@article {pmid40333994,
year = {2025},
author = {Chen, H and LaFlamme, CW and Wang, YD and Blan, AW and Koehler, N and Mendonca Moraes, R and Olszewski, AR and Almanza Fuerte, EP and Bonkowski, ES and Bajpai, R and Lavado, A and Pruett-Miller, SM and Mefford, HC},
title = {Patient-derived models of UBA5-associated encephalopathy identify defects in neurodevelopment and highlight potential therapeutic avenues.},
journal = {Science translational medicine},
volume = {17},
number = {797},
pages = {eadn8417},
doi = {10.1126/scitranslmed.adn8417},
pmid = {40333994},
issn = {1946-6242},
mesh = {Humans ; Organoids/metabolism/pathology ; *Ubiquitin-Activating Enzymes/genetics/metabolism ; *Brain Diseases/genetics/pathology/therapy ; Endoplasmic Reticulum/metabolism ; *Models, Biological ; Male ; Female ; Unfolded Protein Response ; CRISPR-Cas Systems/genetics ; Neurons/metabolism/pathology ; },
abstract = {UBA5 encodes for the E1 enzyme of the UFMylation cascade, which plays an essential role in endoplasmic reticulum (ER) homeostasis. The clinical phenotypes of UBA5-associated encephalopathy include developmental delays, epilepsy, and intellectual disability. To date, there is no humanized neuronal model to study the cellular and molecular consequences of UBA5 pathogenic variants. We developed and characterized patient-derived cortical organoid cultures from two patients with compound heterozygous variants in UBA5. Both shared the same missense variant, which encodes a hypomorphic allele (p.A371T), along with a nonsense variant (p.G267* or p.A123fs*4). Single-cell RNA sequencing of 100-day organoids identified defects in GABAergic interneuron development. We demonstrated aberrant neuronal firing and reduction in size of patient-derived organoids. Mechanistically, we showed that ER homeostasis is perturbed along with an exacerbated unfolded protein response pathway in engineered U87-MG cells and patient-derived organoids expressing UBA5 pathogenic variants. We also assessed two potential therapeutic modalities that augmented UBA5 protein abundance to rescue aberrant molecular and cellular phenotypes. We assessed SINEUP, a long noncoding RNA that augments translation efficiency, and CRISPRa, a modified CRISPR-Cas9 approach to augment transcription efficiency to increase UBA5 protein production. Our study provides a humanized model that allows further investigations of UBA5 variants in the brain and highlights promising approaches to alleviate cellular aberrations for this rare, developmental disorder.},
}

Humans
Organoids/metabolism/pathology
*Ubiquitin-Activating Enzymes/genetics/metabolism
*Brain Diseases/genetics/pathology/therapy
Endoplasmic Reticulum/metabolism
*Models, Biological
Male
Female
Unfolded Protein Response
CRISPR-Cas Systems/genetics
Neurons/metabolism/pathology

@article {pmid40333840,
year = {2025},
author = {Chang, J and Yang, X and Zhang, T and Sun, H and Cheng, H and Jia, Z and Li, Y and Ma, S and Sun, T and Cao, J},
title = {High-Throughput Screening to Identify Novel Compounds Affecting the Genome Editing Efficiency of CRISPR System.},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {8},
pages = {},
pmid = {40333840},
issn = {1420-3049},
support = {202203021212372//Applied Basic Research Program of Shanxi Province/ ; 82170523//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/drug effects ; HEK293 Cells ; *High-Throughput Screening Assays/methods ; },
abstract = {Genome editing is a promising therapeutic strategy for genetic disorders by modifying the genome precisely, especially the CRISPR/Cas9 system. However, a major limitation of CRISPR/Cas9 in gene therapy is the biosafety issues caused by off-target effects. Compounds that can modulate the genome editing efficiency of the CRISPR/Cas9 system, especially those reducing the off-target effects, are potentially useful pharmacological tools for improving the effectiveness and safety of genome editing. Here, we performed high-throughput screening in HEK 293FT cells to discover compounds that decrease or increase the genome editing efficiency of the CRISPR/Cas9 system from 9930 compounds. After two rounds of screening, we identified that CP-724714, a ErbB2 (HER2) tyrosine kinase inhibitor, decreased the CRISPR/Cas9 efficiency and reduced the off-target effects by suppressing the efficiency of CRISPR/Cas9, and was thus named a CRISPR decelerator (or inhibitor), while Clofarabine, a DNA synthesis inhibitor, increased the efficiency of CRISPR/Cas9, and was named a CRISPR accelerator. We further identified four compounds (Tranilast, Cerulenin, Rosolic acid and Resveratrol) that affected the efficiency of single-strand annealing (SSA) repair. Among them, Tranilast, Cerulenin and Rosolic acid are potential SSA decelerators, while Resveratrol is a potential SSA accelerator. These identified compounds may be useful in optimizing mammalian genetic manipulation techniques.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/drug effects
HEK293 Cells
*High-Throughput Screening Assays/methods

