. Developing diagnostic tools using CRISPR/Cas and Ago systems could transform molecular diagnostics, such tools promise to be easily accessible worldwide. They are essential for precise identification and strategic containment of infectious disease transmission.},
}

@article {pmid40763014,
year = {2025},
author = {Chen, L and Zhou, X and Huang, C and Zhang, Y and Xin, C and Hong, J and Wang, Y},
title = {Engineered Un1Cas12f1 with boosted gene-editing activity and expanded genomic coverage.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {32},
pages = {e2501292122},
doi = {10.1073/pnas.2501292122},
pmid = {40763014},
issn = {1091-6490},
support = {2023YFA0915000//Nation Key Research and Development Program of China/ ; 82273967//National Natural Science Foundation of China/ ; 2021QN020576//Department of Science and Technology of Guangdong Province/ ; 82425015//MOST | NSFC | National Science Fund for Distinguished Young Scholars (NSF for Distinguished Young Scholars)/ ; 82171102//National Natural Science Foundation of China/ ; 82271044//National Natural Science Fundation of China/ ; QWF158001//National Youth talent support program/ ; 22Y21900900//Shanghai Medical Innovation Research Program/ ; 24SG11//(the "Dawn" Program of) Shanghai Municipal Education Commission/ ; 24J22800500//Shanghai Science and Technology Innovation Action Plan for Cell and Gene Therapy/ ; 24CL2900802//Shanghai Science and Technology Innovation Action Plan for Advanced Materials/ ; 20254Z0019//Shanghai Municipal Commission of Health/ ; },
mesh = {Animals ; *Gene Editing/methods ; Mice ; Dependovirus/genetics ; Humans ; CRISPR-Cas Systems/genetics ; Genetic Vectors/genetics ; PTEN Phosphohydrolase/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; HEK293 Cells ; },
abstract = {Compact programmable nucleases provide versatile genome editing tools with therapeutic potential, particularly when delivered via adeno-associated virus (AAV) vectors. However, their limited editing efficacy and stringent protospaceradjacent motif (PAM) requirements impose significant limitations in practical application. Here, we engineered MiniCasUltra, an optimized Un1Cas12f1 variant, through rational mutagenesis. MiniCasUltra exhibits sixfold higher editing activity than Un1Cas12f1, minimal off-target effects (on/off-target ratio > 10), and an expanded PAM preference (5'-WBTR). Using a single AAV vector encoding MiniCasUltra and two single-guide RNAs, we achieved simultaneous editing of two disease-causing genes (Pten and Fah) in mouse liver, with indel rates of 15.82% and 29.39%, respectively- significantly surpassing CasMINI V3.1 (3.45% and 10.98%). Furthermore, AAV delivery of MiniCasUltra targeting a noncanonical 5'-TCTG PAM site in human vascularendothelial growth factor A reduced choroidal neovascularization (CNV) lesions in a laser-induced CNV mouse model of neovascular age-related macular degeneration, a leading global cause of blindness. The broad and effective targeting capabilities of MiniCasUltra, coupled with its compact size, highlight its potential for in vivo genome editing and therapeutic interventions.},
}

Animals
*Gene Editing/methods
Mice
Dependovirus/genetics
Humans
CRISPR-Cas Systems/genetics
Genetic Vectors/genetics
PTEN Phosphohydrolase/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Genetic Therapy/methods
HEK293 Cells

@article {pmid40761563,
year = {2025},
author = {Saraswat, P and Ranjan, R},
title = {Unlocking the potential of CRISPR tools and databases for precision genome editing.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1563711},
pmid = {40761563},
issn = {1664-462X},
abstract = {Recent breakthroughs in CRISPR/Cas genome editing have transformed molecular biology research and offer significant potential across biotechnology and medicine. This has created a broad spectrum of computational tools and databases that aim to optimize each phase of the genome-edited workflow, from guide RNA design and off-target prediction through screening analysis and biological validation. Here, we survey major CRISPR tools and analyse their features in the context of precision genome editing. CRISPOR and CHOPCHOP versatile platform that provides robust guide RNA design for several species, integrated off-target scoring, and intuitive genomic locus visualization. This review gives an overview of these new resources that have been developed, grouped based on their functionalities like design of guide RNA, off-target predictions, genome-wide screens, and visualizations of the data. Furthermore, we discuss new trends in database development like their integration with genome browsers and implementation of machine learning. This review thus gives a useful overview of the dynamic field of CRISPR/Cas genome editing tools. It also serves as a helpful guide for researchers looking to utilize these tools in their research.},
}

@article {pmid40760280,
year = {2025},
author = {Nguyen, PDT and Nguyen, NH},
title = {Revolutionizing molecular plant breeding through genetic engineering of in vivo haploid induction genes.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {791},
pmid = {40760280},
issn = {1573-4978},
mesh = {*Haploidy ; *Plant Breeding/methods ; Gene Editing/methods ; *Genetic Engineering/methods ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Genes, Plant/genetics ; Genome, Plant ; Oryza/genetics ; },
abstract = {Haploid and doubled haploid plants serve critical functions in plant genetics and breeding studies, as they enable the production of genetically uniform lines (pure lines) and thereby accelerate the development of new cultivars. Nowadays, various key genes regulating haploid induction have been found in maize and other plant species (e.g. Arabidopsis, rice, and wheat). These genes offer insights into the mechanisms underlying in vivo haploid induction and provide targets for genetic manipulation. Recent advancements in gene editing and genetic engineering technologies, such as CRISPR/Cas9, have revolutionized our ability to understand gene functions and precisely modify genomes. By leveraging these technologies, scientists can introduce targeted genetic changes, optimize the haploid induction process, and broaden the range of species amenable to haploid production. The application of gene editing and genetic engineering in haploid production holds immense promise for various fields, including agriculture and biotechnology. This focused review aims to advance our understanding of haploid induction genes as well as the potential applications of gene editing/genetic engineering technologies in haploid production, paving the way for innovative solutions to pressing challenges in future plant breeding and agriculture.},
}

*Haploidy
*Plant Breeding/methods
Gene Editing/methods
*Genetic Engineering/methods
CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
Genes, Plant/genetics
Genome, Plant
Oryza/genetics

@article {pmid40760060,
year = {2025},
author = {Liang, S and Jia, S and Lu, W and Wang, J and Huang, M and Chen, C and Huang, C and Zhou, D and Guo, T and Liu, H},
title = {Generation and characterization of rice OsCENH3 mutants for haploid induction.},
journal = {Plant cell reports},
volume = {44},
number = {8},
pages = {190},
pmid = {40760060},
issn = {1432-203X},
support = {No. 2022YFD1200703//National Key Research and Development Project/ ; No. 2022B0202060006//Guangdong Province Key Research and Development/ ; No. 2024NBZ00003//Guangdong Province Revitalization of Seed Industry/ ; CARS-018//National Rice Industry Technology System of China/ ; },
mesh = {*Oryza/genetics ; *Haploidy ; *Mutation/genetics ; CRISPR-Cas Systems/genetics ; Gene Editing ; *Plant Proteins/genetics/metabolism ; Aneuploidy ; Chromosome Segregation/genetics ; Chromosomes, Plant/genetics ; Histones/genetics/metabolism ; Plants, Genetically Modified ; Pollen/genetics ; },
abstract = {CRISPR/Cas9-mediated modification of OsCENH3 induces aneuploidy but fails to trigger haploid formation in rice, underscoring limited efficiency and the need for strategy refinement. The centromeric histone H3 variant (CENH3) is essential for kinetochore assembly and accurate chromosome segregation during cell division. Alterations in CENH3 have been shown to trigger haploid induction in various plant species; however, its utility in rice remains largely unexplored. In this study, six OsCENH3 mutant lines were generated through CRISPR/Cas9-mediated genome editing, complemented by a GFP-tagged construct. To assess their haploid induction potential, both self pollination and outcrossing with the cultivar Nipponbare (NIP) were performed. Flow cytometry analysis revealed that none of the mutants produced true haploids. However, two aneuploid individuals (Het-C1-1-1 and Het-C1-5-1) were identified among the hybrid progeny derived from tail domain mutants C1-1 and C1-5, suggesting partial chromosome missegregation. These aneuploid plants exhibited reduced pollen viability, abnormal morphology, and compromised agronomic performance. In addition, significantly elevated rates of embryo/endosperm abortion were observed across different crosses, which far exceeded the frequency of aneuploid production, implying that early-stage chromosomal instability may result in embryo lethality. Collectively, while specific OsCENH3 mutations can induce limited chromosomal instability, their haploid induction efficiency remains low in rice. Further refinement of editing strategies and exploration of favorable genetic backgrounds will be essential for developing effective centromere-based haploid induction systems. This study provides a theoretical framework and technical reference for engineering haploid inducers in rice through centromere manipulation.},
}

*Oryza/genetics
*Haploidy
*Mutation/genetics
CRISPR-Cas Systems/genetics
Gene Editing
*Plant Proteins/genetics/metabolism
Aneuploidy
Chromosome Segregation/genetics
Chromosomes, Plant/genetics
Histones/genetics/metabolism
Plants, Genetically Modified
Pollen/genetics

@article {pmid40719616,
year = {2025},
author = {Zhou, X and Zhang, R and Cai, L and Fu, H and Wang, D and Yuan, R and Yuan, Y},
title = {Efficient Entropy-Driven Cross-Double-Loop Reaction Cascade Asymmetric CRISPR/Cas12a Cleavage for Ultrasensitive Electrochemical Biosensing.},
journal = {Analytical chemistry},
volume = {97},
number = {30},
pages = {16355-16363},
doi = {10.1021/acs.analchem.5c02014},
pmid = {40719616},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *Entropy ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism ; DNA, Single-Stranded/chemistry ; Limit of Detection ; Electrodes ; DNA/analysis ; Bacterial Proteins ; },
abstract = {The limited signal amplification efficiency of the conventional CRISPR/Cas12a-cleavage system was primarily due to the structural constraints of crRNA and single-trigger activation. Herein, an efficient target-induced entropy-driven cross-double-loop strand displacement reaction (SDR) cascade asymmetric CRISPR/Cas12a cleavage was developed to construct an ultrasensitive and reliable signal-off electrochemical biosensor. The desirable entropy-driven modulation could spontaneously undergo a cross-double-loop reaction that possessed self-accelerating ability, effectively improving the rate of chain replacement and avoiding the usage of extra fuel chains with generation of two abundant distinct DNA outputs, significantly improving target conversion efficiency. More importantly, all the targets and two distinct DNA outputs could simultaneously act as activators in the asymmetric CRISPR/Cas12a system, which cooperatively bound to both split and full-sized crRNAs to accomplish the highly efficient discharge of ferrocene-labeled single-stranded DNA (Fc-reporter) on the electrode, thereby markedly improving the detection sensitivity and reliably compared to that of traditional ones. The experimental results suggested that the proposed biosensor had a wide linear range spanning from 1 fM to1 nM with a detection limit as low as 0.23 fM. By integrating entropy-driven amplification with CRISPR-enhanced signal transduction, this work established a versatile and robust analytical tool for early cancer diagnosis and precision biomolecular detection.},
}

*Biosensing Techniques/methods
*Entropy
*Electrochemical Techniques/methods
*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism
*Endodeoxyribonucleases/metabolism
DNA, Single-Stranded/chemistry
Limit of Detection
Electrodes
DNA/analysis
Bacterial Proteins

@article {pmid40698640,
year = {2025},
author = {Dai, X and Meng, C and Huang, S and Wang, Y and He, J and Chen, Z and Ji, Y and Tai, J and Zhang, J and Ni, H and Zhuang, Z and Chen, J and Zhang, H and Qu, J and Shao, Y},
title = {Attomolar Nucleic Acid Detection Using CRISPR Enhanced Phase-Sensitive Surface Plasmon Resonance Imaging.},
journal = {Analytical chemistry},
volume = {97},
number = {30},
pages = {16296-16303},
doi = {10.1021/acs.analchem.5c01772},
pmid = {40698640},
issn = {1520-6882},
mesh = {*Surface Plasmon Resonance/methods ; *SARS-CoV-2/genetics/isolation & purification ; Gold/chemistry ; Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems ; *DNA, Viral/analysis/genetics ; Humans ; COVID-19/diagnosis/virology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA, Single-Stranded/chemistry ; Limit of Detection ; },
abstract = {Driven by the growing need for real-time, amplification-free, and label-free nucleic acid detection in clinical diagnostics and pathogen surveillance, traditional methods often fall short due to limited sensitivity, a narrow dynamic range, and difficulties in detecting low-concentration nucleic acids and single-nucleotide mutations. To address these challenges, we developed a clustered regularly interspaced short palindromic repeats (CRISPR) enhanced Phase-interrogation Surface Plasmon Resonance imaging (CRISPR-PSPRi) sensor that employs phase delay modulation for highly sensitive extraction of SPR phase signals and a wavelength scanning strategy to extend its dynamic range. By harnessing CRISPR-Cas12a for target DNA recognition and activating trans-cleavage to cleave ssDNA-linked gold nanoparticle probes, our platform converts extremely weak signals from low-concentration DNA into readily detectable cleavage signals. Achieving a sensitivity of 1.436 × 10[-6] RIU and a dynamic range of 0.0111 RIU, this system successfully detects specific DNA from the SARS-CoV-2 Omicron BA.2 variant and monkeypox virus, and it can detect single-nucleotide mutations down to 1 aM. This breakthrough offers a real-time, high-throughput, and ultrasensitive nucleic acid detection approach, promising significant advancements in clinical diagnostics and pathogen monitoring.},
}

*Surface Plasmon Resonance/methods
*SARS-CoV-2/genetics/isolation & purification
Gold/chemistry
Metal Nanoparticles/chemistry
*CRISPR-Cas Systems
*DNA, Viral/analysis/genetics
Humans
COVID-19/diagnosis/virology
*Clustered Regularly Interspaced Short Palindromic Repeats
DNA, Single-Stranded/chemistry
Limit of Detection

@article {pmid40689881,
year = {2025},
author = {Xu, X and Tang, K and Yang, L and Wu, Y and Gu, Y and Li, Y},
title = {Split crRNA-Mediated Regulation of CRISPR-Cas12a Enables Construction of Biochemical Circuits for Sensitive Molecular Diagnostics.},
journal = {Analytical chemistry},
volume = {97},
number = {30},
pages = {16645-16651},
doi = {10.1021/acs.analchem.5c03196},
pmid = {40689881},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *RNA/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/analysis ; Humans ; *Bacterial Proteins/genetics/metabolism ; *Molecular Diagnostic Techniques/methods ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR) system represents a fundamental tool for biotechnological applications. However, the ability to engineer intrinsic components of the CRISPR system to regulate its activity remains limited. Here, we present a strategy for the regulation of CRISPR-Cas12a by engineering an enhanced split CRISPR RNA (split en-crRNA) variant. Reassembly of this variant autonomously restores the full activity of Cas12a nuclease without the need for chemical modifications or exogenous activation stimuli. Leveraging this strategy, we developed CREST (CRISPR Reactions Elicited by Split en-crRNA Transcription) for molecular diagnostics. CREST integrates biochemical circuits that ingeniously harness isothermal amplification to trigger the on-demand synthesis and reassembly of a universal split en-crRNA, addressing longstanding challenges in CRISPR diagnostics, including the low efficiency of one-pot assays, protospacer adjacent motif restriction, the need for crRNA reoptimization, and limited reagent stability. The CREST assay enables robust detection of attomolar DNAs or RNAs within 15 to 20 min and demonstrates capability for identifying bacterial infections in patients. This study provides new insights into Cas12a regulation, which is expected to advance the development of controllable CRISPR technologies for diagnostics as well as broader applications such as genome editing and programmable gene expression.},
}

*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/genetics/metabolism
*Endodeoxyribonucleases/genetics/metabolism
*RNA/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
DNA/analysis
Humans
*Bacterial Proteins/genetics/metabolism
*Molecular Diagnostic Techniques/methods

@article {pmid40505899,
year = {2025},
author = {Bai, Y and Gao, BJ and Shi, XY and Hu, TH and Shi, LD and Duan, JX and Zhang, ZH and Shang, GH and Xu, J and Li, DL and He, ZQ and Li, K},
title = {The tyrosine hydroxylase regulates organs development via affecting hormones level in Gryllus bimaculatus.},
journal = {International journal of biological macromolecules},
volume = {319},
number = {Pt 2},
pages = {145159},
doi = {10.1016/j.ijbiomac.2025.145159},
pmid = {40505899},
issn = {1879-0003},
mesh = {Animals ; *Tyrosine 3-Monooxygenase/genetics/metabolism ; *Juvenile Hormones/metabolism ; Mutation ; Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; Phenotype ; Pigmentation ; },
abstract = {Tyrosine hydroxylase (TH) is the initial enzyme in the melanin biosynthesis pathway and catalyzes the conversion of tyrosine to dihydroxyphenylalanine (DOPA). Extensive research has demonstrated that TH exhibits evolutionarily conserved function in insects, regulating multiple physiological pathways including integumentary repair mechanisms, cuticular sclerotization processes, and melanization in response to pathogenic challenges. However, few studies have addressed the role of TH in Gryllus bimaculatus (G. bimaculatus), a hemimetabolous model insect. In this study, we investigated the in vivo role of GbTH gene by using CRISPR/Cas9 gene-editing tool to generate mosaic mutants (GbTH F0) and homozygous mutants (GbTH[-/-]). The GbTH F0 mutants displayed less pigmented phenotypes, meanwhile the pronotum became thinner, leading to defects in chirping and motor behaviors. The GbTH[-/-] mutants exhibited pleiotropic developmental abnormalities, characterized by complete lethality at the nymphal stage. Phenotypic analysis revealed compromised cuticular integrity with impaired chitin matrix deposition in pre-nymphal exoskeletons, accompanied by significantly enhanced desiccation susceptibility and progressive body mass reduction in mutant nymphs. RNA-sequencing transcriptome analysis indicated that GbTH was involved in regulation the synthesis and coordination of juvenile hormone (JH) and ecdysone (20E). Furthermore, GbTH knockout significantly reduced the expression of the lipid transport protein NPC1b. This suppression led to impaired cholesterol uptake, which consequently disrupted steroid hormone biosynthesis and ultimately compromised key developmental transitions, including molting cycles and metamorphic progression. Collectively, these findings establish that GbTH serves as a master regulatory enzyme for the fine-tuning of developmental processes in hemimetabolic insects, extending beyond its canonical function in cuticular pigmentation regulation.},
}

Animals
*Tyrosine 3-Monooxygenase/genetics/metabolism
*Juvenile Hormones/metabolism
Mutation
Gene Expression Regulation, Developmental
CRISPR-Cas Systems
Phenotype
Pigmentation

@article {pmid40503836,
year = {2025},
author = {Liu, W and Wang, Y and Zhao, J and Zhang, X and Li, Z and Jia, H and Zhu, C and Zhang, L and Pan, L and Zhang, Z},
title = {Identification of Mycobacterium tuberculosis intracellular survival-related virulence factors via CRISPR-based eukaryotic-like secretory protein mutant library screen.},
journal = {Microbiology spectrum},
volume = {13},
number = {8},
pages = {e0076725},
doi = {10.1128/spectrum.00767-25},
pmid = {40503836},
issn = {2165-0497},
support = {32394013//National Natural Science Foundation of China/ ; 82172279//National Natural Science Foundation of China/ ; 82402641//National Natural Science Foundation of China/ ; 82070012//National Natural Science Foundation of China/ ; G2024-2-007//Beijing High-level Public Health Project/ ; },
mesh = {*Mycobacterium tuberculosis/genetics/pathogenicity/metabolism ; Macrophages/microbiology/immunology ; *Virulence Factors/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Animals ; Clustered Regularly Interspaced Short Palindromic Repeats ; Mice ; CRISPR-Cas Systems ; Mutation ; Tuberculosis/microbiology ; Humans ; Gene Library ; RAW 264.7 Cells ; Gene Editing ; },
abstract = {UNLABELLED: Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), remains a serious infectious disease posing significant global health challenges. A critical evolutionary feature of M.tb is its genome encoding a set of eukaryotic-like secretory proteins, which facilitate intracellular survival by manipulating host immune responses. However, the specific eukaryotic-like secretory proteins that facilitate M.tb intracellular survival and their regulatory mechanisms on host immunity remain uncharacterized. In this study, a mutant library comprising 137 potential eukaryotic-like secretory proteins was constructed using clustered regularly interspaced short palindromic repeats (CRISPR)-non-homologous end joining genome editing technology. Subsequently, macrophages were infected with the mutant library, and CRISPR sequencing enabled preliminary identification of virulence factors associated with bacterial intracellular persistence. To validate the screen, two genes (Rv0066c and Rv3139) exhibiting the most pronounced reduction in intracellular survival rates when mutated were selected for the construction of large-fragment knockout strains (ΔRv0066c and ΔRv3139). Subsequent macrophage infection assays reconfirmed the impaired intracellular survival of these two mutants. RNA-seq analysis was conducted to characterize host gene expression profiles during ΔRv0066c-infected macrophage interactions. RNA-seq analysis of macrophages infected with wild-type and ΔRv0066c strains identified 138 differentially expressed genes, with 75 upregulated and 63 downregulated in ΔRv0066c. Gene ontology clustering of these differentially expressed genes highlighted molecular functions related to chemokine binding, chemokine-mediated signaling pathways, Ras protein signal transduction, and calcineurin-mediated signaling. Collectively, this work established a potential eukaryotic-like secretory protein mutant library and identified two novel M.tb effectors governing intracellular survival, providing potential new targets for anti-TB drug development.

IMPORTANCE: Eukaryotic-like secretory proteins that subvert host immunity to enable intracellular persistence are a key evolutionary adaptation of Mycobacterium tuberculosis (M.tb). In this study, we established a mutant library targeting 137 potential eukaryotic-like secretory proteins through clustered regularly interspaced short palindromic repeats (CRISPR)-non-homologous end joining genome editing technology. The library was subjected to macrophage infection assays, and CRISPR sequencing enabled identification of M.tb persistence-associated virulence determinants. Validation screens highlighted two genes (Rv0066c and Rv3139) that displayed the most significant intracellular survival defects to generate large-fragment knockout strains (ΔRv0066c and ΔRv3139). Macrophage infection experiments reconfirmed the compromised intracellular viability of both mutants. RNA-seq profiling of ΔRv0066c-infected macrophages identified 138 differentially expressed genes, with functional enrichment in chemokine signaling, Ras protein signal transduction, and calcineurin-mediated signaling. To conclude, this study identified two novel M.tb effectors contributing to intracellular survival as potential new targets for anti-TB drug development.},
}

*Mycobacterium tuberculosis/genetics/pathogenicity/metabolism
Macrophages/microbiology/immunology
*Virulence Factors/genetics/metabolism
*Bacterial Proteins/genetics/metabolism
Animals
Clustered Regularly Interspaced Short Palindromic Repeats
Mice
CRISPR-Cas Systems
Mutation
Tuberculosis/microbiology
Humans
Gene Library
RAW 264.7 Cells
Gene Editing

@article {pmid40399576,
year = {2025},
author = {Gu, P and Yuan, Y and Qi, L and Lan, F},
title = {RPA-CRISPR Cas13a-Based Point-of-Care Testing established for rapid detection of Methicillin-Resistant Staphylococcus aureus (MRSA) resistance genes.},
journal = {European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology},
volume = {44},
number = {8},
pages = {1959-1966},
pmid = {40399576},
issn = {1435-4373},
mesh = {*Methicillin-Resistant Staphylococcus aureus/genetics/isolation & purification/drug effects ; Humans ; *Bacterial Proteins/genetics ; Penicillin-Binding Proteins/genetics ; Staphylococcal Infections/microbiology/diagnosis ; *CRISPR-Cas Systems ; *Point-of-Care Testing ; Polymorphism, Single Nucleotide ; *Nucleic Acid Amplification Techniques/methods ; Recombinases/metabolism ; Sensitivity and Specificity ; Mutation ; },
abstract = {PURPOSE: This study aimed to develop a streamlined and efficient method for detecting single nucleotide polymorphism (SNP), specifically the S643N mutation in the mecA gene of Staphylococcus aureus (S. aureus). Detecting this mutation is crucial for guiding appropriate antibiotic therapy, avoiding antibiotic misuse, and enhancing infection control measures, especially in the cases of methicillin-resistant S. aureus (MRSA) infections.