@article {pmid40333207,
year = {2025},
author = {Ouyang, Y and Xia, Y and Tang, X and Qin, L and Xia, S},
title = {Trans-Kingdom sRNA Silencing in Sclerotinia sclerotiorum for Crop Fungal Disease Management.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {4},
pages = {},
pmid = {40333207},
issn = {2076-0817},
support = {31971836//the National Nature Science Foundation of China/ ; },
mesh = {*Ascomycota/genetics/pathogenicity ; *Plant Diseases/microbiology/prevention & control ; *RNA Interference ; *Crops, Agricultural/microbiology ; Gene Silencing ; Disease Management ; *RNA, Fungal/genetics ; },
abstract = {Sclerotinia sclerotiorum is a globally widespread and vast destructive plant pathogenic fungus that causes significant yield losses in crops. Due to the lack of effective resistant germplasm resources, the control of diseases caused by S. sclerotiorum largely relies on chemical fungicides. However, excessive use of these chemicals not only causes environmental concerns but also leads to the increased development of resistance in S. sclerotiorum. In contrast, trans-kingdom sRNA silencing-based technologies, such as host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS), offer novel, effective, and environmentally friendly methods for the management of S. sclerotiorum infection. This review summarizes recent advances in the identification of S. sclerotiorum pathogenic genes, target gene selection, categories, and application of trans-kingdom RNA interference (RNAi) technologies targeting this pathogen. Although some challenges, including off-target effects and the efficiency of external sRNA uptake, exist, recent findings have proposed solutions for further improvement. Combined with the latest developments in CRISPR/Cas gene editing and other technologies, trans-kingdom RNAi has significant potential to become a crucial tool in the control of sclerotinia stem rot (SSR), mitigating the impact of S. sclerotiorum on crop production.},
}

*Ascomycota/genetics/pathogenicity
*Plant Diseases/microbiology/prevention & control
*RNA Interference
*Crops, Agricultural/microbiology
Gene Silencing
Disease Management
*RNA, Fungal/genetics

@article {pmid40333073,
year = {2025},
author = {Saenz-Garcia, JL and Souza-Melo, N and Miranda, JS and Borges, B and Pacheco-Lugo, LA and Quintero-Solano, JM and Moretti, N and Wheeler, R and Soares-Medeiros, LC and DaRocha, WD},
title = {Kharon Is Crucial for Trypanosoma cruzi Morphology but Does Not Impair In Vitro Infection.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {4},
pages = {},
pmid = {40333073},
issn = {2076-0817},
support = {2020221000012//Fundação Araucária/ ; 2018/09948-0//FAPESP/ ; 2020/07870-4//FAPESP/ ; 2022/03075-0//FAPESP/ ; },
mesh = {*Trypanosoma cruzi/genetics/metabolism/growth & development ; *Protozoan Proteins/genetics/metabolism ; *Chagas Disease/parasitology ; Animals ; Cytoskeleton/metabolism ; CRISPR-Cas Systems ; },
abstract = {Chagas disease, caused by Trypanosoma cruzi, is a neglected tropical disease with few options for treatment and no available vaccine. Deletion mutants for live attenuated vaccines, particularly deletions of proteins related to the cytoskeleton, have been widely tested in related parasites but candidates have not been tested in T. cruzi. Kharon is one such protein, identified as being associated with the cytoskeleton in Leishmania and essential for amastigote replication. Here we investigated the T. cruzi Kharon ortholog (TcKharon) to test if it has orthologous function and thus potential in generating a live attenuated vaccine. In silico analysis predicted TcKharon to be an intrinsically disordered protein, consistent with its ortholog feature, and GFP fusion protein revealed that TcKharon is associated with the cytoskeleton of epimastigotes. CRISPR-Cas9-mediated gene disruption impaired epimastigote proliferation and cytokinesis, resulting in altered nucleus-to-kinetoplast ratios and pronounced morphological defects, particularly in the posterior cell region. Despite these abnormalities, TcKharon[-/-] mutants retained the ability to differentiate into metacyclic trypomastigotes and exhibited in vitro infection rates comparable to wild-type parasites. Our data show that TcKharon is crucial for cell morphology. However, in contrast to close related parasites, TcKharon is not essential for in vitro infectivity.},
}

*Trypanosoma cruzi/genetics/metabolism/growth & development
*Protozoan Proteins/genetics/metabolism
*Chagas Disease/parasitology
Animals
Cytoskeleton/metabolism
CRISPR-Cas Systems

@article {pmid40311322,
year = {2025},
author = {He, Z and Cole, KD and He, HJ},
title = {A novel immortalization method for immortalizing human primary CD8[+] T cells by inserting a single copy of human telomerase reverse transcriptase via CRISPR/Cas9.},
journal = {Tissue & cell},
volume = {95},
number = {},
pages = {102908},
doi = {10.1016/j.tice.2025.102908},
pmid = {40311322},
issn = {1532-3072},
mesh = {Humans ; *Telomerase/genetics/metabolism ; *CD8-Positive T-Lymphocytes/cytology/metabolism ; *CRISPR-Cas Systems/genetics ; },
abstract = {BACKGROUND: Existing cell immortalization methods made the cells obtain oncogenesis phenotype and/or caused the cells gain and/or lose chromosomes. Immortalized normal human T cells lines provide critical in vitro models for basic research and therapeutic products development.

METHODS: We developed a novel method utilizing a CRISPR/Cas9 system to replace the exon 2 of the cell cycle inhibitor gene CDKN2A (encoding p16 and p14 proteins) with a single copy of human telomerase reverse transcriptase (hTERT) to immortalize human primary CD8[+] T cells (hCD8[+]T-TERT).

RESULTS: By using Cas9 protein and low donor DNA copies/cell, we successfully immortalized hCD8[+]T cells with a single copy of hTERT transgene, which also avoided uncontrolled insertion of Cas9 gene and guide RNA vector. Human primary CD8[+] cells from independent donors were immortalized and expanded more than 2.6 × 10[7] times. Characterization of one of the immortalized CD8[+] T-TERT cell lines revealed that the cells retained most of the cell surface markers and normal karyotype. The CD8[+] T-TERT cells also retained the dependence of IL-2 and CD3/CD28 activator for survival and expansion.

CONCLUSION: We established a stable immortalized cell lines using the novel immortalization method, and the immortalized CD8[+] T cells had a phenotype consistent with T cells.},
}

Humans
*Telomerase/genetics/metabolism
*CD8-Positive T-Lymphocytes/cytology/metabolism
*CRISPR-Cas Systems/genetics

@article {pmid40214251,
year = {2025},
author = {Mao, S and Yang, X and Wang, Y and Chen, F and Jiang, H and Wang, Y and Kang, Y and Li, S},
title = {Ultrasensitive and highly specific detection of the Brucella genus and B. melitensis by CRISPR/Cas12b-multiple cross displacement amplification technique.},
journal = {Journal of clinical microbiology},
volume = {63},
number = {5},
pages = {e0153224},
doi = {10.1128/jcm.01532-24},
pmid = {40214251},
issn = {1098-660X},
support = {82273758//National Natural Science Foundation of China/ ; gzwkj2024-055//Health Commission of Guizhou Province/ ; Guo Jikong Zong Ren Han [2024]122//National Disease Control and Prevention Administration/ ; QJJ [2022]019//Foundation of Key Laboratory of Microbiology and Parasitology of Education Department of Guizhou/ ; },
mesh = {*Brucellosis/diagnosis/microbiology ; Sensitivity and Specificity ; *Brucella/genetics/isolation & purification/classification ; *Brucella melitensis/genetics/isolation & purification ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *CRISPR-Cas Systems ; Animals ; *Molecular Diagnostic Techniques/methods ; DNA Primers/genetics ; },
abstract = {Brucellosis is caused by members of the Brucella spp. and remains one of the world's major zoonotic diseases. Brucella melitensis (B. melitensis) as the most contagious Brucella species cannot be ignored as an essential source of infection for brucellosis, especially in countries/regions dominated by animal husbandry. Thus, the identification of the Brucella genus and B. melitensis is crucial for rapid diagnosis of brucellosis to control disease transmission and clinical treatment. Here, we developed the CRISPR/Cas12b nuclease combined with a multiple cross displacement amplification (MCDA) assay (CRISPR-MCDA) for highly specific and sensitive detection of Brucella genus and B. melitensis in clinical applications. Two sets of specific primers were designed targeting the specific gene of Brucella genus (Bcsp31) and B. melitensis (BMEII0466), respectively. The CRISPR-MCDA assay showed high specificity and sensitivity in 28 non-Brucella isolates and 64 clinical samples. The detection limit of CRISPR-MCDA assay was 2 copies/μL in the plasmid dilution template, and the whole detection process took within 90 minutes with nanoparticle-based lateral flow biosensor (LFB) to validate experimental results. Taken together, the CRISPR-MCDA-LFB assay is a visual, sensitive, and highly specific detection technique that can be used as an attractive potential identification tool for Brucella genus and B. melitensis.IMPORTANCEThe prevention and control of Brucellosis urgently require rapid and accurate diagnostic methods. This work validates a new method for the simultaneous detection of Brucella genus and B. melitensis. The method can effectively reduce the chances of contamination and provides a more rapid, sensitive, and specific on-site detection of Brucella. It also offers a solution for the rapid screening of Brucellosis in resource-limited environments, which is crucial for effective disease prevention and control. This technology can also be widely applied to the rapid detection of other pathogens beyond Brucella.},
}