METHODS: We employed the RPA-CRISPR Cas13a method to detect the S643N mutation. This approach integrated recombinase polymerase amplification (RPA) with CRISPR Cas13a-mediated detection into a single-step procedure, significantly reducing detection time compared to the conventional two-step process.

RESULTS: The established RPA-CRISPR Cas13a-Based Point-of-Care Testing method achieved high sensitivity, detecting as few as 10 copies per reaction and successfully differentiating between wild-type and mutant mecA genes. The one-step procedure streamlined the workflow and reduced detection time to less than 30 min, while delivering results that were consistent with the conventional two-step method.

CONCLUSION: The one-step RPA-CRISPR Cas13a method significantly facilitates the rapid and accurate detection of single nucleotide mutations, such as the S643N mutation in the mecA gene. This advancement holds substantial clinical value by guiding precise antibiotic therapy and improving the management of S. aureus infections.},
}

*Methicillin-Resistant Staphylococcus aureus/genetics/isolation & purification/drug effects
Humans
*Bacterial Proteins/genetics
Penicillin-Binding Proteins/genetics
Staphylococcal Infections/microbiology/diagnosis
*CRISPR-Cas Systems
*Point-of-Care Testing
Polymorphism, Single Nucleotide
*Nucleic Acid Amplification Techniques/methods
Recombinases/metabolism
Sensitivity and Specificity
Mutation

@article {pmid40393858,
year = {2025},
author = {Tenjo-Castaño, F and Rout, SS and Dey, S and Montoya, G},
title = {Unlocking the potential of CRISPR-associated transposons: from structural to functional insights.},
journal = {Trends in genetics : TIG},
volume = {41},
number = {8},
pages = {660-677},
doi = {10.1016/j.tig.2025.04.005},
pmid = {40393858},
issn = {0168-9525},
mesh = {*DNA Transposable Elements/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Animals ; Transposases/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated transposons (CASTs) are emerging genome-editing tools that enable RNA-guided DNA integration without inducing double-strand breaks (DSBs). Unlike CRISPR-associated (Cas) nucleases, CASTs use transposon machinery to insert large DNA segments with high precision, potentially reducing off-target effects and bypassing DNA damage responses. CASTs are categorized into classes 1 and 2, each employing distinct mechanisms for DNA targeting and integration. Recent structural insights have elucidated how CASTs recognize target sites, recruit transposases, and mediate insertion. These advances position CASTs as promising tools for genome engineering in bacteria and possibly in mammalian cells. Key challenges remain in enhancing efficiency and specificity, particularly for therapeutic use. Ongoing research aims to evolve CAST systems for precise, large-scale genome editing in human cells.},
}

*DNA Transposable Elements/genetics
Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Animals
Transposases/genetics

@article {pmid40335327,
year = {2025},
author = {Chen, W and Choi, J},
title = {Molecular circuits for genomic recording of cellular events.},
journal = {Trends in genetics : TIG},
volume = {41},
number = {8},
pages = {647-659},
doi = {10.1016/j.tig.2025.04.004},
pmid = {40335327},
issn = {0168-9525},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genomics/methods ; *Synthetic Biology/methods ; Animals ; *Gene Regulatory Networks ; Cell Lineage/genetics ; Genome ; },
abstract = {Advances in precise genome editing are enabling genomic recordings of cellular events. Since the initial demonstration of CRISPR-based genome editing, the field of genomic recording has witnessed key strides in lineage recording, where clonal lineage relationships among cells are indirectly recorded as synthetic mutations. However, methods for directly recording and reconstructing past cellular events are still limited, and their potential for revealing new insights into cell fate decisions has yet to be realized. The field needs new sensing modules and genetic circuit architectures that faithfully encode past cellular states into genomic DNA recordings to achieve such goals. Here we review recently developed strategies to construct diverse sensors and explore how emerging synthetic biology tools may help to build molecular circuits for genomic recording of diverse cellular events.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Genomics/methods
*Synthetic Biology/methods
Animals
*Gene Regulatory Networks
Cell Lineage/genetics
Genome

@article {pmid39865841,
year = {2025},
author = {Choi, BJ and Kim, BR and Choi, HJ and Kim, OH and Kim, SJ},
title = {Enhanced membrane protein production in HEK293T cells via ATF4 gene knockout: A CRISPR-Cas9 mediated approach.},
journal = {Biomolecules & biomedicine},
volume = {25},
number = {9},
pages = {1961-1971},
doi = {10.17305/bb.2024.11519},
pmid = {39865841},
issn = {2831-090X},
mesh = {Humans ; HEK293 Cells ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques ; *Activating Transcription Factor 4/genetics/metabolism ; *Membrane Proteins/genetics/biosynthesis/metabolism ; Gene Editing ; Cadherins/genetics/metabolism/biosynthesis ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {HEK293T cells are extensively utilized for therapeutic protein production due to their human origin, which enables accurate post-translational modifications. This study aimed to enhance membrane protein production in HEK293T cells by knocking out the ATF4 gene using CRISPR-Cas9 technology. The ATF4 gene was edited by infecting HEK293T cells with a lentivirus carrying optimized single-guide RNA (ATF4-KO-3) and Cas9 genes. Comparative evaluations were conducted using all-in-one and two-vector systems. Genome sequencing and membrane protein productivity of ATF4-knockout (KO) cells were compared to wild-type (WT) cells using next-generation sequencing (NGS) and a membrane protein isolation kit, respectively. Single-cell analysis confirmed gene editing patterns, with NGS verifying the intended deletions. Membrane protein production was also assessed indirectly via flow cytometry, analyzing cells expressing Membrane-GFP. Compared to WT cells, ATF4-KO cells exhibited a significant increase in membrane protein production, with a 52.2 ± 19.0% improvement. Gene editing efficiency was compared between the two delivery systems, with the two-vector system demonstrating higher efficiency based on T7 endonuclease I assays. Western blot analysis confirmed ATF4 suppression and increased expression of membrane proteins, including E-cadherin and CD63. Quantitative analysis via PAGE revealed a 77.2 ± 30.6% increase in purified membrane protein yields, consistent with the observed enhancements. Flow cytometry using Membrane-GFP further demonstrated a 22.9 ± 9.7% increase in productivity. In summary, ATF4 knockout significantly enhances membrane protein production in HEK293T cells, offering potential improvements in biopharmaceutical manufacturing by enabling more efficient protein synthesis.},
}

Humans
HEK293 Cells
*CRISPR-Cas Systems/genetics
*Gene Knockout Techniques
*Activating Transcription Factor 4/genetics/metabolism
*Membrane Proteins/genetics/biosynthesis/metabolism
Gene Editing
Cadherins/genetics/metabolism/biosynthesis
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40760018,
year = {2025},
author = {Haider, D and Bauer, R and Grempels, A and Roscher, R and Aslan, CC and Mauerer, S and Spellerberg, B},
title = {The stress of carrying CRISPR-Cas.},
journal = {Virulence},
volume = {16},
number = {1},
pages = {2541701},
doi = {10.1080/21505594.2025.2541701},
pmid = {40760018},
issn = {2150-5608},
mesh = {*CRISPR-Cas Systems ; *Stress, Physiological ; *Streptococcus anginosus/genetics/radiation effects/physiology/growth & development ; Ultraviolet Rays ; Hydrogen Peroxide ; Microbial Viability/radiation effects ; Hot Temperature ; Plasmids ; CRISPR-Associated Protein 9/genetics ; Bacterial Proteins/genetics ; },
abstract = {Streptococcus anginosus (S. anginosus) is a commensal that can cause severe invasive bacterial infections. A considerable percentage of S. anginosus strains harbour CRISPR-Cas systems, which apart from being a bacterial immunity system, can play an important role in adaptation to environmental stress. The functionality of S. anginosus CRISPR-Cas systems has not been investigated previously. To address this, we created a set of deletion mutants in the CRISPR-Cas type II-A system of the S. anginosus SK52 type strain, targeting the nuclease Cas9 and CRISPR array. We were able to confirm CRISPR-Cas activity by testing these strains in a plasmid clearance assay. Furthermore, the impact of the CRISPR-Cas system on the viability of S. anginosus was investigated under various stress conditions, such as UV light, hydrogen peroxide exposure, and high temperatures in wild-type S. anginosus and CRISPR-Cas mutant strains. Under these conditions, survival was significantly lower in the strains carrying cas9. Bacterial growth and metabolic activity in Alamar Blue assays were also negatively affected by the presence of cas9 in S. anginosus. In summary, we found that the presence of a functional CRISPR-Cas system in S. anginosus leads to measurable metabolic and fitness costs in the wild-type strain. Carrying cas9 was associated with an impaired stress response in our experiments and may explain why many strains of this species lack CRISPR-Cas.},
}

*CRISPR-Cas Systems
*Stress, Physiological
*Streptococcus anginosus/genetics/radiation effects/physiology/growth & development
Ultraviolet Rays
Hydrogen Peroxide
Microbial Viability/radiation effects
Hot Temperature
Plasmids
CRISPR-Associated Protein 9/genetics
Bacterial Proteins/genetics

@article {pmid40759662,
year = {2025},
author = {Leprince, A and Lefrançois, J and Millen, AM and Magill, D and Horvath, P and Romero, DA and Rousseau, GM and Moineau, S},
title = {Strengthening phage resistance of Streptococcus thermophilus by leveraging complementary defense systems.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7142},
doi = {10.1038/s41467-025-62408-3},
pmid = {40759662},
issn = {2041-1723},
mesh = {*Streptococcus thermophilus/virology/genetics/immunology ; *CRISPR-Cas Systems/genetics ; *Bacteriophages/genetics/physiology ; *Streptococcus Phages/genetics/physiology ; },
abstract = {CRISPR-Cas and restriction-modification systems represent the core defense arsenal in Streptococcus thermophilus, but their effectiveness is compromised by phages encoding anti-CRISPR proteins (ACRs) and other counter-defense strategies. Here, we explore the defensome of 263 S. thermophilus strains to uncover other anti-phage systems. The defense landscape of S. thermophilus is enriched by 21 accessory defense systems, 13 of which have never been investigated in this species. Experimental validation of 17 systems with 14 phages reveals a range of anti-phage activities, highlighting both broad and narrow specificities across the five viral genera infecting S. thermophilus. Synergies are observed when combining CRISPR immunity with accessory systems. We also assess the fitness cost associated with the chromosomal integration of these systems in their native context and find no impact under laboratory or industrial conditions. These findings underscore the potential of these accessory defense systems to enhance the resistance of S. thermophilus, particularly against ACR-encoding phages.},
}

*Streptococcus thermophilus/virology/genetics/immunology
*CRISPR-Cas Systems/genetics
*Bacteriophages/genetics/physiology
*Streptococcus Phages/genetics/physiology

@article {pmid40758882,
year = {2025},
author = {Vera, AO and Truex, NL and Sreekanth, V and Pentelute, BL and Choudhary, A and Raines, RT},
title = {Protective antigen-mediated delivery of an anti-CRISPR protein for precision genome editing.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {32},
pages = {e2426960122},
doi = {10.1073/pnas.2426960122},
pmid = {40758882},
issn = {1091-6490},
support = {R01 GM137606/GM/NIGMS NIH HHS/United States ; R35 GM148220/GM/NIGMS NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; *Bacterial Toxins/genetics/metabolism ; *Antigens, Bacterial/genetics/metabolism ; HEK293 Cells ; CRISPR-Associated Protein 9 ; },
abstract = {Precise control over the dosage of Cas9-based technologies is essential because off-target effects, mosaicism, chromosomal aberrations, immunogenicity, and genotoxicity can arise with prolonged Cas9 activity. Type II anti-CRISPR proteins (Acrs) inhibit and control Cas9 but are generally impermeable to the cell membrane due to their size and anionic charge. Moreover, existing Acr delivery methods are long-lived and operate within hours (e.g., viral and nonviral vectors) or require external devices (e.g., electroporation), limiting therapeutic applications. To address these problems, we developed a protein-based anti-CRISPR delivery platform, LFN-Acr/PA, which delivers Acrs into cells within minutes. LFN-Acr/PA is a nontoxic, two-component protein system derived from anthrax toxin, where protective antigen (PA) proteins bind receptors widespread in human cells, forming a pH-triggered endosomal pore that an engineered Acr (LFN-Acr) binds and uses to enter the cell. In the presence of PA, LFN-Acr enters human cells (e.g., immortalized cell lines, embryonic stem cells, and 3D cell cultures) at concentrations as low as 2.5 pM to inhibit up to 95% of Cas9-mediated knockout, knock-in, transcriptional activation, and base editing. Timing LFN-Acr delivery reduces off-target base editing and increases Cas9 specificity by 41%. LFN-Acr/PA is the most potent known cell-permeable CRISPR-Cas inhibition system, significantly improving the utility of CRISPR for genome editing.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems
*Bacterial Toxins/genetics/metabolism
*Antigens, Bacterial/genetics/metabolism
HEK293 Cells
CRISPR-Associated Protein 9

@article {pmid40758756,
year = {2025},
author = {Angst, P and Thivolle, A and Haden, Z and Wale, N and Ebert, D},
title = {Genomic analysis of the zooplankton-associated pathogenic bacterium Spirobacillus cienkowskii reveals its functional and metabolic capacities.},
journal = {Microbial genomics},
volume = {11},
number = {8},
pages = {},
doi = {10.1099/mgen.0.001463},
pmid = {40758756},
issn = {2057-5858},
mesh = {Animals ; *Genome, Bacterial ; *Zooplankton/microbiology ; Genomics ; Daphnia/microbiology ; Whole Genome Sequencing ; CRISPR-Cas Systems ; Carotenoids/metabolism ; },
abstract = {Genomic information can yield new insights into the molecular and physiological mechanisms that underpin pathogen virulence and transmission. We decode the genome of Spirobacillus cienkowskii Metchnikoff 1889, a gram-negative bacterium and one of the first described parasites of Daphnia. We use long-read sequencing and extensive annotation to assemble the complete circular genome of 2.81 Mbp with 2,486 protein-coding genes. In addition to antiviral systems, including CRISPR-Cas and restriction-modification systems, we describe the likely molecular basis of the unusual red phenotype of S. cienkowskii, which results from carotenoid production. We further describe genetic modules that may mediate this bacterium's interactions with its host and environment. Our study provides insight into the metabolic and functional capacities of a parasite through the assessment of its genome. More generally, it demonstrates what can be learnt by applying recent advances in high-throughput sequencing to the study of parasites.},
}

Animals
*Genome, Bacterial
*Zooplankton/microbiology
Genomics
Daphnia/microbiology
Whole Genome Sequencing
CRISPR-Cas Systems
Carotenoids/metabolism

@article {pmid40758696,
year = {2025},
author = {Thavorasak, T and Santajit, S and Tunyong, W and Kong-Ngoen, T and Reamtong, O and Ampawong, S and Saelim, N and Srisai, T and Aiumurai, P and Pumirat, P and Chaicumpa, W and Indrawattana, N},
title = {CRISPR/Cas9-targeted smpB mutation revealing roles in biofilm formation, motility, and antibiotic susceptibility in Acinetobacter baumannii.},
journal = {PloS one},
volume = {20},
number = {8},
pages = {e0329638},
doi = {10.1371/journal.pone.0329638},
pmid = {40758696},
issn = {1932-6203},
mesh = {*Biofilms/drug effects/growth & development ; *Acinetobacter baumannii/genetics/drug effects/pathogenicity/physiology ; *CRISPR-Cas Systems/genetics ; *Bacterial Proteins/genetics/metabolism ; *Anti-Bacterial Agents/pharmacology ; Mutation ; Animals ; Microbial Sensitivity Tests ; Virulence/genetics ; Gene Editing ; Acinetobacter Infections/microbiology/drug therapy ; },
abstract = {BACKGROUND: Acinetobacter baumannii is a multidrug-resistant pathogen and a major cause of hospital-acquired infections worldwide. Its ability to survive in harsh environments and evade antibiotic treatments underscores the urgent need for new therapeutic targets. Emerging evidence suggests that the small protein B (SmpB) may also play broader roles in bacterial virulence, including regulation of biofilm formation, motility, and stress adaptation. However, the specific contributions of SmpB to these pathogenic traits in A. baumannii remain poorly defined. Addressing this knowledge gap is essential for evaluating SmpB as a potential antimicrobial target and developing new strategies to combat multidrug-resistant infections.

METHODS: CRISPR/Cas9-mediated gene editing was used to generate a targeted smpB mutant in A. baumannii. The smpB mutant was assessed for growth, biofilm formation, motility, antibiotic susceptibility, and virulence. Biofilm was quantified via crystal violet staining and microscopy, while motility was examined using swimming, swarming, and twitching assays. Antibiotic susceptibility was evaluated using disk diffusion. Virulence was tested in the Galleria mellonella infection model. Proteomic analysis was performed to identify changes in protein expression associated with smpB disruption.

RESULTS: CRISPR/Cas9-mediated editing successfully introduced a C212T nucleotide substitution in the smpB gene, resulting in an A89G amino acid change. Growth curve analysis showed no significant difference between the wild-type and smpB mutant strains under nutrient-rich conditions. However, the mutant exhibited a significant reduction in biofilm formation (p = 0.0079) and impaired twitching motility, while swimming and swarming motility remained unaffected. Antibiotic susceptibility testing revealed increased sensitivity to ceftizoxime, piperacillin/tazobactam, and gentamicin, alongside decreased susceptibility to cefepime, tetracycline, and spectinomycin. In the G. mellonella infection model, the smpB mutant showed reduced virulence, with 84% larval survival compared to 72% in the wild type (p = 0.4183). Proteomic analysis revealed downregulation of key stress response and virulence-associated proteins, including GroEL, DnaK, RecA, and PirA, while proteins involved in ribosome maturation and transcription, such as RimP and RpoA, were upregulated. STRING network analysis supported the broad regulatory role of SmpB in biofilm formation, motility, stress adaptation, and pathogenesis.

CONCLUSION: This study demonstrates that SmpB is a key regulator of biofilm formation, twitching motility, antibiotic response, and virulence in A. baumannii. While not essential for growth under optimal conditions, smpB disruption impairs multiple pathogenic traits and alters stress-related proteomic pathways. These findings highlight the potential of SmpB as a novel antimicrobial target, offering a promising strategy to weaken bacterial virulence without promoting resistance. Targeting the trans-translation system may pave the way for innovative therapies against multidrug-resistant A. baumannii.},
}

*Biofilms/drug effects/growth & development
*Acinetobacter baumannii/genetics/drug effects/pathogenicity/physiology
*CRISPR-Cas Systems/genetics
*Bacterial Proteins/genetics/metabolism
*Anti-Bacterial Agents/pharmacology
Mutation
Animals
Microbial Sensitivity Tests
Virulence/genetics
Gene Editing
Acinetobacter Infections/microbiology/drug therapy

@article {pmid40758649,
year = {2025},
author = {Qu, L and Meng, L and Sun, X and Cui, W and Shi, J and Wang, Y and Hu, Q and Xu, S and Sun, B and Liang, C},
title = {CRISPR/Cas-Based Electrochemical Biosensor for Human Immunodeficiency Virus-1 Nucleic Acid Amplification-Free Detection to the Attomolar Level.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c00363},
pmid = {40758649},
issn = {2379-3694},
abstract = {Aiming at realizing field detection during HIV-1 patient screening in developing countries and point of care testing (POCT) of virus levels in HIV-1 patients receiving medication, an urgent demand for portable nucleic acid detection technology with low cost and sensitivity is raised. To solve this, a CRISPR/Cas13a-based electrochemical detection platform by a multiple combined crRNAs strategy is developed. This sensing platform is based on the Ion Current Rectification regulation through a Porous Anodic Alumina membrane decorated with ssRNA chains, which are trans-cleavage substrates for activated Cas13a and become shorter when the target gene exists, resulting in an altered motion of transmembrane ions. This platform was proven to be feasible and ultrasensitive for tracing the HIV-1 virus by multiple applicable crRNAs combination, and its clinical sample detection limit arrives at 60 copies/μL for HIV-1 patients' serum samples. Importantly, this method is amplification-free, and its sensitivity is beyond the nonamplification fluorescence-based nucleic acid strategy by one order of magnitude. This sensor is facile to use and cost-effective, and can also be extended to other RNA-based pathogenic microorganism detections. This POCT method facilitates self-monitoring of drug resistance in HIV-1 patients, regardless of medication status.},
}

@article {pmid40758593,
year = {2025},
author = {Carreira de Paula, J and Solano Parada, J and Rosel Miñarro, JF and García Olmedo, P and Orantes, FJ and Osuna, A and de Pablos Torró, LM},
title = {On-site DNA Detection of Trypanosomatid Parasites and Nosema ceranae Through Alkaline Lysis Coupled to RPA/CRISPR/Cas12a System.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {221},
pages = {},
doi = {10.3791/68874},
pmid = {40758593},
issn = {1940-087X},
mesh = {*Nosema/genetics/isolation & purification ; Animals ; Bees/parasitology/microbiology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *DNA, Protozoan/analysis/genetics ; Recombinases/chemistry/metabolism/genetics ; *DNA, Fungal/analysis/genetics ; *Trypanosomatina/genetics/isolation & purification ; },
abstract = {Early on-site detection of microscopic pathogens is essential for the effective management and health of honeybee colonies. Currently, the gold standard for molecular detection of bee pathogens is qPCR or RT-qPCR. Here, we present a rapid, sensitive, and cost-effective alternative for field-deployable DNA pathogen detection. This method combines direct, amplification-ready cell lysis of worker bee abdomens with recombinase polymerase amplification (RPA), CRISPR/Cas12a-mediated trans-cleavage of reporter probes, and detection via lateral flow assays. We successfully validated this protocol in honeybees infected with Nosema ceranae and Lotmaria passim. The described protocol can be performed using a simple heat block or at room temperature and is potentially applicable to any DNA-based pathogen or gut microbiota of the honeybee. The entire process takes approximately 180 min and achieves a sensitivity comparable to qPCR, detecting as few as 96 parasite copies/µL. In conclusion, this approach offers a promising tool for reliable and rapid field diagnosis of honeybee infections without the need for complex laboratory equipment, making it ideal for on-site colony surveillance.},
}

*Nosema/genetics/isolation & purification
Animals
Bees/parasitology/microbiology
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*DNA, Protozoan/analysis/genetics
Recombinases/chemistry/metabolism/genetics
*DNA, Fungal/analysis/genetics
*Trypanosomatina/genetics/isolation & purification

@article {pmid40756515,
year = {2025},
author = {Hendi, NN and Nemer, G},
title = {SDR42E1 modulates vitamin D absorption and cancer pathogenesis: insights from an in vitro model.},
journal = {Frontiers in endocrinology},
volume = {16},
number = {},
pages = {1585859},
pmid = {40756515},
issn = {1664-2392},
mesh = {Humans ; *Vitamin D/metabolism ; HCT116 Cells ; *Neoplasms/metabolism/pathology/genetics ; *Vitamin D Deficiency/genetics/metabolism ; Proteomics ; Cell Proliferation ; CRISPR-Cas Systems ; *Colorectal Neoplasms/metabolism/genetics/pathology ; Signal Transduction ; },
abstract = {INTRODUCTION: Vitamin D is a pleiotropic hormone essential for bone health and overall physiological function. Despite its significance, vitamin D deficiency remains widespread and is often influenced by genetic factors.

METHODS: This study investigates the role of SDR42E1, a gene encoding a short-chain dehydrogenase/reductase enzyme, in vitamin D regulation and sterol metabolism. Using CRISPR/Cas9 gene-editing, we generated an SDR42E1 knock-in model in HCT116 colorectal cells, which exhibit high endogenous SDR42E1 expression, harboring a nonsense variant associated with vitamin D deficiency.

RESULTS: Integrated transcriptomic and proteomic analyses revealed significant dysregulation of sterol absorption and metabolism (fold change (FC) = 1.8, P = 0.007) and cancer-related signaling pathways (FC = -1.7, P = 0.02). Notably, key differentially expressed genes included upregulated LRP1B and ABCC2, alongside downregulated WNT16 and SLC7A5. Proteomic profiling confirmed alterations in cell proliferation-related proteins, including reduced ALDOA expression (FC = -0.37, P = 0.0005). Functionally, SDR42E1 deficiency reduced cell viability by 53% (P = 0.0001), an effect reversed by transient SDR42E1 overexpression with restoring ABCC2 expression.

CONCLUSION: These findings establish SDR42E1 as a key modulator of vitamin D-related pathways and highlight its potential as a therapeutic target for addressing vitamin D deficiency and associated pathologies, including cancer.},
}

Humans
*Vitamin D/metabolism
HCT116 Cells
*Neoplasms/metabolism/pathology/genetics
*Vitamin D Deficiency/genetics/metabolism
Proteomics
Cell Proliferation
CRISPR-Cas Systems
*Colorectal Neoplasms/metabolism/genetics/pathology
Signal Transduction

@article {pmid40755799,
year = {2025},
author = {Kong, H and Yin, Y and Zeng, N and Zhu, Y and Cui, Y},
title = {Rnf32 is not essential for spermatogenesis and male fertility in mice.},
journal = {PeerJ},
volume = {13},
number = {},
pages = {e19794},
pmid = {40755799},
issn = {2167-8359},
mesh = {Animals ; *Spermatogenesis/genetics ; Male ; Mice ; *Fertility/genetics ; Mice, Knockout ; *Ubiquitin-Protein Ligases/genetics/metabolism ; Sperm Motility/genetics ; Testis/metabolism ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Ring finger motifs are found in a variety of proteins with diverse functions, often involved in protein-DNA or protein-protein interactions. The Rnf32-encoded protein contains two such motifs and is predominantly expressed in the testes and ovaries, suggesting that its expression may be regulated by elements within the Rnf32 promoter region. Rnf32 is active during spermatogenesis, mainly in spermatocytes and spermatids, indicating a potential role in sperm development.

METHODS: We established an Rnf32 knockout (Rnf32 [-/-]) mouse model using CRISPR/Cas9 technology. Gene expression was analyzed via reverse transcription quantitative polymerase chain reaction (RT-qPCR). Testicular and epididymal phenotypes were assessed through histological and immunofluorescence staining, and fertility and sperm motility were evaluated.

RESULTS: Here, we successfully established an Rnf32 knockout mouse model using CRISPR/Cas9 technology. Surprisingly, male Rnf32 [-/-] mice exhibited normal fertility, with no significant differences in testicular and epididymal histology, spermatogenesis, sperm count, or motility compared to Rnf32 [+/+] mice. These findings suggest that Rnf32 may not be essential for male fertility in mice, and its potential functions warrant further investigation.},
}

Animals
*Spermatogenesis/genetics
Male
Mice
*Fertility/genetics
Mice, Knockout
*Ubiquitin-Protein Ligases/genetics/metabolism
Sperm Motility/genetics
Testis/metabolism
CRISPR-Cas Systems

@article {pmid40754411,
year = {2025},
author = {Kiga, K},
title = {[The Need for Phage Therapy in Combating Antimicrobial Resistance].},
journal = {Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan},
volume = {145},
number = {8},
pages = {679-688},
doi = {10.1248/yakushi.24-00190-4},
pmid = {40754411},
issn = {1347-5231},
mesh = {*Phage Therapy/trends/methods ; Humans ; *Bacteriophages/genetics ; *Drug Resistance, Bacterial ; Genetic Engineering ; CRISPR-Cas Systems ; RNA, Transfer ; *Bacterial Infections/therapy ; *Drug Resistance, Microbial ; Bacteria/virology ; },
abstract = {The escalating crisis of antimicrobial resistance poses a grave threat to global health and medicine in the 21st century. Phage therapy has emerged as a promising alternative to conventional antibiotics in addressing this urgent issue. Phages, unlike traditional antibiotics, leave the healthy microbiome largely undisturbed by selectively targeting and infecting their bacterial host. Additionally, phages can be readily genetically engineered to enhance their efficacy against specific bacterial strains. While some countries are slowly developing new regulations and implementing phage therapy in the clinic, widespread societal adoption remains limited. Phage therapy has the potential to revolutionize infection treatment; however, the unique biological properties of phages necessitate a multifaceted approach for the societal implementation of phage therapy. Recent research has focused on genetically engineering phages to enhance their capabilities or confer novel functions. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have facilitated the development of phages that target specific genes. Furthermore, the emergence of tRNA-carrying phages and phages that inhibit bacterial defense systems represents new classes of genetically engineered phages with enhanced bactericidal properties.},
}

*Phage Therapy/trends/methods
Humans
*Bacteriophages/genetics
*Drug Resistance, Bacterial
Genetic Engineering
CRISPR-Cas Systems
RNA, Transfer
*Bacterial Infections/therapy
*Drug Resistance, Microbial
Bacteria/virology

@article {pmid40745005,
year = {2025},
author = {Ruis, BL and Bielinsky, AK and Hendrickson, EA},
title = {Gene editing and CRISPR-dependent homology-mediated end joining.},
journal = {Experimental & molecular medicine},
volume = {57},
number = {7},
pages = {1409-1418},
doi = {10.1038/s12276-025-01442-z},
pmid = {40745005},
issn = {2092-6413},
support = {CA266254//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R35GM148181//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {*Gene Editing/methods ; Humans ; *DNA End-Joining Repair ; *CRISPR-Cas Systems ; Animals ; DNA Breaks, Double-Stranded ; },
abstract = {Gene editing is the intentional modification of a genetic locus in a living cell and is used for two general applications of great importance and wide interest. One is the inactivation of genes ('knockouts'), a process utilized to delineate the loss-of-function phenotype(s) of a particular gene. The second application ('knock-ins') is essentially the process of gene therapy, which predominately involves correcting a pre-existing mutated allele(s) of a gene back to wild-type to ameliorate some pathological phenotype associated with the mutation. Importantly, although these applications are conceptually exact reciprocal opposites of one another, they are achieved via mechanistically different pathways. In the case of knockouts, breakage (usually in the form of double-stranded breaks) of the chromosomal DNA at the site of targeting is used to engage a repair process (nonhomologous end joining) that is error prone. The ensuing repair frequently results in insertions/deletions at the cleavage site, which, in turn, results in out-of-frame mutations and, hence, a knockout of the gene in question. In the case of knock-ins, breakage (again, usually in the form of double-stranded breaks) of the DNA is used to engage a repair process (homology-dependent repair/recombination) in which homologous sequences between an incoming donor DNA (containing new genetic information) and the chromosomal DNA are exchanged. Although homology-directed repair was known to predominate in bacteria and lower eukaryotes, the competing process of nonhomologous end joining predominates in higher eukaryotes and was presumed to prevent the use of knock-in gene editing in human somatic cells in culture. A series of molecular and technical advances disproved this notion but still resulted in a process that was cumbersome, labor intensive, highly inefficient and slow. In 2013, however, a new RNA-programmable nuclease, CRISPR-Cas9 was described that has revolutionized the field and made gene editing accessible to anyone with even a rudimentary knowledge of molecular biology. Thus, gene editing in a wide variety of model organisms, as well as human somatic cells in culture, has become not only extremely feasible but also extremely facile, and it harbingers a golden age for directed mutagenesis, directed evolution and improvements in gene therapy.},
}

*Gene Editing/methods
Humans
*DNA End-Joining Repair
*CRISPR-Cas Systems
Animals
DNA Breaks, Double-Stranded

@article {pmid40745003,
year = {2025},
author = {Kwon, J and Park, Y and Ha, T},
title = {Unveiling the invisible genomic dynamics.},
journal = {Experimental & molecular medicine},
volume = {57},
number = {7},
pages = {1400-1408},
doi = {10.1038/s12276-025-01434-z},
pmid = {40745003},
issn = {2092-6413},
mesh = {Humans ; *CRISPR-Cas Systems ; *Genomics/methods ; Chromatin/genetics/metabolism ; Animals ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR-based imaging technologies have emerged as powerful tools for visualizing specific genomic loci, providing groundbreaking insights into chromatin structure and dynamics. Here, in this Review, we discuss the development and recent advances in these techniques, highlighting key strategies such as signal amplification, background reduction, multiplexing and enhanced genomic resolution. By engineering Cas proteins and guide RNAs, and incorporating peptide and aptamer tags, researchers have remarkably improved the sensitivity, specificity and resolution of CRISPR-based imaging, enabling the detection of nonrepetitive genomic regions and single-nucleotide polymorphisms. Recent studies have further pushed the boundaries of CRISPR-based imaging with the introduction of degron-mediated fluorogenic labeling and light-controllable background reduction. Despite remaining challenges, such as the bulkiness of signal amplification systems, limitations in guide RNA design and the effects of fixation on chromatin-protein interactions, CRISPR-based imaging holds great promise for advancing our understanding of chromatin dynamics, genomic interactions and their roles in various biological processes.},
}

Humans
*CRISPR-Cas Systems
*Genomics/methods
Chromatin/genetics/metabolism
Animals
Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40744998,
year = {2025},
author = {Lee, J and Kweon, J and Kim, Y},
title = {Emerging trends in prime editing for precision genome editing.},
journal = {Experimental & molecular medicine},
volume = {57},
number = {7},
pages = {1381-1391},
doi = {10.1038/s12276-025-01463-8},
pmid = {40744998},
issn = {2092-6413},
mesh = {*Gene Editing/methods/trends ; Humans ; CRISPR-Cas Systems ; Animals ; Genome ; },
abstract = {Prime editing is an advanced genome editing technology that enables precise genetic modifications without inducing double-strand breaks or requiring donor DNA templates. Prime editing has rapidly become a versatile tool, supporting a wide range of genetic modifications, including point mutations, insertions and deletions. Here we examine the evolution of prime editing technologies, detailing advancements from the initial prime editing systems to recent innovations that enhance editing efficiency. Through structural modifications and improved delivery methods, prime editing has expanded its applicability across eukaryotic systems. By enabling access to previously challenging mutations, prime editing opens new avenues for therapeutic development and precision genetic research, with efficiency, specificity and accessibility expected to shape its future impact in genome engineering.},
}

*Gene Editing/methods/trends
Humans
CRISPR-Cas Systems
Animals
Genome

@article {pmid40720544,
year = {2025},
author = {Walsh, B and Summers, RL and Rangel, GW and Hagenah, LM and Mok, S and Llinás, M and Fidock, DA and Wirth, DF and Bopp, S},
title = {The plasmepsin-piperaquine paradox persists in Plasmodium falciparum.},
journal = {PLoS pathogens},
volume = {21},
number = {7},
pages = {e1012779},
doi = {10.1371/journal.ppat.1012779},
pmid = {40720544},
issn = {1553-7374},
mesh = {*Plasmodium falciparum/drug effects/genetics/enzymology ; *Aspartic Acid Endopeptidases/genetics/metabolism ; *Antimalarials/pharmacology ; *Quinolines/pharmacology ; *Drug Resistance/genetics ; *Protozoan Proteins/genetics/metabolism ; Humans ; *Malaria, Falciparum/drug therapy/parasitology ; Artemisinins/pharmacology ; CRISPR-Cas Systems ; Piperazines ; },
abstract = {Malaria remains a pressing global health challenge, with rising drug resistance threatening current treatment strategies. Partial resistance to dihydroartemisinin-piperaquine (DHA-PPQ) has emerged in Southeast Asia, particularly in Plasmodium falciparum strains from Cambodia. While artemisinin partial resistance is associated with mutations in kelch13, reduced PPQ sensitivity has been linked to increased copy numbers of the aspartic protease genes plasmepsin II and III and mutations in the chloroquine resistance transporter. In this study, we demonstrate the effective use of CRISPR-Cas9 technology to generate single knockouts (KO) of plasmepsin II and plasmepsin III, as well as a double KO of both genes, in two isogenic Cambodian parasites with varying numbers of plasmepsin gene copies. The deletion of plasmepsin II and/or III increased parasite sensitivity to PPQ. We explored several hypotheses to understand how an increased plasmepsin gene copy number might influence parasite survival under high PPQ pressure. Our findings indicate that protease inhibitors have a minimal impact on parasite susceptibility to PPQ. Additionally, parasites with higher plasmepsin gene copy numbers did not exhibit significantly increased hemoglobin digestion, differences in peptide composition, nor did they produce different amounts of free heme following PPQ treatment compared to wildtype (single copy) parasites. Interestingly, hemoglobin digestion was slowed in parasites with plasmepsin II deletions. We also found that culturing parasites with different plasmepsin II and III copies in amino acid-limited media had little impact on parasite sensitivity to high-dose PPQ. By treating parasites with modulators of digestive vacuole (DV) homeostasis, we found that changes in DV pH potentially affect their response to PPQ. Our research highlights the crucial role of increased plasmepsin II and III gene copy numbers in modulating response to PPQ and begins to uncover the molecular and physiological mechanisms underlying the contribution of plasmepsin II and III amplification to PPQ resistance in Cambodian parasites.},
}

*Plasmodium falciparum/drug effects/genetics/enzymology
*Aspartic Acid Endopeptidases/genetics/metabolism
*Antimalarials/pharmacology
*Quinolines/pharmacology
*Drug Resistance/genetics
*Protozoan Proteins/genetics/metabolism
Humans
*Malaria, Falciparum/drug therapy/parasitology
Artemisinins/pharmacology
CRISPR-Cas Systems
Piperazines

@article {pmid40640612,
year = {2025},
author = {Yu, H and Qian, W and Song, Y and Welch, JD},
title = {PerturbNet predicts single-cell responses to unseen chemical and genetic perturbations.},
journal = {Molecular systems biology},
volume = {21},
number = {8},
pages = {960-982},
pmid = {40640612},
issn = {1744-4292},
support = {R01HG010883//HHS | NIH | National Human Genome Research Institute (NHGRI)/ ; U01HG011952//HHS | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {*Single-Cell Analysis/methods ; Humans ; GATA1 Transcription Factor/genetics/chemistry/metabolism ; CRISPR-Cas Systems ; *Computational Biology/methods ; Gene Expression Regulation/drug effects ; Mutation, Missense ; },
abstract = {Chemical and genetic perturbations, such as those induced by small molecules and CRISPR, effect complex changes in the molecular states of cells. Despite advances in high-throughput single-cell perturbation screening technology, the space of possible perturbations is far too large to measure exhaustively. Here, we introduce PerturbNet, a flexible deep generative model designed to predict the distribution of cell states induced by unseen chemical or genetic perturbations. PerturbNet accurately predicts gene expression changes in response to unseen small molecules based on their chemical structures while also accounting for key covariates such as dosage and cell type. Moreover, PerturbNet accurately predicts the distribution of single-cell gene expression states following CRISPR activation or CRISPR interference by leveraging gene functional annotations. Our approach significantly outperforms previous methods, particularly for predicting the effects of perturbing completely unseen genes. Finally, we demonstrate for the first time that amino acid sequence embeddings can be used to predict gene expression changes induced by missense mutations. We use PerturbNet to predict the effects of all point mutations in GATA1 and nominate variants that significantly impact the cell state distribution of human hematopoietic stem cells. Using a crystal structure of GATA1 bound to DNA, we validate that these large-effect variants occur in the core DNA-contact region of GATA1 and tend to involve large changes in amino acid side-chain volume.},
}

*Single-Cell Analysis/methods
Humans
GATA1 Transcription Factor/genetics/chemistry/metabolism
CRISPR-Cas Systems
*Computational Biology/methods
Gene Expression Regulation/drug effects
Mutation, Missense

@article {pmid40633258,
year = {2025},
author = {Liberty, JT and Lin, H and Sipos, Y and Ihedioha, OC and Kwaji, MJ},
title = {Synergizing gene editing and cellular agriculture for a sustainable and healthy food future.},
journal = {Current opinion in biotechnology},
volume = {94},
number = {},
pages = {103334},
doi = {10.1016/j.copbio.2025.103334},
pmid = {40633258},
issn = {1879-0429},
mesh = {*Gene Editing/methods ; Humans ; *Agriculture/methods ; CRISPR-Cas Systems ; Food Supply ; Food Security ; Animals ; },
abstract = {The intersection of gene editing and cellular agriculture is transforming food production by offering sustainable, ethical alternatives to conventional agriculture. Cellular agriculture uses tissue engineering and fermentation technologies to produce animal-free food, whereas gene editing tools like CRISPR-Cas9 optimize cellular efficiency, nutritional value, and sustainability. While some researchers emphasize the environmental and food security benefits, others raise concerns about high costs, regulatory challenges, and consumer acceptance. This paper critically examines existing literature, compares breakthroughs and controversies, and provides an expert perspective on the challenges and opportunities in gene-edited cellular agriculture. By tackling key scientific, economic, regulatory, and ethical issues, this article presents a roadmap for responsibly advancing these technologies and integrating them into global food systems. To our knowledge, this is the first work to explore how gene editing and cellular agriculture can be synergized to advance sustainability, food security, and global health.},
}

*Gene Editing/methods
Humans
*Agriculture/methods
CRISPR-Cas Systems
Food Supply
Food Security
Animals

@article {pmid40616517,
year = {2025},
author = {Spataru, I and Mahoro, F and Badea, I},
title = {Development of Inorganic and Hybrid Nanosystems for Delivery of CRISPR-Based Gene Editors.},
journal = {Molecular pharmaceutics},
volume = {22},
number = {8},
pages = {4373-4393},
doi = {10.1021/acs.molpharmaceut.5c00251},
pmid = {40616517},
issn = {1543-8392},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Nanoparticles/chemistry ; Animals ; *Drug Delivery Systems/methods ; Liposomes/chemistry ; Lipids/chemistry ; Gene Transfer Techniques ; Transfection/methods ; },
abstract = {The CRISPR-Cas9 system is a gene editing tool, replacing specific target sequences of eukaryotic DNA via a distinct molecular pathway, with minimal off-target effects. In this manner, the effects are long-lasting and potentially require only one dose of medication to be effective. The package of bacterial nucleases is susceptible to the same degradative pathways as other nucleic acid therapeutics. Similarly, the CRISPR-Cas9 system is incapable of traversing biological membranes on its own. Therefore, a delivery mechanism is needed for effective transfection. In recent literature reviews, the focus has been on viral and lipid-based drug delivery systems, with little attention paid to solid-core nanoparticles, such as gold or silica nanoparticles, which present unique physicochemical properties and delivery opportunities. While viral delivery systems are efficient carriers, they can be highly immunogenic and unstable, and the production of high-viral titers is limited. Lipid-based drug delivery systems, such as liposomes, possess good shelf life, encapsulation, and transfection efficiency, but their biological stability and biodistribution profile limit their in vivo use. Thus, the arsenal of delivery agents could be complemented by solid-core nanoparticles. Their unique structural properties could lead to improved delivery strategies to mitigate disease outcomes and promote organ-specific delivery.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Nanoparticles/chemistry
Animals
*Drug Delivery Systems/methods
Liposomes/chemistry
Lipids/chemistry
Gene Transfer Techniques
Transfection/methods

@article {pmid40613419,
year = {2025},
author = {Rochussen, AM and Ma, CY and Griffiths, GM},
title = {Transcriptional adaptation after deletion of Cdc42 in primary T cells.},
journal = {Journal of cell science},
volume = {138},
number = {15},
pages = {},
doi = {10.1242/jcs.263826},
pmid = {40613419},
issn = {1477-9137},
support = {217100/WT_/Wellcome Trust/United Kingdom ; 220543/WT_/Wellcome Trust/United Kingdom ; 102163/WT_/Wellcome Trust/United Kingdom ; //University of Cambridge/ ; },
mesh = {*cdc42 GTP-Binding Protein/genetics/metabolism ; Animals ; *Transcription, Genetic ; *T-Lymphocytes, Cytotoxic/metabolism/immunology ; Mice ; *Gene Deletion ; CRISPR-Cas Systems/genetics ; *T-Lymphocytes/metabolism ; Mice, Inbred C57BL ; },
abstract = {Cdc42 is a Rho family GTPase known for its central role in cell polarity and cytoskeletal regulation. To understand the role of Cdc42 in polarised secretion from cytotoxic T lymphocytes (CTLs) we used CRISPR/Cas9 gene deletion. Although Cdc42-deleted CTLs initially showed reduced cytotoxicity for up to 2 days after CRISPR-mediated deletion, full secretion and cytotoxicity was rapidly restored and even enhanced while CDC42 protein remained absent. In contrast, chemical inhibition of CDC42 using CASIN consistently decreased secretion in wild-type cells, but had no impact on Cdc42-deleted CTLs, confirming the specificity of this inhibitor. Comparative proteomics and transcriptomics of CTLs after Cdc42 deletion revealed transcriptional changes that could support improved T cell function, including compensation via other Rho GTPases. Targeting the promoter region of Cdc42 did not trigger transcriptional adaptation, consistent with a nonsense-mediated decay mechanism of genetic compensation. Our work highlights the importance of taking orthogonal approaches to study protein function and reveals the remarkable robustness of primary T cells to adapt to loss of an essential gene.},
}

*cdc42 GTP-Binding Protein/genetics/metabolism
Animals
*Transcription, Genetic
*T-Lymphocytes, Cytotoxic/metabolism/immunology
Mice
*Gene Deletion
CRISPR-Cas Systems/genetics
*T-Lymphocytes/metabolism
Mice, Inbred C57BL