*Brucellosis/diagnosis/microbiology
Sensitivity and Specificity
*Brucella/genetics/isolation & purification/classification
*Brucella melitensis/genetics/isolation & purification
*Nucleic Acid Amplification Techniques/methods
Humans
*CRISPR-Cas Systems
Animals
*Molecular Diagnostic Techniques/methods
DNA Primers/genetics

@article {pmid40211002,
year = {2025},
author = {Cheng, Y and Hu, M and Yang, B and Jensen, TB and Zhang, Y and Yang, T and Yu, R and Ma, Z and Radda, JSD and Jin, S and Zang, C and Wang, S},
title = {Perturb-tracing enables high-content screening of multi-scale 3D genome regulators.},
journal = {Nature methods},
volume = {22},
number = {5},
pages = {950-961},
pmid = {40211002},
issn = {1548-7105},
support = {DP2GM137414//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; 5T32GM007205//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; 2T32GM007499//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R35GM133712//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01HG013503//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {Humans ; *Chromatin/genetics/chemistry/metabolism ; *Genome, Human ; CRISPR-Cas Systems ; In Situ Hybridization, Fluorescence/methods ; },
abstract = {Three-dimensional (3D) genome organization becomes altered during development, aging and disease, but the factors regulating chromatin topology are incompletely understood and currently no technology can efficiently screen for new regulators of multi-scale chromatin organization. Here, we developed an image-based high-content screening platform (Perturb-tracing) that combines pooled CRISPR screens, a cellular barcode readout method (BARC-FISH) and chromatin tracing. We performed a loss-of-function screen in human cells, and visualized alterations to their 3D chromatin folding conformations, alongside perturbation-paired barcode readout in the same single cells. We discovered tens of new regulators of chromatin folding at different length scales, ranging from chromatin domains and compartments to chromosome territory. A subset of the regulators exhibited 3D genome effects associated with loop extrusion and A-B compartmentalization mechanisms, while others were largely unrelated to these known 3D genome mechanisms. Finally, we identified new regulators of nuclear architectures and found a functional link between chromatin compaction and nuclear shape. Altogether, our method enables scalable, high-content identification of chromatin and nuclear topology regulators that will stimulate new insights into the 3D genome.},
}

Humans
*Chromatin/genetics/chemistry/metabolism
*Genome, Human
CRISPR-Cas Systems
In Situ Hybridization, Fluorescence/methods