@article {pmid40371725,
year = {2025},
author = {Kalniņa, Z and Liekniņa, I and Koteloviča, S and Petrovska, R and Žvinys, G and Petrosiute, A and Zubrienė, A and Laugalis, MT and Skeltona, V and Jansons, J and Kreishmane, M and Čapkauskaitė, E and Matulis, D and Tārs, K},
title = {Development of 4T1 breast cancer mouse model system for preclinical carbonic anhydrase IX studies.},
journal = {FEBS open bio},
volume = {15},
number = {8},
pages = {1285-1302},
doi = {10.1002/2211-5463.70052},
pmid = {40371725},
issn = {2211-5463},
support = {1.1.1.2/VIAA/4/20/728//European Regional Development Fund (ERDF)/ ; },
mesh = {Animals ; *Carbonic Anhydrase IX/metabolism/genetics ; Mice ; Female ; Cell Line, Tumor ; Mice, Inbred BALB C ; Disease Models, Animal ; *Triple Negative Breast Neoplasms/pathology/metabolism/genetics ; Humans ; CRISPR-Cas Systems ; },
abstract = {Triple-negative breast cancer (TNBC) is the most aggressive type of breast cancer, for which targeted treatment is currently lacking. Carbonic anhydrase IX (CAIX) is a known cancer target due to its selective overexpression in hypoxia, a hallmark of many solid cancers including TNBC. This study aimed to develop a robust murine TNBC cell line 4T1-based model system that could be used in the comprehensive preclinical evaluation of targeting CAIX. The model is based on the original 4T1 breast cancer cell line and two genetically edited versions of it-one with biallelic CRISPR/Cas9-mediated Car9 inactivation and another with constitutively expressed Car9, thus ensuring negative and positive controls for CAIX production in the model system, respectively. The generated cell lines were validated for CAIX production and characterised functionally in vitro and in vivo after orthotopic implantation in syngeneic BALB/c mice. Results demonstrated significantly reduced primary tumour growth and metastatic progression rates in animals with CAIX-deficient tumours, while the CAIX-expressing tumours had vascularised phenotypes with prominent central areas of coagulative necrosis. The differential CAIX expression levels in the model were preserved during tumour growth in syngeneic mice, as verified by in vivo imaging using a novel high-affinity CAIX-specific near-infrared (NIR) fluorescent imaging probe, GZ22-4. Constitutive overexpression of autologous CAIX did not elicit specific autoantibody responses in vivo, demonstrating the suitability of this model for evaluating the efficacy of anti-CAIX vaccination as a therapeutic strategy. The in vivo study was repeated as an independent experiment and demonstrated good robustness of the developed model.},
}

Animals
*Carbonic Anhydrase IX/metabolism/genetics
Mice
Female
Cell Line, Tumor
Mice, Inbred BALB C
Disease Models, Animal
*Triple Negative Breast Neoplasms/pathology/metabolism/genetics
Humans
CRISPR-Cas Systems

@article {pmid40367935,
year = {2025},
author = {Egashira, S and Maehara, K and Tanaka, K and Nakamura, M and Takemoto, T and Ohkawa, Y and Harada, A},
title = {Histone H2B isoform H2bc27 is expressed in the developing brain of mouse embryos.},
journal = {Journal of biochemistry},
volume = {178},
number = {2},
pages = {109-119},
doi = {10.1093/jb/mvaf026},
pmid = {40367935},
issn = {1756-2651},
support = {//Autonomous Medical Research Center in Kyushu University/ ; //CURE:JPMXP1323015486/ ; //MRCI for High Depth Omics/ ; //MEXT Promotion of Development of a Joint Usage/Research System Project:Pan-Omics DDRIC/ ; JP18H05528, JP24H02323, JP23H00372, JP22H04676 and JP22K19275//JSPS KAKENHI/ ; JP22H04696 and JP23H04288//JSPS KAKENHI/ ; JP21H05292, JP23K27087 and JP23H02394//JSPS KAKENHI/ ; JP23gm1810008//AMED-CREST/ ; JP22ama121017j0001//AMED BINDS/ ; JPMJPR2026//JST PRESTO/ ; JPMJCR23N3//JST CREST/ ; },
mesh = {Animals ; Mice ; *Histones/genetics/metabolism ; *Brain/metabolism/embryology ; Protein Isoforms/genetics/metabolism ; Mice, Knockout ; *Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; *Embryo, Mammalian/metabolism ; Mice, Inbred C57BL ; },
abstract = {Histones bind directly to DNA and play a role in regulating gene expression in part by influencing chromatin structure. The DNA sequences of these histone genes are quite similar, which has hindered individual analyses. The exact function of the 13 different isoforms of histone H2B remains unclear. In this study, we performed a comprehensive gene expression analysis of the H2B isoforms, focusing on tissue specificity. Our results revealed that the H2bc27 gene exhibited brain-specific expression in mice at E14.5. We generated mice lacking the H2bc27 gene using the CRISPR/Cas9 system. While the phenotype of H2bc27 knockout mouse brains was not different from that of wild-type mouse brains, transcriptome analysis indicated that H2bc27 is associated with regulating the expression of several functional genes involved in mouse brain development. The methods used in this study may serve to facilitate comprehensive H2B isoform analysis.},
}

Animals
Mice
*Histones/genetics/metabolism
*Brain/metabolism/embryology
Protein Isoforms/genetics/metabolism
Mice, Knockout
*Gene Expression Regulation, Developmental
CRISPR-Cas Systems
*Embryo, Mammalian/metabolism
Mice, Inbred C57BL

@article {pmid40756867,
year = {2025},
author = {Samson, C and du Rand, A and Hunt, J and Whitford, W and Jacobsen, J and Sheppard, H},
title = {A bioinformatic analysis of gene editing off-target loci altered by common polymorphisms, using 'PopOff'.},
journal = {Journal of the Royal Society of New Zealand},
volume = {55},
number = {6},
pages = {2440-2463},
pmid = {40756867},
issn = {1175-8899},
abstract = {Gene editing therapies are designed to minimise off-target editing. However, it is not widespread practice for common polymorphisms to be considered when identifying potential off-target sites in silico. Nevertheless, genetic variants should be included as they have the potential to alter existing, or to generate new, off-target sites. To facilitate the consideration of common polymorphisms when designing targeted gene therapies we developed PopOff, a web-based tool that integrates minor allele frequencies from the gnomAD variant database into an off-target analysis. We used PopOff to analyse predicted off-target loci from guide RNAs used in four clinical trials and thirty-four research publications. From an analysis of sixty guides, we identified that approximately 20% of off-target loci overlap with a common polymorphism. Of these sites, 6.93% contained variants that reduce the level of mismatch between the off-target locus and guide, and therefore may increase off-target cleavage. In addition, we identified that 0.34% of common polymorphisms generated novel PAM sites, resulting in off-target loci that standard workflows would miss. Our findings demonstrate that common polymorphisms should be considered when designing guides to maximise the safety of CRISPR-based gene therapies. However, this may be problematic in populations where the breadth of genetic diversity remains uncharacterised.},
}

@article {pmid40753163,
year = {2025},
author = {Wolkoff, M and Yoshida, M and Lowmiller, T and Sim, C and Meuti, ME},
title = {The cycle gene is essential for both daily responses and seasonal reproduction in the Northern house mosquito, Culex pipiens.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {28279},
doi = {10.1038/s41598-025-06637-y},
pmid = {40753163},
issn = {2045-2322},
support = {IOS-1944323//National Science Foundation/ ; 2023-67013-39915//U.S. Department of Agriculture/ ; R01AI188567/NH/NIH HHS/United States ; },
mesh = {Animals ; *Culex/genetics/physiology ; Reproduction/genetics ; Seasons ; Female ; Photoperiod ; *Circadian Rhythm/genetics ; Circadian Clocks/genetics ; Male ; *Insect Proteins/genetics/metabolism ; Diapause, Insect/genetics ; },
abstract = {The insect circadian clock synchronizes daily activities, such as feeding, eclosion, and mating to diurnal light:dark cycles, while the photoperiodic clock synchronizes development and reproduction to the appropriate season. Despite mounting evidence that the circadian and photoperiodic timekeeping systems are linked, it is unclear whether the circadian clock as a whole or individual genes within the circadian clock are responsible for measuring daylength, releasing hormones and/or inducing diapause phenotypes. To interrogate the role of a core circadian transcription factor, cycle, in mediating both seasonal and daily behaviors we used CRISPR/Cas-9 genome editing to introduce a deletion mutation into cycle in the Northern house mosquito, Culex pipiens. Females homozygous for this deletion exhibited high rates of reproductive arrest characteristic of an overwintering diapause even when reared in summer-like conditions, while a minority remained reproductive even when reared in winter-like conditions. Nearly all (79%) homozygotes lacked robust behavioral rhythms in constant darkness, while 52% of heterozygotes carrying one wild-type copy of the cycle gene lacked robust rhythmicity in constant darkness. Heterozygotes and homozygotes were more active in constant darkness than WT mosquitoes, but this difference was abolished under light:dark conditions. Lastly, the daily transcription profiles of various circadian genes, especially timeless and vrille, were disrupted in homozygous females even when they were reared under long and short days with light:dark cycles. These results demonstrate that cycle is essential for coordinating both daily activity and transcript abundance and seasonal diapause in Cx. pipiens.},
}

Animals
*Culex/genetics/physiology
Reproduction/genetics
Seasons
Female
Photoperiod
*Circadian Rhythm/genetics
Circadian Clocks/genetics
Male
*Insect Proteins/genetics/metabolism
Diapause, Insect/genetics

@article {pmid40460689,
year = {2025},
author = {Liu, T and Wang, Y and Liao, Z and Li, X and Tang, S and Zhang, Z and Chu, M and Wei, L},
title = {RPA-CRISPR/Cas12a detection based on HCMV-UL123 gene: A way with higher detection rate than clinical detection methods.},
journal = {Journal of infection and public health},
volume = {18},
number = {9},
pages = {102845},
doi = {10.1016/j.jiph.2025.102845},
pmid = {40460689},
issn = {1876-035X},
mesh = {Humans ; *Cytomegalovirus/genetics/isolation & purification ; Sensitivity and Specificity ; *Cytomegalovirus Infections/diagnosis/virology ; *CRISPR-Cas Systems ; DNA Primers/genetics ; *Molecular Diagnostic Techniques/methods ; *Viral Proteins/genetics ; DNA, Viral/genetics ; },
abstract = {OBJECTIVE: Human cytomegalovirus (HCMV), a prevalent double-stranded DNA enveloped virus, poses a threat to immunocompromised individuals. The current clinical detection methods are insufficient in sensitivity, highlighting the need for more effective approaches.

METHODS: We designed and screened RPA primers and crRNA based on the UL123 gene of HCMV. Evaluate the HCMV-RPA-CRISPR detection method using cloned plasmids and the whole-genome samples of HCMV-infected cells. Conduct RPA-CRISPR/Cas12a reactions with 48 clinical samples and compare the results with those of PCR-Fluorescent Probe Method in clinical applications and the qPCR method for detecting the UL123 gene.

RESULTS: The optimized RPA-CRISPR system exhibited high sensitivity and specificity for HCMV detection. The positive rate of clinical sample detection was approximately 20.5 % (6/48) higher than that of the clinical detection method.

CONCLUSION: Currently, the sensitivity and early detection of HCMV in clinical settings are still limited. The UL123 gene of HCMV is characterized by high transcription in the early stage and high conservation. The RPA-CRISPR/Cas12a technology exhibits high sensitivity in detecting the HCMV UL123 gene, and it is expected to provide a more effective method for the early specific detection of HCMV infection.},
}

Humans
*Cytomegalovirus/genetics/isolation & purification
Sensitivity and Specificity
*Cytomegalovirus Infections/diagnosis/virology
*CRISPR-Cas Systems
DNA Primers/genetics
*Molecular Diagnostic Techniques/methods
*Viral Proteins/genetics
DNA, Viral/genetics

@article {pmid40752531,
year = {2025},
author = {Liu, Q and Zhuo, R and He, W and Li, C},
title = {The new SCCmec type methicillin-resistant Staphylococcus aureus carried CRISPR-Cas system isolated from a pig in China.},
journal = {Microbial pathogenesis},
volume = {},
number = {},
pages = {107943},
doi = {10.1016/j.micpath.2025.107943},
pmid = {40752531},
issn = {1096-1208},
abstract = {Methicillin-resistant Staphylococcus aureus (MRSA) lineages circulate globally in healthcare, community, and livestock-associated (LA) settings. Nine MRSA isolates were recovered from swine in China, all exhibiting resistance to ampicillin and multidrug resistance phenotypes. Among eight ST9-t899 isolates, SCCmec type XII(9C2) predominated. However, we identified a novel staphylococcal cassette chromosome mec (SCCmec) type, designated XIII (9A), in an LA-MRSA strain (LS45). Structural analysis revealed SCCmec XIII(9A) comprises a CRISPR-Cas system (cas10-csm2-csm3-csm4-csm5-csm6). Functional analysis demonstrated this CRISPR-Cas system provided partial protection against phage infection at low multiplicities of infection (MOIs ≤10[-7]), but conferred no detectable immunity against spacer-matched plasmids, with no significant change in cas10 expression during plasmid challenge. The co-location of this novel SCCmec element and a functional CRISPR-Cas system within an LA-MRSA strain demonstrates that S. aureus can maintain a defense system active against phages while accommodating SCCmec-mediated horizontal gene transfer. These findings provide new insights into the genomic adaptations of MRSA across different hosts.},
}

@article {pmid40752020,
year = {2025},
author = {Camborde, L and Jaturapaktrarak, C and Gouzy, J and Lopez-Roques, C and Krajaejun, T and Gaulin, E and Badis, Y},
title = {Adenine Phosphoribosyltransferase Is a Universal Counter-Selectable Marker for DNA-Free Genome Editing in Oomycetes.},
journal = {Molecular plant-microbe interactions : MPMI},
volume = {},
number = {},
pages = {},
doi = {10.1094/MPMI-05-25-0063-TA},
pmid = {40752020},
issn = {0894-0282},
abstract = {CRISPR-Cas genome editing is a powerful tool for understanding the pathogenicity of oomycetes, a group that includes several destructive plant parasites. While few Phytophthora species have benefited from plasmid-based transformation methods for gene overexpression and RNAi silencing, these techniques remain inefficient for other oomycete genera such as Pythium and Aphanomyces. Here, we explored the applicability of DNA-free endogenous counter-selection in filamentous oomycetes, using CRISPR-Cas9 ribonucleoproteins (RNPs). We used biolistics to deliver RNPs targeting the Adenine phosphoribosyltransferase (APT) gene, and generated selectable 2-fluoroadenine-resistant mutants in Aphanomyces, Pythium, and Phytophthora species. Targeted mutagenesis resulted in various deletions at the expected cut-sites, confirming efficient genome editing. Knockout mutants exhibited no alterations in growth or virulence, making APT a suitable selectable marker gene for oomycete research. Whole genome comparative analyses on CRISPR-edited mutants revealed no or very few additional mutations in A. euteiches and P. oligandrum, and substantial off-target effects in P. capsici. This one-step approach circumvents the need for protoplast generation and can be broadly applied to oomycetes producing zoospores or oospores.},
}

@article {pmid40750796,
year = {2025},
author = {Li, Z and Ee, A and Amaya, L and Hamad, JL and Yadav, PK and Wang, SK and Chang, HY and Wender, PA},
title = {Discrete Immolative Guanidinium Transporters deliver mRNA to specific organs and red blood cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7055},
doi = {10.1038/s41467-025-62200-3},
pmid = {40750796},
issn = {2041-1723},
support = {R01CA245533//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Animals ; *RNA, Messenger/metabolism/administration & dosage/chemistry/genetics ; *Erythrocytes/metabolism ; Mice ; Female ; *Guanidine/chemistry/metabolism ; Humans ; Lung/metabolism ; *Drug Delivery Systems/methods ; Spleen/metabolism ; Cell Survival ; },
abstract = {RNA medicine is an emerging groundbreaking technology for the prevention and treatment of disease. However, tools to deliver messenger RNA (mRNA) and other polyanions (circRNA, saRNA, pDNA, CRISPR-Cas, reprogramming factors) are required to advance current RNA therapies and address next generation challenges. Existing delivery systems often suffer from laborious syntheses, limited organ selectivity, formulation complexity, and undesired inflammatory responses. Here, we report novel mRNA delivery systems termed Discrete Immolative Guanidinium Transporters (DIGITs), which are synthesized convergently in as few as 4 steps. Unlike most cationic (ammonium) delivery systems, DIGITs are based on cationic guanidinium moieties, which complex mRNA at acidic pH and undergo irreversible neutralization at physiological pH to enable efficient RNA release. Systematic evaluation of structural variations and formulations have led to DIGIT/mRNA complexes that selectively target lung, spleen, and immature red blood cells in peripheral blood in female mice model. DIGIT/mRNA delivery systems show minimal toxicity based on cell viability and biochemical assays, supporting their future utility in biomedical applications.},
}

Animals
*RNA, Messenger/metabolism/administration & dosage/chemistry/genetics
*Erythrocytes/metabolism
Mice
Female
*Guanidine/chemistry/metabolism
Humans
Lung/metabolism
*Drug Delivery Systems/methods
Spleen/metabolism
Cell Survival

@article {pmid40750194,
year = {2025},
author = {Li, T and Chen, S and Chen, X and Yang, S and Huang, H and Xu, J and Liu, Y and Zhang, J and Cao, L and Kang, Z and Li, M and Wang, H},
title = {CRISPR-based sensing platform for the Group B streptococcus screening in pregnant women.},
journal = {Analytica chimica acta},
volume = {1370},
number = {},
pages = {344390},
doi = {10.1016/j.aca.2025.344390},
pmid = {40750194},
issn = {1873-4324},
mesh = {Female ; *Streptococcus agalactiae/isolation & purification/genetics ; Pregnancy ; Humans ; *Streptococcal Infections/diagnosis ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Nucleic Acid Amplification Techniques ; *Prenatal Diagnosis ; },
abstract = {BACKGROUND: Group B Streptococcus (GBS) is a major cause of perinatal infections. Prenatal screening is critical to prevent maternal and neonatal GBS infections, reduce adverse outcomes, and guide clinical interventions. While bacterial culture is the gold standard, it is time-consuming and delays decision-making. Rapid molecular tests like PCR are sensitive and specific but require expensive equipment and skilled personnel. Point-of-care tests (e.g., Xpert GBS LB) offer speed and sensitivity but remain costly and underutilized. This study aims to develop a portable CRISPR/Cas13a-based device for rapid, on-site GBS detection.

RESULTS: A palm-sized CRISPR detection platform, PalmCS, was developed. PalmCS includes two key components: an integrated tube for nucleic acid extraction, gene amplification, and CRISPR-based reaction, and a multifunctional device for thermal regulation, fluorescence detection, and automatic result interpretation. The sealed plug needle valve controls fluid flow for sample input and result output. By optimizing crRNA selection, Cas13a/crRNA ribonucleoprotein (RNP) complex concentrations, and isothermal recombinase polymerase amplification primers, we established a one-pot CRISPR/Cas13a-RPA method for GBS detection. Results showed that 3 M guanidinium salt combined with 5 % Tween 20 achieved the highest extraction efficiency. The system extracted nucleic acids from samples in 5 min at room temperature, demonstrating its potential for rapid diagnostics. PalmCS was validated using 40 clinical samples, achieving a limit of detection (LOD) of 20 copies/reaction, 97.5 % sensitivity, and 100 % specificity.

SIGNIFICANCE AND NOVELTY: This study used a one-step nucleic acid extraction combined with a closed one-pot RPA-CRISPR reaction for rapid, sensitive, and specific GBS detection. This provides a new platform for prenatal screening in pregnant women. In resource-limited settings with emergency deliveries, PalmCS enables on-site GBS testing, allowing antibiotic administration during delivery for positive cases to significantly reduce neonatal infection risk.},
}

Female
*Streptococcus agalactiae/isolation & purification/genetics
Pregnancy
Humans
*Streptococcal Infections/diagnosis
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Nucleic Acid Amplification Techniques
*Prenatal Diagnosis

@article {pmid40750193,
year = {2025},
author = {Zhu, C and Li, J and Yu, H and Xu, K and He, X and Liu, Z and Chen, J},
title = {CRISPR/Cas13a combined with reverse transcription-multienzyme isothermal rapid amplification for hepatitis B virus RNA detection.},
journal = {Analytica chimica acta},
volume = {1370},
number = {},
pages = {344389},
doi = {10.1016/j.aca.2025.344389},
pmid = {40750193},
issn = {1873-4324},
mesh = {*Hepatitis B virus/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; *RNA, Viral/genetics/analysis/blood ; Humans ; *CRISPR-Cas Systems/genetics ; Reverse Transcription ; Limit of Detection ; Molecular Diagnostic Techniques ; },
abstract = {BACKGROUND: Hepatitis B virus (HBV) infection represents a major global public health challenge. The covalently closed circular DNA (cccDNA), which serves as the key reservoir for viral persistence, currently requires invasive liver biopsy for clinical monitoring. Recent studies have identified serum HBV pregenomic RNA (pgRNA) and its splicing variants emerging as potential noninvasive and reliable biomarkers for tracking disease progression and forecasting prognosis in chronic HBV infection. Precise detection of pgRNA is therefore essential for informed clinical decision-making and optimized patient management.

RESULTS: In this study, we developed a rapid, accurate, and clinically applicable method for HBV RNA detection by integrating CRISPR/Cas13a with reverse transcription-multienzyme isothermal rapid amplification (RT-MIRA). This innovative platform enabled simultaneous amplification of two pgRNA targets within a single-tube RT-MIRA reaction, allowing dual-target detection via one-step amplification. The optimized system achieved efficient isothermal amplification with a detection sensitivity of 10[3] copies/mL for both total and spliced pgRNA and exhibited no cross-reactivity with other common clinical viruses. Validation using both RT-qPCR and the RT-MIRA-Cas13a assay on clinical samples from 48 HBV-infected patients demonstrated a positive prediction value of 97.2 % and a negative predictive value of 100 %. These results conclusively validate the assay's reliability for clinical application in detecting and quantifying both total pgRNA and its splicing variants.

SIGNIFICANCE: This study presents a rapid and user-friendly HBV RNA detection platform capable of accurately identifying both total and spliced pgRNA. The RT-MIRA-Cas13a assay demonstrates excellent clinical potential for prognosis assessment and disease monitoring in HBV infection. Its robust performance and operational simplicity suggest strong suitability for point-of-care applications, potentially transforming chronic HBV monitoring through accessible nucleic acid testing.},
}

*Hepatitis B virus/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
*RNA, Viral/genetics/analysis/blood
Humans
*CRISPR-Cas Systems/genetics
Reverse Transcription
Limit of Detection
Molecular Diagnostic Techniques

@article {pmid40748632,
year = {2025},
author = {Zhao, Z and Li, Y and Xiu, L and Wang, F and Peng, J},
title = {Development of a CRISPR/Cas-Based Detection Platform for Tracking Decreased Susceptibility to Cephalosporins in Neisseria gonorrheae.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c01400},
pmid = {40748632},
issn = {1520-6882},
abstract = {Gonorrhea has become an escalating public health issue due to the rapid emergence of antimicrobial resistance (AMR). Developing efficient and accurate detection of resistant strains is urgently needed for their management and treatment. We have developed the Multiplex Integrated RPA-CRISPR/Cas12a detection Assay (MIRCA) for simultaneous detection of Neisseria gonorrheae (Ng) and mutations with decreased susceptibility to cephalosporins. MIRCA enables multiplex detection of Ng and single-nucleotide polymorphisms in resistance-associated genes within 40 min, with high specificity and sensitivity (10-20 copies/reaction). Clinical evaluation showed 100% concordance with qPCR for Ng identification and Sanger sequencing for FC428 strain tracking. For predicting decreased-susceptibility strains with A501 mutations, MIRCA achieved 98.33% agreement with Sanger sequencing. Simulated tests demonstrated 100% consistency between MIRCA results in centrifuge tubes and microfluidic chips. This robust and cost-effective approach addresses current challenges in AMR surveillance. Its integration with microfluidic chip offers an affordable and user-friendly diagnostic solution, making it highly valuable for timely infectious disease diagnosis and resistance monitoring. It also holds significant potential for point-of-care testing in resource-limited areas.},
}

@article {pmid40516634,
year = {2025},
author = {Wu, Y and Wang, Y and Wang, Y and Qiu, H and Yuan, X and Xiong, H and Zou, Y},
title = {Exploring synthetic lethality in cancer therapy: CRISPR-Cas9 technology offers new hope.},
journal = {Biochimica et biophysica acta. Reviews on cancer},
volume = {1880},
number = {4},
pages = {189370},
doi = {10.1016/j.bbcan.2025.189370},
pmid = {40516634},
issn = {1879-2561},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/therapy/drug therapy ; *Synthetic Lethal Mutations ; *Gene Editing/methods ; Drug Resistance, Neoplasm/genetics ; *Genetic Therapy/methods ; Animals ; },
abstract = {Synthetic lethality (SL) is a breakthrough concept in cancer therapy that describes a scenario in which the simultaneous inactivation of two genes leads to cell death, whereas inactivation of either gene alone does not. The rise of clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated nuclease 9 (Cas9) technology has provided a new tool for exploring this phenomenon, enabling genome editing and screening. This review evaluates the advancements achieved by CRISPR technology in identifying novel therapeutic targets and comprehending the processes of drug resistance using the concept of SL in cancer cells. This review explores the fundamental concept of SL and its application in cancer therapy, highlighting how the CRISPR-Cas9 system functions and how CRISPR-based screening can be leveraged to identify synthetic lethal genes and investigate the mechanisms of drug resistance. We summarize important research in related fields from recent years, demonstrating the role of CRISPR screening in revealing cancer cellular pathways and identifying new drug targets. We also summarize the clinical trials of related drugs currently underway, and anticipate that with the continuous development of CRISPR technology, its integration with cancer genetics and immuno-oncology will bring new hope to patients with drug-resistant cancers.},
}

Humans
*CRISPR-Cas Systems/genetics
*Neoplasms/genetics/therapy/drug therapy
*Synthetic Lethal Mutations
*Gene Editing/methods
Drug Resistance, Neoplasm/genetics
*Genetic Therapy/methods
Animals

@article {pmid40425316,
year = {2025},
author = {Rao, Z and Zhang, M and Huang, S and Wu, C and Zhou, Y and Zhang, W and Lin, X and Zhao, D},
title = {Cancer driver topologically associated domains identify oncogenic and tumor-suppressive lncRNAs.},
journal = {Genome research},
volume = {35},
number = {8},
pages = {1842-1858},
pmid = {40425316},
issn = {1549-5469},
mesh = {*RNA, Long Noncoding/genetics/metabolism ; Humans ; *Neoplasms/genetics ; Gene Expression Regulation, Neoplastic ; *Oncogenes ; *Genes, Tumor Suppressor ; Computational Biology/methods ; CRISPR-Cas Systems ; },
abstract = {Cancer long noncoding RNAs (lncRNAs) have been identified by experimental and in silico methods. However, current approaches for identifying cancer lncRNAs are not sufficient and effective. To uncover them, we focus on the core cancer driver lncRNAs, which directly interact with cancer driver protein-coding genes (PCGs). We investigate various aspects of cancer lncRNAs, including their expression patterns, genomic locations, and direct interactions with cancer driver PCGs, and developed a pipeline to identify candidate cancer driver lncRNAs. Finally, we validate the reliability of potential cancer driver lncRNAs through functional analysis of bioinformatics data and CRISPR-Cas9 knockout experiments. We find that cancer lncRNAs are more concentrated in cancer driver topologically associated domains (CDTs), and CDT is an important feature in identifying cancer lncRNAs. Moreover, cancer lncRNAs show a high tendency to be coexpressed with and bind to cancer driver PCGs. Utilizing these distinctive characteristics, we develop a pipeline CAncer Driver Topologically Associated Domains (CADTAD) to identify candidate cancer driver lncRNAs in pan-cancer, including 256 oncogenic lncRNAs, 177 tumor-suppressive lncRNAs, and 75 dual-function lncRNAs, as well as in three individual cancer types, and validate their cancer-related functions. More importantly, the function of 10 putative cancer driver lncRNAs in prostate cancer is subsequently validated to influence cancer phenotype through cell studies. In light of these findings, our study offers a new perspective from the 3D genome to study the roles of lncRNAs in cancer. Furthermore, we provide a valuable set of potential lncRNAs that could deepen our understanding of the oncogenic mechanism of cancer driver lncRNAs.},
}

*RNA, Long Noncoding/genetics/metabolism
Humans
*Neoplasms/genetics
Gene Expression Regulation, Neoplastic
*Oncogenes
*Genes, Tumor Suppressor
Computational Biology/methods
CRISPR-Cas Systems

@article {pmid40745870,
year = {2025},
author = {Sui, Z and Chen, B and Zhao, J and Yang, H and Guo, L and Xu, J},
title = {Self-Sustaining miRNA Amplification Diagnostics via Catalytic Hairpin Assembly and Split Activator-Initiated CRISPR/Cas12a Hybrid Feedback Circuit with Target Regeneration and Recycling.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c02845},
pmid = {40745870},
issn = {1520-6882},
abstract = {To meet the growing demand for ultrasensitive diagnostics, representative hybrid platforms integrating nonenzymatic isothermal nucleic acid amplification such as catalytic hairpin assembly (CHA) with CRISPR/Cas systems have been developed. However, two major challenges remain: background leakage from spontaneous hairpin hybridization and inherent fluorescence from conventional ssDNA reporters. Here, we present a self-sustaining isothermal biosensing platform that addresses these limitations by combining CHA with a split activator-initiated CRISPR/Cas12a feedback circuit for the ultrasensitive detection of miRNA-155, a key biomarker of breast cancer. In our design, miRNA-155 initiates CHA to form a DNA duplex, which, along with the miRNA, acts as split activators to trigger CRISPR/Cas12a. Cas12a cleaves a ds-loop DNA reporter, releasing fluorescence and regenerating the target. This dual-recognition mechanism ensures strict target dependence, reduces background noise, and, with the reporter design, minimizes leakage. The released miRNA reactivates CHA, enabling continuous signal amplification through a self-sustaining feedback loop involving successive CHA and Cas12a trans-cleavage cycles, enhancing detection sensitivity. Via these features, the platform achieves attomolar sensitivity and excellent specificity, even distinguishing single-base miRNA variants. Direct detection of endogenous miRNA-155 in serum samples from breast cancer patients demonstrated clear differentiation from healthy controls. This strategy provides a robust molecular detection platform for the accurate and ultrasensitive detection of low-abundance miRNAs in biomedical studies.},
}

@article {pmid40745619,
year = {2025},
author = {Bezerra, AM and Leite, AB and de Souza Reis, CR and de Lemos Padilha Pitta, JL and Maran, SR and Moretti, NS and Moura, DMN and Rezende, AM},
title = {Impact on the Leishmania mexicana transcriptome due to knockout of genes encoding orthologs of methyltransferases involved in m1A and m5C mRNA modifications.},
journal = {Parasites & vectors},
volume = {18},
number = {1},
pages = {315},
pmid = {40745619},
issn = {1756-3305},
support = {IBPG-1257-2.1319//Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco/ ; Financial Code 001//Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/ ; 23/16672-0//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2021/13477-6//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2018/09948-0//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 314103/2021-0//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; 400739/2019-4//Instituto Aggeu Magalhães/FIOCRUZ - CNPq/ ; },
mesh = {*Leishmania mexicana/genetics/enzymology/growth & development ; *Methyltransferases/genetics/metabolism ; *Transcriptome ; *RNA, Messenger/metabolism/genetics ; Protozoan Proteins/genetics/metabolism ; Gene Knockout Techniques ; CRISPR-Cas Systems ; *RNA Processing, Post-Transcriptional ; },
abstract = {BACKGROUND: Chemical modifications of mRNAs constitute an alternative mechanism for gene expression regulation, which involves proteins responsible for adding, recognizing and removing these modifications. While orthologs of enzymes involved in adding m1A (TRMT6/TRMT61A) and m5C (NSUN2) modifications are present in trypanosomatid species, a clear understanding of their biological role in these parasites is necessary.

METHODS: To shed light on this, we genetically manipulated the TRMT61A and NSUN2 protein-encoding genes in the Leishmania mexicana species using the CRISPR-Cas9 editing technique and analyzed the impact on cell growth and differentiation as well as the global gene expression profile.

RESULTS: Deletion of the genes investigated here caused changes in the normal pattern of L. mexicana differentiation, and functional analyses of differentially expressed genes in the mutants unveiled significant biological effects. For the TRMT61A gene, transcripts related to nucleotide metabolism, translation, protein folding and refolding were affected. For the NSUN2 genes, enrichment analysis indicated impacts on biological processes mostly related to nucleotide metabolism and DNA binding.

CONCLUSIONS: Our findings provide insights into the role of these methyltransferases orthologs in the regulation of trypanosomatid transcriptome, contributing to our understanding of gene expression control in this parasite.},
}

*Leishmania mexicana/genetics/enzymology/growth & development
*Methyltransferases/genetics/metabolism
*Transcriptome
*RNA, Messenger/metabolism/genetics
Protozoan Proteins/genetics/metabolism
Gene Knockout Techniques
CRISPR-Cas Systems
*RNA Processing, Post-Transcriptional

@article {pmid40745004,
year = {2025},
author = {Cheng, H and Jeong, E and Cho, SW},
title = {Applications of multiplexed CRISPR-Cas for genome engineering.},
journal = {Experimental & molecular medicine},
volume = {},
number = {},
pages = {},
pmid = {40745004},
issn = {2092-6413},
abstract = {The CRISPR-Cas system has become a worldwide genome editing tool for various organisms. Its precision and efficiency have facilitated basic research, drug discovery and therapeutic interventions. In contrast to other genome editing agents, CRISPR-Cas is modulated by a short guide RNA. Due to its simplicity, CRISPR-Cas is recognized as the best candidate for multiplexed genome editing. With simultaneous targeting, efficient knockout of genes with large deletions is possible. In addition, CRISPR-Cas can induce complex structural variations, such as inversions, translocations and duplications. Moreover, by utilizing engineered CRISPR-Cas proteins specialized for direct repression or activation of gene expression, one can perform multiplexed epigenetic editing. Lastly, multiplexed targeting enables killing of specific types of cells by accumulating stress mediated by simultaneous DNA damages. Here we discuss how CRISPR-based editing technologies for multiple targets are applied in recent studies.},
}

@article {pmid40568870,
year = {2025},
author = {Narendradev, ND and Marathe, S and Baboo, S and McClatchy, DB and Diedrich, JK and Jain, P and Purwar, R and Yates, JR and Srinivasula, SM},
title = {Quantitative Proteomic Analysis Reveals JMJD6 and DNAJB11 as Endogenous Substrates of E3 Ligase RFFL.},
journal = {Journal of proteome research},
volume = {24},
number = {8},
pages = {3913-3930},
doi = {10.1021/acs.jproteome.5c00086},
pmid = {40568870},
issn = {1535-3907},
mesh = {Humans ; *Proteomics/methods ; *Ubiquitin-Protein Ligases/metabolism/genetics ; *HSP40 Heat-Shock Proteins/metabolism/genetics ; *Jumonji Domain-Containing Histone Demethylases/metabolism/genetics ; Ubiquitination ; Proteolysis ; HEK293 Cells ; Proteasome Endopeptidase Complex/metabolism ; Gene Knockout Techniques ; CRISPR-Cas Systems ; },
abstract = {The ubiquitin-proteasome system contributes to protein quality control, involving E3 ligases that ubiquitinate proteins and leading to their degradation. The dysregulation of protein degradation results in the abnormal accumulation of proteins and is implicated in the pathology of diverse diseases, making targeted protein degradation a promising therapeutic strategy. Here, we focus on RFFL, an endosome-associated RING E3 ligase involved in mitochondrial homeostasis and the clearance of misfolded cystic fibrosis transmembrane conductance regulator proteins. Using label-free quantitative mass spectrometry based proteomics for interactome and differential expression analyses, we systematically investigated and identified putative substrates of RFFL. For more confident identification, we performed these analyses on three cell lines that we generated: an RFFL knockout cell line generated using CRISPR/Cas9, another cell line rescuing RFFL expression when complemented with KO cells with stably expressing RFFL cDNA, and wild-type cells. We validated JMJD6 and DNAJB11 as substrates of endogenous RFFL, providing orthogonal validation and confidence in our screening approach. We demonstrated that RFFL ubiquitinates and degrades JMJD6 and DNAJB11 via the proteasomal pathway using in vivo assays. Interestingly, we also discovered a hitherto unknown role of RFFL in lipid metabolism. Collectively, this study provides the first comprehensive and unbiased analysis of RFFL substrates employing multiple complementary approaches.},
}

Humans
*Proteomics/methods
*Ubiquitin-Protein Ligases/metabolism/genetics
*HSP40 Heat-Shock Proteins/metabolism/genetics
*Jumonji Domain-Containing Histone Demethylases/metabolism/genetics
Ubiquitination
Proteolysis
HEK293 Cells
Proteasome Endopeptidase Complex/metabolism
Gene Knockout Techniques
CRISPR-Cas Systems

@article {pmid40462261,
year = {2025},
author = {Liu, Z and Liu, L and Liu, S and Ba, Y and Zuo, A and Xu, H and Zhang, Y and Wang, S and Wang, L and Han, X},
title = {Genome-Scale CRISPR-Cas9 Analysis Reveals Tumor Heterogeneity and Identifies NDC80 as Novel Biomarker in HCC.},
journal = {Journal of gastroenterology and hepatology},
volume = {40},
number = {8},
pages = {2078-2090},
doi = {10.1111/jgh.17016},
pmid = {40462261},
issn = {1440-1746},
support = {221100310100//Major Science and Technology Projects of Henan Province/ ; xzy022024018//Basic Scientific Research Business of Central Universities/ ; },
mesh = {Humans ; *Carcinoma, Hepatocellular/genetics/pathology/mortality ; *Liver Neoplasms/genetics/pathology/mortality ; *Biomarkers, Tumor/genetics ; *CRISPR-Cas Systems/genetics ; Prognosis ; Cell Proliferation/genetics ; Female ; Male ; Cell Line, Tumor ; Cell Movement/genetics ; *Nuclear Proteins/genetics ; Gene Expression Regulation, Neoplastic ; },
abstract = {BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is a malignant tumor with a poor prognosis and is characterized by severe intratumoral heterogeneity. Identifying key genomic features and more reliable classifications is helpful for clinical management.

METHODS: Cancer essential genes (CEGs) were identified using genome-scale CRISPR-Cas9 and univariate Cox regression analyses. Based on gene expression, nonnegative matrix factorization (NMF) was used to generate distinct molecular subtypes. The nearest template prediction (NTP) algorithm was used to validate the accuracy and robust classifications among three independent cohorts, including GSE14520, GSE54236, and ICGC-LIRI. Specifically, potential biomarkers were screened for clinical transformation based on their prognostic characteristics and biological function features. EdU, colony formation, and Transwell assays were utilized to confirm the effect of biomarkers in vitro.

RESULTS: The C1 subtype had the worst prognosis and was characterized by advanced AJCC stages and high genomic instability. The NTP approach confirmed that the molecular subtypes were practical, robust, and reproducible. We further identified NDC80 as a gene specifically expressed in C1 subtype, indicative of prognosis solely for this subtype. Based on overrepresentation analysis (ORA), it was found that the biological function of NDC80 was mainly enriched in proliferation. In vitro cellular assays verified that promoted tumor growth and migration.

CONCLUSIONS: Our study identified three robust molecular subtypes and revealed tumor heterogeneity. Meanwhile, the potential biomarker NDC80 served as a characteristic gene of the C1 subtype, correlating with poor prognosis and promoting tumor growth and migration, providing new insights for prognostic treatment strategies in HCC.},
}

Humans
*Carcinoma, Hepatocellular/genetics/pathology/mortality
*Liver Neoplasms/genetics/pathology/mortality
*Biomarkers, Tumor/genetics
*CRISPR-Cas Systems/genetics
Prognosis
Cell Proliferation/genetics
Female
Male
Cell Line, Tumor
Cell Movement/genetics
*Nuclear Proteins/genetics
Gene Expression Regulation, Neoplastic

@article {pmid40327605,
year = {2025},
author = {Lee, S and Kim, K and Jeong, HJ and Choi, S and Cheng, H and Kim, D and Heo, S and Mun, J and Kim, M and Lee, E and Choi, YJ and Lee, SG and Lee, EA and Jang, Y and Lim, K and Kim, HS and Jeong, E and Myung, SJ and Jung, DB and Yu, CS and Song, IH and Corces, MR and Kang, JH and Myung, K and Kwon, T and Park, TE and Joo, J and Cho, SW},
title = {Combining Multiplexed CRISPR/Cas9-Nickase and PARP Inhibitors Efficiently and Precisely Targets Cancer Cells.},
journal = {Cancer research},
volume = {85},
number = {15},
pages = {2890-2904},
doi = {10.1158/0008-5472.CAN-24-2938},
pmid = {40327605},
issn = {1538-7445},
support = {IBS-R022-D1//Institute for Basic Science (IBS)/ ; RS-2024-00399800//National Research Foundation of Korea (NRF)/ ; RS-2023-00213043//National Research Foundation of Korea (NRF)/ ; RS-2024-00509412//National Research Foundation of Korea (NRF)/ ; RS-2023-00209822//National Research Foundation of Korea (NRF)/ ; 2021R1A6A1A03040260//National Research Foundation of Korea (NRF)/ ; HU20C0094//Ministry of Science and ICT, South Korea (MSIT)/ ; HI19C1095//Ministry of Health and Welfare (MOHW)/ ; //POSCO TJ Park Foundation (포스코청암재단)/ ; 1.240016.01//Ulsan National Institute of Science and Technology (UNIST)/ ; },
mesh = {Humans ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; *CRISPR-Cas Systems ; Animals ; Mice ; *Neoplasms/genetics/drug therapy/pathology/therapy ; Xenograft Model Antitumor Assays ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; DNA Repair/drug effects ; DNA Breaks, Double-Stranded/drug effects ; Female ; },
abstract = {UNLABELLED: Triggering cancer cell death by inducing DNA damage is the primary aim of radiotherapy; however, normal cells are also damaged. In this study, we showed that delivery of only four synthetic guide RNAs with Cas9 endonuclease efficiently induced simultaneous DNA double-strand breaks, resulting in efficient cell death in a cell type-specific manner. Off-target effects of Cas9 endonuclease were prevented by using Cas9-nickase to induce DNA single-strand breaks and blocking their repair with PARP inhibitors (PARPi). When recombinant Cas9-nickase protein and multiple synthetic guide RNAs were delivered with PARPis into cultured cells, in vivo xenografts, and patient-derived cancer organoids via lipid nanoparticles, cancer cells were unable to tolerate the induced DNA damage even in the presence of a functional BRCA2 gene. This approach has the potential to expand the use of PARPis with verified safety and thus is a potentially powerful tool for personalized genome-based anticancer therapy.