@article {pmid40140468,
year = {2025},
author = {Rodríguez-Sánchez, A and Quijada-Álamo, M and Pérez-Carretero, C and Herrero, AB and Arroyo-Barea, A and Dávila-Valls, J and Rubio, A and García de Coca, A and Benito-Sánchez, R and Rodríguez-Vicente, AE and Hernández-Rivas, JM and Hernández-Sánchez, M},
title = {SAMHD1 dysfunction impairs DNA damage response and increases sensitivity to PARP inhibition in chronic lymphocytic leukemia.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {10446},
pmid = {40140468},
issn = {2045-2322},
support = {JCYL-EDU/1868/2022//Predoctoral research fellowship 2022 from Junta de Castilla y León/ ; },
mesh = {Humans ; *Leukemia, Lymphocytic, Chronic, B-Cell/genetics/drug therapy/metabolism/pathology ; *SAM Domain and HD Domain-Containing Protein 1/genetics/metabolism ; *DNA Damage ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use ; Mutation ; Female ; Male ; BRCA1 Protein/metabolism/genetics ; Middle Aged ; Aged ; CRISPR-Cas Systems ; Cell Line, Tumor ; },
abstract = {Chronic lymphocytic leukemia (CLL) is a clinically and genetically heterogenous disease. Recent next-generation sequencing (NGS) studies have uncovered numerous low-frequency mutated genes in CLL patients, with SAMHD1 emerging as a candidate driver gene. However, the biological and clinical implications of SAMHD1 mutations remain unclear. Using CRISPR/Cas9, we generated CLL models to investigate the impact of SAMHD1 deficiency on pathogenesis and explore therapeutic strategies. Moreover, we performed NGS in treatment-naïve CLL patients to characterize SAMHD1 mutations and employed RNA-sequencing to evaluate their clinical significance. Our study shows that SAMHD1 inactivation impairs the DNA damage response by reducing homologous recombination efficiency through BRCA1 and RAD51 dysregulation. Importantly, SAMHD1 colocalizes with BRCA1 at DNA damage sites in CLL cells. This research also identifies that SAMHD1-mutated cells are more sensitive to PARP inhibition. Clinically, SAMHD1 dysfunction negatively impacts clinical outcome of CLL cases: SAMHD1 mutations reduce failure-free survival (median 46 vs 57 months, p = 0.033), while low SAMHD1 expression associates with shorter time to first treatment (median 47 vs 77 months; p = 0.00073). Overall, this study elucidates that SAMHD1 dysfunction compromises DNA damage response mechanisms, potentially contributing to unfavorable clinical outcomes in CLL, and proposes PARP-inhibitors as a potential therapeutic approach for SAMHD1-mutated CLL cells.},
}

Humans
*Leukemia, Lymphocytic, Chronic, B-Cell/genetics/drug therapy/metabolism/pathology
*SAM Domain and HD Domain-Containing Protein 1/genetics/metabolism
*DNA Damage
*Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use
Mutation
Female
Male
BRCA1 Protein/metabolism/genetics
Middle Aged
Aged
CRISPR-Cas Systems
Cell Line, Tumor

@article {pmid40135601,
year = {2025},
author = {Yee, PS and Chai, AWY and Yee, SM and Ooi, S and Tan, YH and Garnett, MJ and Ng, SK and Cheong, SC},
title = {Interferon-Inducible ADAR1 p150 Is Essential for the Survival of Oral Squamous Cell Carcinoma.},
journal = {Molecular carcinogenesis},
volume = {64},
number = {6},
pages = {1066-1077},
doi = {10.1002/mc.23910},
pmid = {40135601},
issn = {1098-2744},
support = {//This study was funded by the Dr. Siti Hasmah Mohd Ali Professorial Chair appointment (PV061-2019) and Fundamental Research Grant Scheme (FRGS/1/2022/SKK10/USM/02/14)./ ; },
mesh = {Humans ; *Adenosine Deaminase/genetics/metabolism ; *RNA-Binding Proteins/genetics/metabolism ; *Mouth Neoplasms/genetics/pathology/metabolism ; Cell Line, Tumor ; *Carcinoma, Squamous Cell/genetics/pathology/metabolism ; Gene Expression Regulation, Neoplastic/drug effects ; Apoptosis ; Cell Survival/genetics ; CRISPR-Cas Systems ; eIF-2 Kinase/metabolism/genetics ; Interferon-Induced Helicase, IFIH1/metabolism/genetics ; *Squamous Cell Carcinoma of Head and Neck/genetics/pathology ; },
abstract = {We identified ADAR1 as one of the top essential genes for oral squamous cell carcinoma (OSCC) survival from our genome-wide CRISPR/Cas9 screen in OSCC cell lines. In this study, we confirm that ADAR1-knockout (KO) inhibits cell viability and colony forming ability, and induces apoptosis. We report that IFN-β treatment sensitizes less-dependent cell lines to ADAR1 KO-induced cell lethality. Overexpression of ADAR1-p150, but not ADAR1-p110, rescued cell lethality upon ADAR1 KO, confirming that the IFN-inducible p150 is responsible for OSCC survival. Using a deaminase inactive mutant, we demonstrate that the editing function of ADAR1 is important for OSCC survival. Furthermore, we show that ADAR1 KO-induced cell death is mediated by both PKR and MDA5. We compute gene signatures of ADAR1 dependency in OSCC tumors, and found that those with high ADAR1 dependency score are associated with well or moderate differentiation, likely due to high PKR expression or activation. While a majority of ADAR1-dependent tumors exhibit a low T cell-inflamed gene expression profile, ADAR1 KO upregulates PD-L1, a marker of anti-PD1 response, indicating that ADAR1 inhibition may enhance immunotherapy response in OSCC. Collectively, these findings suggest that targeting ADAR1-p150 not only induces OSCC cell death but could induce a favorable response to anti-PD1.},
}