SIGNIFICANCE: Targeting cancer-specific variants with CRISPR/Cas9-nickase induces cancer-specific cell death in combination with DNA repair pathway inhibitors, demonstrating the potential of CRISPR cancer therapy for treating a broad range of cancers.},
}

Humans
*Poly(ADP-ribose) Polymerase Inhibitors/pharmacology
*CRISPR-Cas Systems
Animals
Mice
*Neoplasms/genetics/drug therapy/pathology/therapy
Xenograft Model Antitumor Assays
RNA, Guide, CRISPR-Cas Systems/genetics
Cell Line, Tumor
DNA Repair/drug effects
DNA Breaks, Double-Stranded/drug effects
Female

@article {pmid40192983,
year = {2025},
author = {Bao, W and Fan, W and Zhang, Y and Lan, F and Ma, S},
title = {Circular CRISPR Edits Human Pluripotent Stem Cells for Disease Modeling.},
journal = {Stem cell reviews and reports},
volume = {21},
number = {5},
pages = {1512-1522},
pmid = {40192983},
issn = {2629-3277},
support = {82400587//National Natural Science Foundation of China/ ; 2022M720498//China Postdoctoral Science Foundation/ ; B2302048//Shenzhen Medical Research Fund/ ; },
mesh = {Humans ; *Gene Editing/methods ; *Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *RNA, Circular/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The CRISPR system has been widely used for human pluripotent stem cell (hPSC) disease modeling. Circular RNA can effectively reduce RNA immunogenicity and improve RNA stability, thus contributing to in vivo DNA editing. In this study, we briefly describe the process of circularizing guide RNA and CRISPR base editing elements and using them to establish stem cell disease models. Our work provides step-by-step guidance for constructing gene point editing cell lines, offering a reliable, low-immunogenic alternative for disease modeling and therapeutic research.},
}

Humans
*Gene Editing/methods
*Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*RNA, Circular/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid40745002,
year = {2025},
author = {Park, EJ and Kim, H},
title = {Live genome imaging by CRISPR engineering: progress and problems.},
journal = {Experimental & molecular medicine},
volume = {},
number = {},
pages = {},
pmid = {40745002},
issn = {2092-6413},
abstract = {CRISPR-Cas-based genome imaging opened a new era of genome visualization in living cells. While genomic loci with repetitive sequences, such as centromeres and telomeres, can be reliably imaged, applying the technique to nonrepetitive genomic loci has remained challenging. Recent advancements in the design of CRISPR RNAs and Cas proteins, the development of novel fluorophores and the combination of CRISPR-Cas with other molecular machinery amplified target-specific signals and suppressed background signals, revolutionizing this unique genome imaging technique and enabling the tracking of genomic loci with a small number of CRISPR-Cas complexes, down to a single complex. Here we review the latest advancements in CRISPR-Cas-based genome imaging techniques and their application to imaging nonrepetitive genomic loci. The challenges that these techniques are currently facing are the cellular toxicity and genomic instability induced by the expression of CRISPR-Cas and its interference with DNA metabolism, which impacts DNA replication and genome maintenance. Recently reported adverse effects of CRISPR-Cas-based genome labeling are discussed here, along with perspectives on how to overcome the problem.},
}

@article {pmid40745001,
year = {2025},
author = {Varshney, GK and Burgess, SM},
title = {CRISPR-based functional genomics tools in vertebrate models.},
journal = {Experimental & molecular medicine},
volume = {},
number = {},
pages = {},
pmid = {40745001},
issn = {2092-6413},
support = {ZIAHG000183-24//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R24OD034438//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
abstract = {The advent of CRISPR-Cas technologies has revolutionized functional genomics by enabling precise genetic manipulations in various model organisms. In popular vertebrate models, including mice and zebrafish, CRISPR has been adapted to high-throughput mutagenesis workflows, knock-in alleles and large-scale screens, bringing us closer to understanding gene functions in development, physiology and pathology. The development of innovative technologies, such as base editors, capable of single-nucleotide modifications, and prime editors, offering precision edits without double-strand breaks, exemplifies the expanding toolkit. In addition to gene editing, transcriptional modulation, that is, CRISPR interference and CRISPR activation systems, can elucidate the mechanisms of gene regulation. Newer methods, such as MIC-Drop and Perturb-seq, which increase screening throughput in vivo, hold significant promise to improve our ability to dissect complex biological processes and mechanisms. Furthermore, CRISPR-based gene therapies for treating sickle cell disease and other monogenic diseases have already demonstrated their potential for clinical translation. Here this Review covers the transformative impact of CRISPR-based tools in vertebrate models, highlighting their utility in functional genomics research and disease modeling.},
}

@article {pmid40745000,
year = {2025},
author = {Kim, MG and Go, MJ and Kang, SH and Jeong, SH and Lim, K},
title = {Revolutionizing CRISPR technology with artificial intelligence.},
journal = {Experimental & molecular medicine},
volume = {},
number = {},
pages = {},
pmid = {40745000},
issn = {2092-6413},
support = {RS-2023-00210965 and RS-2024-00339116//National Research Foundation of Korea (NRF)/ ; 2V10573 and 2E33791//Korea Institute of Science and Technology (KIST)/ ; },
abstract = {Genome engineering has made remarkable strides, evolving from DNA-binding proteins such as zinc fingers and transcription activator-like effectors to CRISPR-Cas systems. CRISPR technology has revolutionized the field through its simplicity and ability to target specific genome regions via guide RNA and Cas proteins. Progress in CRISPR tools-CRISPR nucleases, base editors and prime editors-has expanded the toolkit to induce targeted insertions or deletions, nucleotide conversions and a wider array of genetic alterations. Nevertheless, variations in editing outcomes across cell types and unintended off-target effects still present substantial hurdles. Artificial intelligence (AI), which has seen rapid advances, provides high-level solutions to these problems. By leveraging large datasets from diverse experiments, AI enhances guide RNA design, predicts off-target activities and improves editing efficiency. In addition, AI aids in discovering and designing novel CRISPR systems beyond natural limitations. These developments provide new modalities essential for the innovation of personalized therapies and help to ensure efficiency, precision and safety. Here we discuss the transformative role of AI in advancing CRISPR technology. We highlight how AI contributes to refining nuclease-based editing, base editing and prime editing. Integrating AI with CRISPR technology enhances existing tools and opens doors to next-generation medicine for gene therapy.},
}

@article {pmid40744946,
year = {2025},
author = {Lysne, DP and Stewart, MH and Susumu, K and Leski, TA and Stenger, DA and Medintz, IL and Díaz, SA and Green, CM},
title = {Quantum dot molecular beacons achieve sub-10 pM CRISPR-Cas detection in field-ready assays.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {27950},
pmid = {40744946},
issn = {2045-2322},
mesh = {*Quantum Dots/chemistry ; *CRISPR-Cas Systems/genetics ; Fluorescence Resonance Energy Transfer/methods ; *Biosensing Techniques/methods ; Limit of Detection ; Humans ; },
abstract = {CRISPR-Cas systems have revolutionized molecular diagnostics through their specificity and programmability, yet their broad adoption is hindered by the reliance on expensive and complex instrumentation. Here, we present an optimized quantum dot (QD) molecular beacon (QD-MB) platform that integrates Förster resonance energy transfer (FRET)-based detection with CRISPR-Cas functionality, achieving sub-picomolar sensitivity without the need for target amplification. By systematically tuning components, including His-tag modifications for improved QD conjugation, nucleic acid hairpin structures for enhanced enzyme interaction, and QD surface passivation strategies, we demonstrate a two-order-of-magnitude improvement in detection sensitivity. Using LwaCas13a and RNA targets, the limit of detection (LOD) decreased to under 1 pM with plate-reader-based fluorescence measurements and below 10 pM with a lamp-and-smartphone setup, establishing the feasibility of portable, field-ready applications. This work highlights the transformative potential of QD-MBs in biosensing and sets a foundation for further advances in CRISPR-based diagnostics and nanotechnology-enabled sensing platforms.},
}

*Quantum Dots/chemistry
*CRISPR-Cas Systems/genetics
Fluorescence Resonance Energy Transfer/methods
*Biosensing Techniques/methods
Limit of Detection
Humans

@article {pmid40744920,
year = {2025},
author = {Dib, C and Queenan, JA and Swartzrock, L and Willner, H and Denis, M and Ahmed, N and Moulana Zada, F and Borges, B and Charlesworth, CT and Lum, T and Yates, BP and Kwon, CY and Scorzo, AV and Davis, SC and Davis, JR and He, R and Xie, J and Gao, G and MacKenzie, TC and Liu, DR and Newby, GA and Czechowicz, AD},
title = {GFP-on mouse model for interrogation of in vivo gene editing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7017},
doi = {10.1038/s41467-025-61449-y},
pmid = {40744920},
issn = {2041-1723},
mesh = {Animals ; *Gene Editing/methods ; *Green Fluorescent Proteins/genetics/metabolism ; Dependovirus/genetics ; Mice ; CRISPR-Cas Systems ; Genetic Vectors/genetics ; Genes, Reporter ; Female ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Codon, Nonsense ; },
abstract = {Gene editing technologies have revolutionized therapies for numerous genetic diseases. However, in vivo gene editing hinges on identifying efficient delivery vehicles for editing in targeted cell types, a significant hurdle in fully realizing its therapeutic potential. A model system to rapidly evaluate systemic gene editing would advance the field. Here, we develop the GFP-on reporter mouse, which harbors a nonsense mutation in a genomic EGFP sequence correctable by adenine base editor (ABE) among other genome editors. The GFP-on system was validated using single and dual adeno-associated virus (AAV9) encoding ABE8e and sgRNA. Intravenous administration of AAV9-ABE8e-sgRNA into adult GFP-on mice results in EGFP expression consistent with the tropism of AAV9. Intrahepatic delivery of AAV9-ABE8e-sgRNA into GFP-on fetal mice restores EGFP expression in AAV9-targeted organs lasting at least six months post-treatment. The GFP-on model provides an ideal platform for high-throughput evaluation of emerging gene editing tools and delivery modalities.},
}

Animals
*Gene Editing/methods
*Green Fluorescent Proteins/genetics/metabolism
Dependovirus/genetics
Mice
CRISPR-Cas Systems
Genetic Vectors/genetics
Genes, Reporter
Female
RNA, Guide, CRISPR-Cas Systems/genetics
Humans
Codon, Nonsense

@article {pmid40744728,
year = {2025},
author = {Sabin, CE and Lauderdale, JD and Menke, DB},
title = {Anolis Lizards as a Model System for Studies of Gene Function in Reptile Development and Evolution.},
journal = {Cold Spring Harbor protocols},
volume = {},
number = {},
pages = {},
doi = {10.1101/pdb.top108535},
pmid = {40744728},
issn = {1559-6095},
abstract = {Anolis lizards are an ecologically diverse group that includes more than 400 described species. These reptiles have been the subject of wide-ranging studies, from speciation and convergent evolution to climate adaptation and tail regeneration. While CRISPR-based gene editing has tremendous potential to reveal new insights into these and other aspects of Anolis biology, the reproductive biology of these reptiles has presented significant barriers to gene editing. Here, we briefly summarize gene editing approaches in vertebrates and discuss some of the major challenges associated with the performance of gene editing in anoles. We then introduce a recently established surgical procedure that enables the injection of CRISPR-Cas into the developing oocytes of female lizards. This approach circumvents the need to manipulate early-stage embryos and permits the production of gene-edited anoles. This method has recently been successfully adapted for use in other reptiles, suggesting that it may be effective in a wide range of species and will broadly enable studies of gene function in reptiles.},
}

@article {pmid40744574,
year = {2025},
author = {Zhang, Z and Wu, S and Long, Y and Huang, W and Bramlett, M and Yang, Y and Wu, Y},
title = {Identification of SfABCC2 as the critical receptor for Cry1Fa and Cry1Ab in Spodoptera frugiperda via CRISPR-mediated gene knockouts.},
journal = {Pesticide biochemistry and physiology},
volume = {213},
number = {},
pages = {106526},
doi = {10.1016/j.pestbp.2025.106526},
pmid = {40744574},
issn = {1095-9939},
mesh = {Animals ; *Spodoptera/genetics/metabolism/drug effects ; *Bacillus thuringiensis Toxins ; *Endotoxins/metabolism/pharmacology ; *Hemolysin Proteins/metabolism/pharmacology ; *Bacterial Proteins/metabolism/pharmacology ; Gene Knockout Techniques ; CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; Insecticide Resistance/genetics ; Insecticides/pharmacology ; Bacillus thuringiensis ; },
abstract = {Insecticidal proteins from Bacillus thuringiensis (Bt) have been widely used to control major agricultural pests through genetically modified (GM) Bt crops. However, the development of resistance in target pests could undermine the effectiveness of Bt crops. Understanding the mechanisms of action of Bt insecticidal proteins and the resistance mechanisms in pests is crucial for developing effective resistance management strategies to sustain the use of Bt crops. In this study, CRISPR/Cas9 gene editing was used to investigate the functional roles of four genes (SfABCC2, SfABCC3, SfCad1, and SfAPN1) that encode putative receptors for Cry1 proteins in the fall armyworm, Spodoptera frugiperda, a globally significant pest. We created five homozygous knockouts, each with a substantial fragment deletion: SfCad1-KO, SfAPN1-KO, SfABCC2-KO, SfABCC3-KO, and SfC2/C3-KO (a double knockout of SfABCC2 and SfABCC3). Bioassay results revealed that SfCad1-KO, SfAPN1-KO, and SfABCC3-KO strains exhibited no resistance to Cry1Fa or Cry1Ab. In contrast, SfABCC2-KO and SfC2/C3-KO strains demonstrated high levels of resistance to Cry1Fa (>3300-fold) and Cry1Ab (>450-fold), demonstrating that SfABCC2 is pivotal to the insecticidal action of these two Bt proteins. As anticipated, all five knockouts generated in this study did not significantly impact susceptibility to Vip3Aa compared with the control strain. Our findings underscore the critical role of SfABCC2 in mediating Cry1Ab and Cry1Fa toxicity in S. frugiperda. Therefore, resistance monitoring program and resistance management tactics should focus on SfABCC2 mutations in field populations of S. frugiperda.},
}

Animals
*Spodoptera/genetics/metabolism/drug effects
*Bacillus thuringiensis Toxins
*Endotoxins/metabolism/pharmacology
*Hemolysin Proteins/metabolism/pharmacology
*Bacterial Proteins/metabolism/pharmacology
Gene Knockout Techniques
CRISPR-Cas Systems
*Insect Proteins/genetics/metabolism
Insecticide Resistance/genetics
Insecticides/pharmacology
Bacillus thuringiensis

@article {pmid40744495,
year = {2025},
author = {Sokirniy, I and Inam, H and Tomaszkiewicz, M and Reynolds, J and McCandlish, D and Pritchard, J},
title = {A side-by-side comparison of variant function measurements using deep mutational scanning and base editing.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf738},
pmid = {40744495},
issn = {1362-4962},
support = {2141650//NSF/ ; T32GM108563/GF/NIH HHS/United States ; R35 GM133613/GF/NIH HHS/United States ; U01CA265709/BC/NCI NIH HHS/United States ; 2033673//NSF RECODE/ ; //Simons Center for Quantitative Biology at Cold Spring Harbor Laboratory/ ; },
mesh = {Humans ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; High-Throughput Nucleotide Sequencing/methods ; *Mutation ; DNA, Complementary/genetics ; CRISPR-Cas Systems ; Molecular Sequence Annotation/methods ; Genetic Variation ; Genomics/methods ; Gene Library ; },
abstract = {Variant annotation is a crucial objective in mammalian functional genomics. Deep mutational scanning (DMS) using saturation libraries of complementary DNAs (cDNAs) is a well-established method for annotating human gene variants, but CRISPR base editing (BE) is emerging as an alternative. However, questions remain about how well high-throughput BE measurements can annotate variant function and the extent of downstream experimental validation required. This study is the first direct comparison of cDNA DMS and BE in the same lab and cell line. We focus on how well short guide RNA (sgRNA) depletion or enrichment is explained by the predicted edits within the editing "window" defined by the sgRNA. The most likely predicted edits enhance the agreement between a "gold standard" DMS dataset and a BE screen. A simple filter for sgRNAs making single edits in their window could sufficiently annotate a large proportion of variants directly from sgRNA sequencing of large pools. When multi-edit guides are unavoidable, directly measuring edits in medium-sized validation pools can recover high-quality variant annotation data. Our data show a surprisingly high degree of correlation between base editor data and gold standard DMS. We suggest that the main variable measured in base editor screens is the desired base edits.},
}

Humans
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
High-Throughput Nucleotide Sequencing/methods
*Mutation
DNA, Complementary/genetics
CRISPR-Cas Systems
Molecular Sequence Annotation/methods
Genetic Variation
Genomics/methods
Gene Library

@article {pmid40744490,
year = {2025},
author = {Cattle, MA and Aguado, LC and Sze, S and Venkittu, S and Wang, Y and Papagiannakopoulos, T and Smith, S and Rice, CM and Schneider, WM and Poirier, JT},
title = {An enhanced Eco1 retron editor enables precision genome engineering in human cells without double-strand breaks.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf716},
pmid = {40744490},
issn = {1362-4962},
support = {P30CA016087//NYU Laura & Isaac Perlmutter Cancer Center/ ; P30CA016087//NYU Laura & Isaac Perlmutter Cancer Center/ ; //Robertson Foundation/ ; R35GM149355/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; DNA Breaks, Double-Stranded ; Zika Virus/genetics ; *Retroelements/genetics ; 3' Untranslated Regions/genetics ; HEK293 Cells ; RNA, Untranslated/genetics/metabolism ; CRISPR-Associated Protein 9/metabolism/genetics ; Genome, Human ; },
abstract = {Retrons are a retroelement class found in diverse prokaryotes that can be adapted to augment CRISPR-Cas9 genome engineering technology to efficiently rewrite short stretches of genetic information in bacteria and yeast. However, efficiency in human cells has been limited by unknown factors. We identified non-coding RNA (ncRNA) instability and impaired Cas9 activity due to 5' sgRNA extension as key contributors to low retron editor efficiency in human cells. We re-engineered the Eco1 ncRNA to incorporate an exoribonuclease-resistant RNA pseudoknot from the Zika virus 3' UTR and devised an RNA processing strategy using Csy4 ribonuclease to minimize 5' sgRNA extension. This strategy increased steady-state ncRNA levels and rescued sgRNA activity, leading to increased templated repair. This work reveals a previously unappreciated role for ncRNA stability in retron editor efficiency in human cells and presents an enhanced Eco1 retron editor capable of precise genome editing in human cells from a single integrated lentivirus and, in the context of the nCas9 H840A nickase, without creating double-strand breaks.},
}

Humans
*Gene Editing/methods
CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
DNA Breaks, Double-Stranded
Zika Virus/genetics
*Retroelements/genetics
3' Untranslated Regions/genetics
HEK293 Cells
RNA, Untranslated/genetics/metabolism
CRISPR-Associated Protein 9/metabolism/genetics
Genome, Human

@article {pmid40743876,
year = {2025},
author = {Gardoul, M and Rached, B and Mbarki, A and Ajdig, M and Belouad, EM and Chouati, T and Chauiyakh, O and Lahcen Ouchari, and Harlé, O and Al-Nakeeb, K and Melloul, M and El Fahime, E},
title = {Comprehensive whole-genome analysis of Streptococcus infantarius strains from Moroccan farmhouse dairy products: Genomic insights into dairy adaptation, safety, and biotechnological potential.},
journal = {International journal of food microbiology},
volume = {442},
number = {},
pages = {111358},
doi = {10.1016/j.ijfoodmicro.2025.111358},
pmid = {40743876},
issn = {1879-3460},
abstract = {Streptococcus infantarius, a lactic acid bacterium prevalent in Moroccan dairy products, holds significant probiotic and biotechnological potential, yet its genomic features remain poorly characterized. This study aims to provide a comprehensive genomic characterization of six S. infantarius isolates (B50-6, B50-7, B51-6, O53-2, T41-8, K85-8) from Moroccan farmhouse dairy products. Whole-genome sequencing and comparative analysis were conducted using advanced bioinformatics tools to assess their genetic diversity, functional attributes, adaptive mechanisms, probiotic properties, safety profile, and potential applications in food fermentation. All strains were confirmed as S. infantarius with average nucleotide identity (ANI) >98.2 % and digital DNA-DNA hybridization (dDDH) >85.9 %. Genomes sizes ranging from 1.81 (K85-8) to 1.91 (T41-8) Mb, with GC content ∼37.4 %. Functional profiling identified 1343, 1343, 1345, 1342, 1359 and 1345 proteins with functional assignments; 519, 521, 536, 520, 542 and 502 hypothetical proteins; and 1752, 1754, 1778, 1752, 1781 and 1740 proteins with COG (Clusters of Orthologous Groups) assignments, in B50-6, B50-7, B51-6, O53-2, T41-8 and K85-8, respectively. Gene enrichment analysis highlighted key metabolic pathways and functional categories relevant to dairy adaptation and biotechnological potential. The total number of genes encoding carbohydrate-active enzymes was 45 (B50-6), 45 (B50-7), 48 (B51-6), 45 (O53-2), 48 (T41-8) and 48 (K85-8). Safety assessments identified 13 (K85-8) to 16 (B51-6) virulence-associated genes per strain. However, no antimicrobial resistance genes or plasmids were detected, while the presence of CRISPR-Cas systems was detected in most strains. A total of 88 mobile genetic elements were identified ranging from 9 (B51-6) to 18 (O53-2) per strain. In addition, prophages were detected in all strains with the exception of T41-8 and K85-8. All Strains exhibited diverse biosynthetic gene clusters (BGCs), including RiPP-like, T3PKS and terpene precursor pathways. These findings position S. infantarius as a relatively stable core genome, metabolically versatile candidate for dairy fermentation, probiotic and biotechnological applications.},
}

@article {pmid40742466,
year = {2025},
author = {Zhang, K and Wang, D and Hu, S and Li, X and Hong, J},
title = {Gene disruption via a transient hypercompact CRISPR-AsCas12f1 system in Kluyveromyces marxianus.},
journal = {Biotechnology letters},
volume = {47},
number = {4},
pages = {84},
pmid = {40742466},
issn = {1573-6776},
support = {202423m10050005//Anhui Provincial Science and Technology Innovation Program/ ; 231100110300//Major Special Science and Technology Project of Henan Province/ ; 32071469//National Natural Science Foundation of China/ ; },
mesh = {*Kluyveromyces/genetics ; *CRISPR-Cas Systems ; *Gene Knockout Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; RNA, Transfer/genetics ; },
abstract = {Kluyveromyces marxianus is an attractive chassis for microbial cell factories due to its rapid growth, thermotolerance, and wide substrate spectrum. However, gene disruption in this organism is challenging primarily due the prevalence of dominant nonhomologous recombination. AsCas12f1, a hypercompact CRISPR-associated protein consisting of 422 amino acids-approximately one-third the size of Cas9 or Cas12a-enables more efficient packaging into delivery vehicles than its larger counterparts. In this study, a gene disruption method using AsCas12f1 was established in K. marxianus through a transient targeting strategy. The integration of tRNA-gRNA into the gRNA construct increased gene disruption efficiency. Additionally, disrupting KmKU70 or KmLIG4 further increased this efficiency, achieving nearly 100%. By combining the disruption of KmKU70 with the AsCas12f1 system, the length of the homologous arm was shortened to 200 bp while maintaining a disruption efficiency of 87.5%. The implementation of the gRNA-tRNA-array system resulted in the successful generation of three single-gene knockout strains from a single transformation, resulting an overall efficiency of 86.4%. This approach leverages the transient transformation of fragments, eliminates the need for extensive time investment in constructing gRNA expression vectors and negates the requirement for the removal of the CRISPR-AsCas12f1 system after gene disruption. This study presents a novel strategy for gene disruption in K. marxianus and demonstrates the applicability of Cas12f in yeast systems.},
}

*Kluyveromyces/genetics
*CRISPR-Cas Systems
*Gene Knockout Techniques/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing/methods
*CRISPR-Associated Proteins/genetics/metabolism
RNA, Transfer/genetics

@article {pmid40741393,
year = {2025},
author = {Wei, J and Motawaa, M and Li, Y},
title = {Thermus thermophilus CRISPR Cas6 Heterologous Expression and Purification.},
journal = {Bio-protocol},
volume = {15},
number = {14},
pages = {e5382},
pmid = {40741393},
issn = {2331-8325},
abstract = {The CRISPR-Cas system of Thermus thermophilus has emerged as a potent biotechnological tool, particularly its Cas6 endonuclease, which plays a crucial role in CRISPR RNA (crRNA) maturation. This protocol details a robust and reproducible method for the high-level expression and purification of recombinant T. thermophilus Cas6 proteins (Cas6-1 and Cas6-2) in E. coli. We describe a streamlined approach encompassing plasmid construction using seamless assembly, optimized bacterial heterologous expression, and multi-step purification leveraging affinity and size-exclusion chromatography. The protocol outlines the generation of both His-tagged and GST-tagged Cas6 variants, enabling flexibility in downstream applications. Key steps, including primer design, PCR optimization, competent cell transformation, and chromatography strategies, are meticulously detailed with critical parameters and troubleshooting guidance to ensure experimental success and high yields of highly pure and active T. thermophilus Cas6 proteins. This protocol is useful for researchers requiring purified T. thermophilus Cas6 for structural studies, biochemical characterization, and the development of CRISPR-based biotechnological tools. Key features • Robust method for expressing and purifying Thermus thermophilus Cas6 proteins in E. coli. • Seamless assembly cloning and dual affinity tagging system: Offers options for both His-tag and GST-tag purification strategies for increased versatility. • Applicable for diverse heterologous expression and purification of well-folding thermostable proteins in mesophilic host chassis cells [E. coli BL21(DE3)].},
}

@article {pmid40739342,
year = {2025},
author = {Ruffolo, JA and Nayfach, S and Gallagher, J and Bhatnagar, A and Beazer, J and Hussain, R and Russ, J and Yip, J and Hill, E and Pacesa, M and Meeske, AJ and Cameron, P and Madani, A},
title = {Design of highly functional genome editors by modelling CRISPR-Cas sequences.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {40739342},
issn = {1476-4687},
abstract = {Gene editing has the potential to solve fundamental challenges in agriculture, biotechnology and human health. CRISPR-based gene editors derived from microorganisms, although powerful, often show notable functional tradeoffs when ported into non-native environments, such as human cells[1]. Artificial-intelligence-enabled design provides a powerful alternative with the potential to bypass evolutionary constraints and generate editors with optimal properties. Here, using large language models[2] trained on biological diversity at scale, we demonstrate successful precision editing of the human genome with a programmable gene editor designed with artificial intelligence. To achieve this goal, we curated a dataset of more than 1 million CRISPR operons through systematic mining of 26 terabases of assembled genomes and metagenomes. We demonstrate the capacity of our models by generating 4.8× the number of protein clusters across CRISPR-Cas families found in nature and tailoring single-guide RNA sequences for Cas9-like effector proteins. Several of the generated gene editors show comparable or improved activity and specificity relative to SpCas9, the prototypical gene editing effector, while being 400 mutations away in sequence. Finally, we demonstrate that an artificial-intelligence-generated gene editor, denoted as OpenCRISPR-1, exhibits compatibility with base editing. We release OpenCRISPR-1 to facilitate broad, ethical use across research and commercial applications.},
}

@article {pmid40739316,
year = {2025},
author = {Callahan, A and Mojumdar, A and Hu, M and Wang, A and Griffith, AA and Huang, N and Chua, XY and Mroz, N and Puterbaugh, RZ and Reilly, SP and Salomon, A},
title = {The phosphatases TCPTP, PTPN22, and SHP1 play unique roles in T cell phosphotyrosine maintenance and feedback regulation of the TCR.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {27747},
doi = {10.1038/s41598-025-12951-2},
pmid = {40739316},
issn = {2045-2322},
support = {R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; R01AI083636/NH/NIH HHS/United States ; },
mesh = {Humans ; *Receptors, Antigen, T-Cell/metabolism ; *Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism/genetics ; Jurkat Cells ; *T-Lymphocytes/metabolism/immunology ; *Protein Tyrosine Phosphatase, Non-Receptor Type 22/metabolism/genetics ; *Phosphotyrosine/metabolism ; Feedback, Physiological ; Signal Transduction ; CRISPR-Cas Systems ; Protein Tyrosine Phosphatase, Non-Receptor Type 1 ; },
abstract = {The protein tyrosine phosphatases (PTPs) TCPTP, PTPN22, and SHP1 are critical regulators of the activating phosphotyrosine (pY) site on the initiating T cell kinase, Lck[Y394]. Still, the broader implications of these phosphatases in T cell receptor (TCR) signalling and T cell biology remain unclear. By combining CRISPR/Cas9 gene editing and mass spectrometry, we evaluate the protein- and pY-level effects of TCPTP, PTPN22, and SHP1 in the Jurkat T cell model system. We find that deletion of each phosphatase corresponds to unique changes in the proteome of T cells, with few large-scale changes to TCR signalling proteins. Notably, PTPN22 and SHP1 deletions have opposing effects on pY abundance globally, while TCPTP deletion modestly elevates pY levels. Finally, we show that TCPTP is indirectly involved in Erk1/2 positive feedback to the TCR. Overall, our work provides evidence for alternative functions of three T cell phosphatases long thought to be redundant.},
}

Humans
*Receptors, Antigen, T-Cell/metabolism
*Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism/genetics
Jurkat Cells
*T-Lymphocytes/metabolism/immunology
*Protein Tyrosine Phosphatase, Non-Receptor Type 22/metabolism/genetics
*Phosphotyrosine/metabolism
Feedback, Physiological
Signal Transduction
CRISPR-Cas Systems
Protein Tyrosine Phosphatase, Non-Receptor Type 1

@article {pmid40738762,
year = {2025},
author = {Gupta, PK and Kumar, S},
title = {Third-generation novel technologies for gene editing.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.07.012},
pmid = {40738762},
issn = {1879-3096},
abstract = {Gene editing technologies have revolutionized the field of biotechnology. CRISPR-Cas methods using RNA-guided enzymes are the most used gene editing tools and have produced gene-edited crops (rice, wheat, corn, etc.) and human therapeutics (Casgevy, approved for commercial use; Vertex Pharmaceuticals). However, these systems have some limitations, including the requirement of a protospacer adjacent motif sequence, generation of undesirable double-strand breaks (DSBs), and the inability to edit long genomic segments. Some of these limitations were partially addressed by the development of second-generation editors, including base editors (BEs) and prime editors (PEs). Third-generation gene editing technologies such as seekRNA and bridgeRNA can overcome most of these limitations and are the subject of this review.},
}

@article {pmid40690516,
year = {2025},
author = {Shigenobu, S and Yoda, S and Ohsawa, S and Suzuki, M},
title = {Refined CRISPR/Cas9 genome editing in the pea aphid uncovers the essential roles of Laccase2 in overwintering egg adaptation.},
journal = {PLoS genetics},
volume = {21},
number = {7},
pages = {e1011557},
doi = {10.1371/journal.pgen.1011557},
pmid = {40690516},
issn = {1553-7404},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Aphids/genetics/physiology ; *Laccase/genetics/metabolism ; Pigmentation/genetics ; *Insect Proteins/genetics ; *Adaptation, Physiological/genetics ; Ovum ; Female ; Seasons ; Diapause/genetics ; Gene Knockout Techniques ; },
abstract = {The production of overwintering eggs is a critical adaptation for winter survival among many insects. Melanization contributes to eggshell pigmentation and hardening, consequently enhancing resistance to environmental stress. The complex life cycle of the pea aphid (Acyrthosiphon pisum), a model hemipteran insect with remarkable reproductive capacity, involves cyclical parthenogenesis. It enables the production of black overwintering eggs that undergo obligate diapause to survive under unfavorable conditions. Laccase2 (Lac2) is essential for cuticle sclerotization and pigmentation in other insects. We hypothesized that Lac2 plays a critical role in aphid eggshell pigmentation and survival during diapause. To test the hypothesis, we used CRISPR/Cas9 ribonucleoprotein microinjections and a novel Direct Parental CRISPR (DIPA-CRISPR) method to knockout Lac2. In Lac2 knockout (KO) crispants (G0), pigment-less eggs correlated with induced indel rates. Additionally, eggshell pigmentation was completely lost in homozygous Lac2 knockouts, leading to embryonic lethality. Observation of late-stage embryos in KO diapause eggs suggested that lethality occurred during late embryogenesis or hatching. Furthermore, eggshell stiffness was significantly reduced in Lac2 KOs, highlighting the role of this gene in eggshell hardening. Moreover, fungal growth was observed in KO eggs. These findings reveal the essential roles of Lac2 in eggshell pigmentation, hardening, late embryonic development, hatching, and fungal protection, which are critical for pea aphid survival during overwintering diapause. This study also advances CRISPR/Cas9-mediated genome editing in pea aphids by addressing the challenges associated with their unique biology, including complex life cycles, obligatory diapause, bacterial endosymbiosis, inbreeding depression, and high nuclease activity. Our optimized protocol achieved efficient targeted mutagenesis and germline transmission, thereby generating stable KO lines. Additionally, we successfully applied DIPA-CRISPR to aphids by inducing mutations via adult oviparous female injections in fertilized eggs. These robust genome-editing protocols will facilitate functional studies in aphids, a key model for research on evolution, ecology, development, and agriculture.},
}

Animals
*CRISPR-Cas Systems/genetics
Gene Editing/methods
*Aphids/genetics/physiology
*Laccase/genetics/metabolism
Pigmentation/genetics
*Insect Proteins/genetics
*Adaptation, Physiological/genetics
Ovum
Female
Seasons
Diapause/genetics
Gene Knockout Techniques

@article {pmid40666904,
year = {2025},
author = {Pindi, C and Ahsan, M and Sinha, S and Palermo, G},
title = {Graph Attention Neural Networks Reveal TnsC Filament Assembly in a CRISPR-Associated Transposon.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {40666904},
issn = {2692-8205},
abstract = {CRISPR-associated transposons (CAST) enable programmable, RNA-guided DNA integration, marking a transformative advancement in genome engineering. A central player in the type V-K CAST system is the AAA+ ATPase TnsC, which assembles into helical filaments on double-stranded DNA (dsDNA) to orchestrate target site recognition and transposition. Despite its essential role, the molecular mechanisms underlying TnsC filament nucleation and elongation remain poorly understood. Here, multiple-microsecond and free energy simulations are combined with deep learning-based Graph Attention Network (GAT) models to elucidate the mechanistic principles of TnsC filament formation and growth. Our findings reveal that ATP binding promotes TnsC nucleation by inducing DNA remodelling and stabilizing key protein-DNA interactions, particularly through conserved residues in the initiator-specific motif (ISM). Furthermore, GNN-based attention analyses identify a directional bias in filament elongation in the 5'→3' direction and uncover a dynamic compensation mechanism between incoming and bound monomers that facilitate directional growth along dsDNA. By leveraging deep learning-based graph representations, our GAT model provides interpretable mechanistic insights from complex molecular simulations and is readily adaptable to a wide range of biological systems. Altogether, these findings establish a mechanistic framework for TnsC filament dynamics and directional elongation, advancing the rational design of CAST systems with enhanced precision and efficiency.},
}

@article {pmid40570715,
year = {2026},
author = {Fang, T and Deng, Y and Chen, M and Luo, T and Ning, T and Chen, G},
title = {Nanoparticles-mediated intratumoral gene editing targeting PD-L1 and Galectin-9 for improved cancer immunotherapy.},
journal = {Biomaterials},
volume = {324},
number = {},
pages = {123511},
doi = {10.1016/j.biomaterials.2025.123511},
pmid = {40570715},
issn = {1878-5905},
mesh = {*Galectins/genetics ; Animals ; *Nanoparticles/chemistry ; *B7-H1 Antigen/genetics ; *Immunotherapy/methods ; Mice ; *Gene Editing/methods ; Humans ; Calcium Phosphates/chemistry ; Cell Line, Tumor ; *Neoplasms/therapy/immunology/genetics ; CRISPR-Cas Systems/genetics ; Female ; Mice, Inbred C57BL ; },
abstract = {PD-L1, a typical immune checkpoint expressed on tumor cells, reduces the effectiveness of T cell-mediated killing, which is further aggravated by Galectin-9 (Gal-9) co-expression through the TIM3/Gal-9 pathway. Although immune checkpoint inhibitors have shown promise in cancer therapy, limitations remain including low response rate, systemic toxicities, and the need of frequent treatments. Here, we described a dual knockout approach targeting PD-L1 and Gal-9 in tumor cells, achieved by nanoparticle-assisted CRISPR-Cas9 delivery, aimed at improved cancer immunotherapy. A calcium phosphate nanoparticle (CaP NP) was engineered for co-delivery of CRISPR-Cas9/sgRNA ribonucleoprotein (RNP) and initiation of anti-tumor immunity. Intratumoral administration of RNP-loaded CaP NPs effectively knocked out PD-L1 and Gal-9 in tumor cells, evoking robust anti-tumor immunity. Additionally, Ca[2+] overload due to the degradation of CaP NPs led to release of damage-associated molecular patterns (DAMPs) signals, further enhancing T-cell-mediated antitumor immune responses. Our results demonstrated that this treatment effectively evoked both local and systemic anti-tumor immune responses, significantly inhibiting the growth of primary and distant tumors in mouse models. Importantly, local treatment also altered the phenotypes of circulating tumor cells, as a substantial of circulating tumor cells originated from RNP-CaP-treated primary tumors and exhibited dual knockouts, which led to reduced lung metastasis.},
}

*Galectins/genetics
Animals
*Nanoparticles/chemistry
*B7-H1 Antigen/genetics
*Immunotherapy/methods
Mice
*Gene Editing/methods
Humans
Calcium Phosphates/chemistry
Cell Line, Tumor
*Neoplasms/therapy/immunology/genetics
CRISPR-Cas Systems/genetics
Female
Mice, Inbred C57BL

@article {pmid40550345,
year = {2025},
author = {Bedir, I and Ozturk, K and Telci, D},
title = {Impact of PLA2G2A rs11573156 C > G Variant on Phospholipase Expression and Metastatic Behavior in Prostate Cancer.},
journal = {Gene},
volume = {964},
number = {},
pages = {149641},
doi = {10.1016/j.gene.2025.149641},
pmid = {40550345},
issn = {1879-0038},
mesh = {Male ; Humans ; *Prostatic Neoplasms/genetics/pathology ; *Group II Phospholipases A2/genetics/metabolism ; Neoplasm Metastasis ; Epithelial-Mesenchymal Transition/genetics ; Gene Expression Regulation, Neoplastic ; PC-3 Cells ; *Polymorphism, Single Nucleotide ; Cell Line, Tumor ; Cell Movement/genetics ; CRISPR-Cas Systems ; Alleles ; },
abstract = {Prostate cancer (PCa) is a major global health concern and a leading cause of cancer-related deaths in men. Despite available treatments, PCa frequently recurs and exhibits high metastatic potential. One key factor in its malignancy is PLA2G2A, a secretory phospholipase A2 enzyme with strong inflammatory properties. Elevated PLA2G2A levels in the bloodstream have been linked to tumor grade, potentially due to the rs11573156 C > G polymorphism in the gene's 5' untranslated region (5'UTR). Previously, we demonstrated that individuals carrying the G allele have a 2.5-fold lower risk of developing metastatic PCa compared to those with the C allele. To further investigate the functional impact of this polymorphism, we employed CRISPR-Cas9 base editing to convert the GC genotype of metastatic PC-3 prostate cancer cells into the homozygous GG genotype. Our findings indicate that the G allele enhances PLA2G2A expression while downregulating genes associated with epithelial-to-mesenchymal transition (EMT), an effect reversed upon siRNA-mediated PLA2G2A silencing. Furthermore, this genetic alteration impaired the migratory capacity of PC-3 cells following interferon-γ (IFN-γ) priming, simulating an inflammatory tumor microenvironment. Notably, siRNA-induced downregulation of PLA2G2A reversed the anti-migratory effects associated with the GG genotype. Our results suggest that increased PLA2G2A expression, driven by the G allele, negatively affects cell proliferation, EMT, and metastatic properties, underscoring its potential anti-tumor role in PCa.},
}

Male
Humans
*Prostatic Neoplasms/genetics/pathology
*Group II Phospholipases A2/genetics/metabolism
Neoplasm Metastasis
Epithelial-Mesenchymal Transition/genetics
Gene Expression Regulation, Neoplastic
PC-3 Cells
*Polymorphism, Single Nucleotide
Cell Line, Tumor
Cell Movement/genetics
CRISPR-Cas Systems
Alleles

@article {pmid38489095,
year = {2025},
author = {Yu, S and Lei, X and Qu, C},
title = {MicroRNA Sensors Based on CRISPR/Cas12a Technologies: Evolution From Indirect to Direct Detection.},
journal = {Critical reviews in analytical chemistry},
volume = {55},
number = {5},
pages = {968-984},
doi = {10.1080/10408347.2024.2329229},
pmid = {38489095},
issn = {1547-6510},
mesh = {*MicroRNAs/analysis/genetics ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; },
abstract = {MicroRNA (miRNA) has emerged as a promising biomarker for disease diagnosis and a potential therapeutic targets for drug development. The detection of miRNA can serve as a noninvasive tool in diseases diagnosis and predicting diseases prognosis. CRISPR/Cas12a system has great potential in nucleic acid detection due to its high sensitivity and specificity, which has been developed to be a versatile tool for nucleic acid-based detection of targets in various fields. However, conversion from RNA to DNA with or without amplification operation is necessary for miRNA detection based on CRISPR/Cas12a system, because dsDNA containing PAM sequence or ssDNA is traditionally considered as the activator of Cas12a. Until recently, direct detection of miRNA by CRISPR/Cas12a system has been reported. In this review, we provide an overview of the evolution of biosensors based on CRISPR/Cas12a for miRNA detection from indirect to direct, which would be beneficial to the development of CRISPR/Cas12a-based sensors with better performance for direct detection of miRNA.},
}

*MicroRNAs/analysis/genetics
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
Humans

@article {pmid40738541,
year = {2025},
author = {Choudhury, PR and Chakravarti, M and Banerjee, S and Baral, R and Bose, A},
title = {B-cell editing: An emerging alternative of precision oncotherapy.},
journal = {Advances in immunology},
volume = {166},
number = {},
pages = {103-135},
doi = {10.1016/bs.ai.2024.10.002},
pmid = {40738541},
issn = {1557-8445},
mesh = {Humans ; *Precision Medicine/methods ; *B-Lymphocytes/immunology/metabolism ; *Neoplasms/therapy/immunology/genetics ; *Gene Editing/methods ; Animals ; *Immunotherapy/methods ; *Cancer Vaccines/immunology/therapeutic use ; CRISPR-Cas Systems ; Antibodies, Monoclonal/therapeutic use/immunology ; },
abstract = {Lately, the urgency of precision medicine in cancer care through immunotherapy has reformed the arena of oncology. Although immunomodulatory therapeutics in cancer have been preliminarily concentrated on T-cells, emerging evidences have suggested that intra-tumoral B-cells and plasma cells have significant contributions in cancer prognosis primarily through the production of antibodies. B-cell oriented cancer vaccines have been used in early clinical trials of breast and other cancers after multiple preclinical studies. Passive immunotherapy via administration of monoclonal antibodies (mAbs) and emergence of anti-idiotypic antibodies have led to considerable advancement in oncotherapy. Endogenous production of mAbs would be of significant benefit in recurrent or residual malignancies and permanent infusion would help in the overcoming of issues related to pharmacodynamic variations observed in case of intravenous inoculations of bi or tri specific mAbs. This has directed towards the development of genome reprogrammed B-cells with the capability of yielding therapeutic mAbs independently. Genetic alteration through clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) nucleases have enabled the introduction of transgenes into B-cell genome thereby stimulating the plasma cells to produce exogenous remedial antibodies. It also facilitates ex vivo B-cell editing to elevate specificities of antigen receptors and generate target specific antibody responses which cannot normally be evoked in patient's immune system. Hence, genome-altered B-cells possess the potential of engineered therapeutics against certain malignancies. Co-operation of B-cells in T-cell based vaccines are ultimate need for vaccine success. In this chapter, the mechanisms, challenges and potential advantages of B-cell editing in cancer immune therapy shall be explored. The prospects of B-cell editing in onco-therapy will be clearly elucidated with all its strength and weaknesses.},
}

Humans
*Precision Medicine/methods
*B-Lymphocytes/immunology/metabolism
*Neoplasms/therapy/immunology/genetics
*Gene Editing/methods
Animals
*Immunotherapy/methods
*Cancer Vaccines/immunology/therapeutic use
CRISPR-Cas Systems
Antibodies, Monoclonal/therapeutic use/immunology

@article {pmid40737091,
year = {2025},
author = {Jain, S and Planells, J and Regadas, I and Barrett, D and von Euler, A and Sinha, I and Lindberg, BG and Hesson, JC and Swacha, P and Gekara, NO and Pelechano, V and Engström, Y and Mannervik, M and Visa, N},
title = {A U3 snoRNA is required for the regulation of chromatin dynamics and antiviral response in Drosophila melanogaster.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
doi = {10.1093/nar/gkaf715},
pmid = {40737091},
issn = {1362-4962},
support = {2019-02335//Swedish Research Council/ ; 2020-01480//Swedish Research Council/ ; 2021-06112//Swedish Research Council/ ; KAW 2021.0167//Wallenberg Academy Fellowship/ ; //Karolinska Institute/ ; 2024-04173//Swedish Research Council/ ; 20 1044 Pj//The Swedish Cancer Society/ ; 23 2963 Pj//The Swedish Cancer Society/ ; 2022-01308_3//Swedish Research Council/ ; CAN 23 3096 Pj//The Swedish Cancer Society/ ; //Stockholm University/ ; //Stockholm University/ ; },
mesh = {Animals ; *Drosophila melanogaster/genetics/virology/immunology ; *RNA, Small Nucleolar/genetics/metabolism ; *Chromatin/genetics/metabolism ; Drosophila Proteins/genetics/metabolism ; Sindbis Virus/immunology/genetics ; Chromatin Assembly and Disassembly/genetics ; Larva/genetics/virology ; CRISPR-Cas Systems ; Trans-Activators ; Cell Cycle Proteins ; },
abstract = {Small nucleolar RNAs (snoRNAs) are prevailing components of the chromatin-associated transcriptome. Here we show that specific snoRNAs are required for the activation of immune response genes and for survival during viral infections in Drosophila melanogaster. We have studied snoRNA:U3:9B, a chromatin-associated snoRNA that binds to a large number of protein coding genes, including immune response genes. We have used CRISPR/Cas9 to delete snoRNA:U3:9B and study its function in vivo. SnoRNA:U3:9B-deficient larvae are viable but failed to develop into pupae when challenged by expression of a Sindbis virus replicon. SnoRNA:U3:9B is localized to immune genes in vivo and the chromatin decompaction and gene activation typically observed at immune genes following infection are abolished in snoRNA:U3:9B-deficient larvae, which suggests that this snoRNA acts locally to regulate chromatin accessibility. Mechanistically, snoRNA:U3:9B is required for the recruitment of the chromatin remodeler Brahma to a set of target immune genes. In summary, these results uncover an antiviral defense mechanism that relies on a snoRNA for the recruitment of a chromatin remodeling factor to immune genes to facilitate immune gene activation.},
}

Animals
*Drosophila melanogaster/genetics/virology/immunology
*RNA, Small Nucleolar/genetics/metabolism
*Chromatin/genetics/metabolism
Drosophila Proteins/genetics/metabolism
Sindbis Virus/immunology/genetics
Chromatin Assembly and Disassembly/genetics
Larva/genetics/virology
CRISPR-Cas Systems
Trans-Activators
Cell Cycle Proteins

@article {pmid40736862,
year = {2025},
author = {Mercer, GD and Ballios, BG and Kertes, PJ},
title = {Mutation Specific Treatments for Inherited Retinal Diseases.},
journal = {Advances in experimental medicine and biology},
volume = {1467},
number = {},
pages = {337-341},
pmid = {40736862},
issn = {0065-2598},
mesh = {Humans ; *Genetic Therapy/methods ; *Retinal Diseases/genetics/therapy ; *Gene Editing/methods ; *Mutation ; CRISPR-Cas Systems ; Animals ; Oligonucleotides, Antisense/therapeutic use/genetics ; RNA Interference ; RNA Editing ; },
abstract = {The next frontier in genetic therapy for IRDs is the correction or silencing of specific mutations. This is of relevance to conditions with dominant negative and gain-of-function disease mechanisms, or with causative genes that are too large for gene replacement using AAV vectors. We discuss two approaches that have reached the stage of human clinical trials: CRISPR-Cas9 based gene editing and post-transcriptional gene silencing using antisense oligonucleotides. Other mutation-specific treatment approaches in pre-clinical development include adenosine deaminases acting on RNA (ADAR)-based RNA editing, RNA interference, and translational read through inducing drugs (TRADs). These have been reviewed elsewhere (e.g., Martinez Velazquez and Ballios 2021).},
}

Humans
*Genetic Therapy/methods
*Retinal Diseases/genetics/therapy
*Gene Editing/methods
*Mutation
CRISPR-Cas Systems
Animals
Oligonucleotides, Antisense/therapeutic use/genetics
RNA Interference
RNA Editing