Humans
*Adenosine Deaminase/genetics/metabolism
*RNA-Binding Proteins/genetics/metabolism
*Mouth Neoplasms/genetics/pathology/metabolism
Cell Line, Tumor
*Carcinoma, Squamous Cell/genetics/pathology/metabolism
Gene Expression Regulation, Neoplastic/drug effects
Apoptosis
Cell Survival/genetics
CRISPR-Cas Systems
eIF-2 Kinase/metabolism/genetics
Interferon-Induced Helicase, IFIH1/metabolism/genetics
*Squamous Cell Carcinoma of Head and Neck/genetics/pathology

@article {pmid40332816,
year = {2025},
author = {Xu, Q and Wang, M and Zeng, J and Sun, H and Wei, X and Jiang, H and Shentu, X and Sun, D},
title = {CRISPR/Cas Technology in Insect Insecticide Resistance.},
journal = {Insects},
volume = {16},
number = {4},
pages = {},
pmid = {40332816},
issn = {2075-4450},
support = {32302354//National Natural Science Foundation of China/ ; LQ24C140002//Zhejiang Provincial Programs for Natural Science/ ; U21A20223//National Natural Science Foundation of China/ ; 2023SNJF034//Zhejiang Provincial Programs for Science and Technology Development/ ; "Pioneer" and "Leading Goose" R&D Program of Zhejiang//"Pioneer" and "Leading Goose" R&D Program of Zhejiang/ ; },
abstract = {Chemicals and biological insecticides play a crucial role as pest management strategies in modern agriculture and forestry. However, their excessive and unreasonable use inevitably leads to varying degrees of resistance among insect populations, which seriously affects the sustainability of insecticide use. One primary reason for this resistance is alterations or mutations in insect gene expression. One class of genes encodes proteins that serve as critical targets for insecticides to exert their toxic effects in insects, while another class of genes encodes proteins involved in the detoxification process of insecticides within insects. Reverse genetics has become a vital research tool for studying the molecular mechanisms underlying changes and mutations in these target genes and their impact on insect resistance. The advent of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and the CRISPR-associated gene Cas as gene-editing technologies has significantly advanced our understanding of how insects adapt to and resist insecticides. This article aims to provide a comprehensive and objective review of the progress made using the CRISPR/Cas system in various arthropods within the field of pest control.},
}

@article {pmid40332635,
year = {2025},
author = {Mukhtiar, A and Ullah, S and Yang, B and Jiang, YQ},
title = {Unlocking genetic potential: a review of the role of CRISPR/Cas technologies in rapeseed improvement.},
journal = {Stress biology},
volume = {5},
number = {1},
pages = {31},
pmid = {40332635},
issn = {2731-0450},
abstract = {Rapeseed (Brassica napus L.) is a globally important oil crop, providing edible vegetable oil and other valuable sources for humans. Being an allotetraploid, rapeseed has a complex genome that has undergone whole-genome duplication, making molecular breeding rather difficult. Fortunately, clustered regularly interspacedshort palindromic repeat (CRISPR)/CRISPR-associated (Cas) technologies have emerged as a potent tool in plant breeding, providing unprecedented accuracy as well as effectiveness in genome editing. This review focuses on the application and progresses of CRISPR/Cas technologies in rapeseed. We discussed the principles and mechanisms of CRISPR/Cas systems focusing on their use in rapeseed improvement such as targeted gene knockout, gene editing and transcriptional regulation. Furthermore, we summarized the regulatory frameworks governing CRISPR-edited crops as well as the challenges and opportunities for their commercialization and adoption. The potential advantages of CRISPR-mediated traits in rapeseed such as increased yield, disease and stress resistance and oil quality are discussed along with biosafety and environmental implications. The purpose of this review is to provide insights into the transformative role of CRISPR/Cas technologies in rapeseed breeding and its potential to address global agricultural challenges while ensuring sustainable crop production.},