@article {pmid40736578,
year = {2025},
author = {Allard-Chamard, X and Rodríguez, EC and Brais, B and Armstrong, GAB},
title = {Loss of dcst2 expression in male zebrafish is not associated with muscle hypertrophy.},
journal = {Molecular genetics and genomics : MGG},
volume = {300},
number = {1},
pages = {74},
doi = {10.1007/s00438-025-02279-x},
pmid = {40736578},
issn = {1617-4623},
support = {Natural Sciences and Engineering Research Council of Canada//Natural Sciences and Engineering Research Council of Canada/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Zebrafish Proteins/genetics/metabolism ; Male ; Hypertrophy/genetics ; CRISPR-Cas Systems ; *Membrane Proteins/genetics/metabolism ; *Muscle, Skeletal/metabolism/pathology ; Frameshift Mutation ; },
abstract = {Recently, a large family of French-Canadians was found to possess above-average strength and muscle hypertrophy that segregated with a single variant in the gene encoding Dendritic Cell-specific Six Transmembrane domain containing protein 2 (DCST2). To investigate the potential role DCST2 has in muscle cell biology we used the CRISPR/Cas9 mutagenic system and generated a 2-nucleotide deletion in exon 3 of zebrafish dcst2 resulting in a frameshift mutation. Homozygous carriers of the mutation displayed reduced transcriptional expression of dcst2 suggesting that our mutation disrupted gene expression. Homozygous mutant dcst2 zebrafish developed normally to adulthood and displayed no differences in motor function using a free-swim and swim tunnel assays. Furthermore, histological examination of muscle cells revealed no differences in slow-twitch or fast-twitch muscle cell cross-sectional area in our mutants. We did observe that male dcst2[-/-] zebrafish were infertile. The data collected here, suggest that dcst2 does not play a role in zebrafish muscle cell biology.},
}

Animals
*Zebrafish/genetics
*Zebrafish Proteins/genetics/metabolism
Male
Hypertrophy/genetics
CRISPR-Cas Systems
*Membrane Proteins/genetics/metabolism
*Muscle, Skeletal/metabolism/pathology
Frameshift Mutation

@article {pmid40735630,
year = {2025},
author = {Wu, L and Yu, Z and Li, P and Wang, Z and Li, Q and Bao, H and García, P and Letarov, A and Zhou, Y and Zhang, H},
title = {Genome MLST scheme for tracing genetic diversity and multidrug resistance of food animal-derived Clostridium perfringens.},
journal = {Current research in food science},
volume = {11},
number = {},
pages = {101149},
pmid = {40735630},
issn = {2665-9271},
abstract = {Clostridium perfringens is an important food-borne anaerobic bacterium that can cause chronic disease in humans and animals. The complex toxins that it produces can cause diarrhea, necrotizing enteritis, and even death. This study aimed to evaluate the presence of antibiotic resistance genes and determinants of toxin production in various strains of C. perfringens, derived from different geographical locations and animal sources, through whole-genome sequencing (WGS) and bioinformatics analysis. Antimicrobial resistance testing revealed a notable prevalence of resistance among isolates, with 39.5 % being resistant to clindamycin and 32.6 % resistant to tetracycline. Additionally, 29.1 % of the isolates were found to be resistant to at least two classes of commonly used antibiotics. TetA(P) and tetB(P), associated with tetracycline resistance, were the most frequently identified resistance genes, present in 93.0 % and 79.0 % of the isolates, respectively. Virulence genes encoding extracellular collagenases (colA and colSI), alpha-toxin (plc), and sialidase (nanH) were detected in 85 isolates, representing 99 % of all sequenced strains. Notably, the gene encoding C. perfringens enterotoxin (cpe) was identified in only one isolate, which originated from chicken. Multi-locus sequence typing revealed that the 86 representative isolates belonged to 49 sequence types (STs), including 33 unique, previously uncharacterized STs. Furthermore, 30.23 % of these STs were grouped into six clonal complexes (CCs). CgSNP analysis of globally circulating isolates demonstrated that the prevalence of the virulence gene cpe was higher in these isolates than in clinical strains. The identification of multi-drug resistance and toxin-encoding genes among the isolates underscores the concerning spread of antimicrobial resistance among C. perfringens affecting both animals and humans. The diversity of CRISPR/Cas system provided addition insight into complex genetic correlations and evolutionary dynamics of pathogenic C. perfringens. Notably, the phage SD72 exhibited a broad inhibitory effect against C. perfringens isolates, irrespective of STs types and antimicrobial resistance (74/86, 80.1 %). These findings highlight the urgent need for enhanced epidemic surveillance of C. perfringens in livestock settings to mitigate the risks of human transmission via environmental or food sources. Additionally, bacteriophages present a promising avenue as bio-antimicrobial agents for controlling the transmission of C. perfringens from animal sources to humans.},
}

@article {pmid40733833,
year = {2025},
author = {Cheng, Y and Wang, Z and Cao, M and Liu, S and Zhang, X and Cheng, C and Cai, C and Zhang, X and Zheng, J},
title = {Structure-activity relationships study on inhibition of CRISPR-Cas9 by glycosaminoglycans.},
journal = {Carbohydrate polymers},
volume = {366},
number = {},
pages = {123912},
doi = {10.1016/j.carbpol.2025.123912},
pmid = {40733833},
issn = {1879-1344},
mesh = {*CRISPR-Cas Systems/drug effects ; Structure-Activity Relationship ; *Heparin/chemistry/pharmacology ; *Chondroitin Sulfates/chemistry/pharmacology ; *Glycosaminoglycans/chemistry/pharmacology ; Molecular Dynamics Simulation ; Gene Editing ; *CRISPR-Associated Protein 9/antagonists & inhibitors/metabolism ; Humans ; },
abstract = {The CRISPR-Cas9 system is a revolutionary genome editing system known for its precision, simplicity and efficiency, playing a crucial role in gene-editing. It has enabled applications ranging from biomedicine to agriculture. However, the uncontrollable activity of Cas9 has raised significant safety concerns in clinical settings, limiting its broader application. Consequently, regulating CRISPR-Cas9 activity holds substantial promise for enhancing the safety and efficacy of gene-editing technologies. In this study, we demonstrated that sulfated glycosaminoglycans (GAGs) exhibit inhibitory effects of Cas9. Specifically, both chondroitin sulfate (CS) and heparin (HP) can inhibit CRISPR/Cas9 activity, with heparin showing a stronger inhibitory effect that correlates positively with its concentration. Molecular dynamics simulations indicate that sulfated heparin residues might inhibit Cas9 function by binding to essential DNA-binding sites, which are crucial for functional interactions, potentially impairing activity. Additionally, higher molecular weight GAGs exhibit enhanced inhibitory effects under the same sulphation. Notably, the sulfation site also influenced activity. C6-sulfation of chondroitin sulfate is more favorable for Cas9 inhibition, and N-sulfation of heparin enhances its inhibitory effect on Cas9 activity. These findings provide valuable insights into the development of carbohydrate-based inhibitors for CRISPR-Cas9, offering a foundation for further exploration in this field.},
}

*CRISPR-Cas Systems/drug effects
Structure-Activity Relationship
*Heparin/chemistry/pharmacology
*Chondroitin Sulfates/chemistry/pharmacology
*Glycosaminoglycans/chemistry/pharmacology
Molecular Dynamics Simulation
Gene Editing
*CRISPR-Associated Protein 9/antagonists & inhibitors/metabolism
Humans

@article {pmid40733529,
year = {2025},
author = {Anastassopoulou, C and Tsakri, D and Panagiotopoulos, AP and Saldari, C and Sagona, AP and Tsakris, A},
title = {Armed Phages: A New Weapon in the Battle Against Antimicrobial Resistance.},
journal = {Viruses},
volume = {17},
number = {7},
pages = {},
doi = {10.3390/v17070911},
pmid = {40733529},
issn = {1999-4915},
mesh = {*Bacteriophages/genetics/physiology ; Humans ; *Phage Therapy/methods ; *Bacterial Infections/therapy/microbiology ; *Bacteria/virology/drug effects ; Drug Resistance, Multiple, Bacterial ; CRISPR-Cas Systems ; Animals ; Anti-Bacterial Agents/pharmacology ; },
abstract = {The increasing prevalence of multidrug-resistant (MDR) bacterial infections necessitates the exploration of alternative antimicrobial strategies, with phage therapy emerging as a viable option. However, the effectiveness of naturally occurring phages can be significantly limited by bacterial defense systems that include adsorption blocking, restriction-modification, CRISPR-Cas immunity, abortive infection, and NAD+ depletion defense systems. This review examines these bacterial defenses and their implications for phage therapy, while highlighting the potential of phages' bioengineering to overcome these barriers. By leveraging synthetic biology, genetically engineered phages can be tailored to evade bacterial immunity through such modifications as receptor-binding protein engineering, anti-CRISPR gene incorporation, methylation pattern alterations, and enzymatic degradation of bacterial protective barriers. "Armed phages", enhanced with antimicrobial peptides, CRISPR-based genome-editing tools, or immune-modulating factors, offer a novel therapeutic avenue. Clinical trials of bioengineered phages, currently SNIPR001 and LBP-EC01, showcase their potential to safely and effectively combat MDR infections. SNIPR001 has completed a Phase I clinical trial evaluating safety in healthy volunteers, while LBP-EC01 is in Phase II trials assessing its performance in the treatment of Escherichia coli-induced urinary tract infections in patients with a history of drug-resistant infections. As "armed phages" progress toward clinical application, they hold great promise for precision-targeted antimicrobial therapies and represent a critical innovation in addressing the global antibiotic resistance crisis.},
}

*Bacteriophages/genetics/physiology
Humans
*Phage Therapy/methods
*Bacterial Infections/therapy/microbiology
*Bacteria/virology/drug effects
Drug Resistance, Multiple, Bacterial
CRISPR-Cas Systems
Animals
Anti-Bacterial Agents/pharmacology

@article {pmid40733516,
year = {2025},
author = {Lin, L and Lui, WY and Ong, CP and Yau, MY and Jin, DY and Yuen, KS},
title = {CRISPR/Cas13-Mediated Inhibition of EBNA1 for Suppression of Epstein-Barr Virus Transcripts and DNA Load in Nasopharyngeal Carcinoma Cells.},
journal = {Viruses},
volume = {17},
number = {7},
pages = {},
doi = {10.3390/v17070899},
pmid = {40733516},
issn = {1999-4915},
support = {UGC/FDS17/M07/22//Research Grants Council of the Hong Kong Special Administrative Region/ ; },
mesh = {Humans ; *Herpesvirus 4, Human/genetics/drug effects ; *Epstein-Barr Virus Nuclear Antigens/genetics/metabolism ; *CRISPR-Cas Systems ; *Nasopharyngeal Carcinoma/virology ; Cell Line, Tumor ; *Nasopharyngeal Neoplasms/virology ; *Viral Load ; *Epstein-Barr Virus Infections/virology/therapy ; DNA, Viral/genetics ; },
abstract = {Epstein-Barr virus (EBV), a double-stranded DNA virus, is implicated in nasopharyngeal carcinoma (NPC), with particularly high incidence in regions such as southern China and Hong Kong. Although NPC is typically treated with radio- and chemotherapy, outcomes remain poor for advanced-stage diagnoses, highlighting the need for targeted therapies. This study explores the potential of CRISPR/CRISPR-associated protein 13 (Cas13) technology to target essential EBV RNA in NPC cells. Previous research demonstrated that CRISPR/Cas9 could partially reduce EBV load, but suppression was incomplete. Here, the combination of CRISPR/Cas13 with CRISPR/Cas9 shows enhanced viral clearance. Long-term EBNA1 suppression via CRISPR/Cas13 reduced the EBV genome, improved CRISPR/Cas9 effectiveness, and identified suitable AAV serotypes for delivery. Furthermore, cotreatment increased NPC cell sensitivity to 5-fluorouracil and cisplatin. These findings underscore the potential of CRISPR/Cas13 as an anti-EBV therapeutic approach, effectively targeting latent EBV transcripts and complementing existing treatments. The study suggests a promising new direction for developing anti-EBV strategies, potentially benefiting therapies for NPC and other EBV-associated malignancies.},
}

Humans
*Herpesvirus 4, Human/genetics/drug effects
*Epstein-Barr Virus Nuclear Antigens/genetics/metabolism
*CRISPR-Cas Systems
*Nasopharyngeal Carcinoma/virology
Cell Line, Tumor
*Nasopharyngeal Neoplasms/virology
*Viral Load
*Epstein-Barr Virus Infections/virology/therapy
DNA, Viral/genetics

@article {pmid40733494,
year = {2025},
author = {Wo, L and Qi, S and Guo, Y and Sun, C and Yin, X},
title = {TRIM5α/Cyclophilin A-Modified MDBK Cells for Lentiviral-Based Gene Editing.},
journal = {Viruses},
volume = {17},
number = {7},
pages = {},
doi = {10.3390/v17070876},
pmid = {40733494},
issn = {1999-4915},
support = {32370162//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Cyclophilin A/genetics/metabolism ; Cattle ; *Lentivirus/genetics ; Cell Line ; CRISPR-Cas Systems ; *Tripartite Motif Proteins/genetics/metabolism ; HIV-1/genetics ; Ubiquitin-Protein Ligases/genetics ; Humans ; },
abstract = {The human immunodeficiency virus 1 (HIV-1)-based lentivirus has been widely used for genetic modification. However, the efficiency of lentiviral-based gene modification in Madin-Darby bovine kidney (MDBK) cells is considerably limited. In this study, we have shown that siRNA-mediated depletion of TRIM5α, a restriction factor in HIV-1 infection, can dramatically enhance HIV-1 infection in MDBK cells. Furthermore, we generated a doxycycline-inducible Cas9-overexpressing MDBK cell line (MDBK-iCas9) suitable for CRISPR/Cas9-mediated editing. On this basis, we created a TRIM5α knock-out MDBK-iCas9 cell line MDBK-iCas9[TRIM5α-/-] without additional genome insertions by combining sgRNA transfection and single-cell cloning. We found that MDBK-iCas9[TRIM5α-/-] displayed greater permissiveness to lentivirus infection compared with MDBK-WT cells. Notably, we found that treatment with the chemical compound cyclosporine A, which directly interacts with cell factor cyclophilin A (CypA), could markedly increase the infectivity of lentivirus in both MDBK-iCas9[TRIM5α-/-] and MDBK-WT cell lines, suggesting that CypA functions independently with TRIM5α as an inhibitor of the lentivirus in bovine cells. Therefore, combining bovine TRIM5α and CypA targeting could remarkably enhance lentivirus infection. In conclusion, our findings highlight a promising gene engineering strategy for bovine cells that can surmount the significant barriers to investigating the interplay between bovine viruses and their host cells.},
}

Animals
*Gene Editing/methods
*Cyclophilin A/genetics/metabolism
Cattle
*Lentivirus/genetics
Cell Line
CRISPR-Cas Systems
*Tripartite Motif Proteins/genetics/metabolism
HIV-1/genetics
Ubiquitin-Protein Ligases/genetics
Humans

@article {pmid40733490,
year = {2025},
author = {Jiang, X and Huang, Y and Jiang, Y and Yang, G and Zheng, X and Gao, S},
title = {CRISPR-Cas12a/RPA Dual-Readout Assay for Rapid Field Detection of Porcine Rotavirus with Visualization.},
journal = {Viruses},
volume = {17},
number = {7},
pages = {},
doi = {10.3390/v17070872},
pmid = {40733490},
issn = {1999-4915},
support = {Grant No: SCKJ-JYRC-2023-29//the Project of Sanya Yazhou Bay Science and Technology City/ ; Grant No: 324QN343.//the Youth Fund Project of Hainan Provincial Natural Science Foundation/ ; },
mesh = {Animals ; Swine ; *Swine Diseases/virology/diagnosis ; *Rotavirus/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Rotavirus Infections/diagnosis/veterinary/virology ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Recombinases/metabolism/genetics ; Capsid Proteins/genetics ; Endodeoxyribonucleases/genetics ; Antigens, Viral/genetics ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; },
abstract = {PoRV is a significant etiological agent of neonatal diarrhea in piglets, resulting in substantial economic losses within the global swine industry due to elevated mortality rates and reduced productivity. To address the urgent need for accessible and rapid diagnostics in resource-limited settings, we have developed a CRISPR/Cas12a-based assay integrated with recombinase polymerase amplification (RPA) for the visual detection of PoRV. This platform specifically targets the conserved VP6 gene using optimized RPA primers and crRNA, harnessing Cas12a's collateral cleavage activity to enable dual-readout via fluorescence or lateral flow dipsticks (LFDs). The assay demonstrates a detection limit of 10[2] copies/μL within 1 h, exhibiting no cross-reactivity with phylogenetically related pathogens such as Transmissible Gastroenteritis Virus (TGEV). By eliminating reliance on thermal cyclers or specialized equipment, this method is fully deployable in swine farms, veterinary clinics, or field environments. The lateral flow format provides immediate colorimetric results that require minimal technical expertise, while the fluorescence mode allows for semi-quantitative analysis. This study presents a robust and cost-effective platform for decentralized PoRV surveillance in swine populations, addressing the critical need for portable diagnostics in resource-limited settings and enhancing veterinary health management.},
}

Animals
Swine
*Swine Diseases/virology/diagnosis
*Rotavirus/genetics/isolation & purification
*CRISPR-Cas Systems
*Rotavirus Infections/diagnosis/veterinary/virology
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
Recombinases/metabolism/genetics
Capsid Proteins/genetics
Endodeoxyribonucleases/genetics
Antigens, Viral/genetics
CRISPR-Associated Proteins/genetics
Bacterial Proteins

@article {pmid40732725,
year = {2025},
author = {Naveed, S and Brown, JK and Mubin, M and Javed, N and Nawaz-Ul-Rehman, MS},
title = {Potential for Duplexed, In-Tandem gRNA-Mediated Suppression of Two Essential Genes of Tomato Leaf Curl New Delhi Virus in Crop Plants.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {7},
pages = {},
doi = {10.3390/pathogens14070679},
pmid = {40732725},
issn = {2076-0817},
support = {6427//Higher Education Commission of Pakistan/ ; },
mesh = {*Begomovirus/genetics ; *Plant Diseases/virology ; *Solanum lycopersicum/virology ; Capsicum/virology ; Nicotiana/virology ; CRISPR-Cas Systems ; Genome, Viral ; Capsid Proteins/genetics ; },
abstract = {Tomato leaf curl New Delhi virus (ToLCNDV) is among the most prevalent and widely distributed begomovirus infecting chili pepper (Capsicum annuum) and tomato in the Indian subcontinent. In this study, a guide RNA (gRNA) sequence-CRISPR-Cas9 approach was used to target and cleave two essential coding regions in the begomovirus genome. The gRNAs were designed to target conserved regions of the ToLCNDV replication-associated protein (rep) gene or ORF AC1, and/or the coat protein (cp) gene or AV1 ORF, respectively. Based on an alignment of 346 representative ToLCNDV genome sequences, all predicted single nucleotide polymorphisms off-target sites were identified and eliminated as potential gRNA targets. Based on the remaining genome regions, four candidate gRNAs were designed and used to build gRNA-Cas9 duplexed constructs, e.g., containing two gRNAs cloned in tandem, in different combinations (1-4). Two contained two gRNAs that targeted the coat protein gene (cp; AV1 ORF), while the other two constructs targeted both the cp and replication-associated protein gene (rep; AC1 ORF). These constructs were evaluated for the potential to suppress ToLCNDV infection in Nicotiana benthamiana plants in a transient expression-transfection assay. Among the plants inoculated with the duplexed gRNA construct designed to cleave ToLCNDV-AV1 or AC1-specific nucleotides, the construct designed to target both the cp (293-993 nt) and rep (1561-2324) showed the greatest reduction in virus accumulation, based on real-time quantitative PCR amplification, and attenuated disease symptoms, compared to plants inoculated with the DNA-A component alone or mock-inoculated, e.g., with buffer. The results demonstrate the potential for gRNA-mediated suppression of ToLCNDV infection in plants by targeting at least two viral coding regions, underscoring the great potential of CRISPR-Cas-mediated abatement of begomovirus infection in numerous crop species.},
}

*Begomovirus/genetics
*Plant Diseases/virology
*Solanum lycopersicum/virology
Capsicum/virology
Nicotiana/virology
CRISPR-Cas Systems
Genome, Viral
Capsid Proteins/genetics

@article {pmid40732701,
year = {2025},
author = {Hanssens, C and Van Cleemput, J},
title = {Applying CRISPR Technologies for the Treatment of Human Herpesvirus Infections: A Scoping Review.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {7},
pages = {},
doi = {10.3390/pathogens14070654},
pmid = {40732701},
issn = {2076-0817},
support = {202409/004//Ghent University/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Herpesviridae Infections/therapy/virology/genetics ; Gene Editing/methods ; *Herpesviridae/genetics ; Antiviral Agents/therapeutic use ; *Genetic Therapy/methods ; Virus Latency/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {BACKGROUND: Human herpesviruses are double-stranded DNA viruses of which eight types have been identified at present. Herpesvirus infection comprises an active lytic phase and a lifelong latency phase with the possibility of reactivation. These infections are highly prevalent worldwide and can lead to a broad spectrum of clinical manifestations, ranging from mild symptoms to severe disease, particularly in immunocompromised individuals. Clustered regularly interspaced palindromic repeats (CRISPR)-based therapy is an interesting alternative to current antiviral drugs, which fail to cure latent infections and are increasingly challenged by viral resistance.

OBJECTIVE: This scoping review aimed to summarize the current state of CRISPR-based antiviral strategies against herpesvirus infections, highlighting the underlying mechanisms, study design and outcomes, and challenges for clinical implementation.

DESIGN: A literature search was conducted in the databases PubMed and Web of Science, using both a general and an individual approach for each herpesvirus.

RESULTS: This scoping review identified five main mechanisms of CRISPR-based antiviral therapy against herpesvirus infections in vitro and/or in vivo. First, CRISPR systems can inhibit the active lytic replication cycle upon targeting viral lytic genes or host genes. Second, CRISPR technologies can remove latent viral genomes from infected cells by targeting viral genes essential for latency maintenance or destabilizing the viral genome. Third, reactivation of multiple latent herpesvirus infections can be inhibited by CRISPR-Cas-mediated editing of lytic viral genes, preventing a flare-up of clinical symptoms and reducing the risk of viral transmission. Fourth, CRISPR systems can purposefully induce viral reactivation to enhance recognition by the host immune system or improve the efficacy of existing antiviral therapies. Fifth, CRISPR technology can be applied to develop or enhance the efficiency of cellular immunotherapy.

CONCLUSIONS: Multiple studies demonstrate the potential of CRISPR-based antiviral strategies to target herpesvirus infections through various mechanisms in vitro and in vivo. However, aspects regarding the delivery and biosafety of CRISPR systems, along with the time window for treatment, require further investigation before broad clinical implementation can be realized.},
}

Humans
*CRISPR-Cas Systems
*Herpesviridae Infections/therapy/virology/genetics
Gene Editing/methods
*Herpesviridae/genetics
Antiviral Agents/therapeutic use
*Genetic Therapy/methods
Virus Latency/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats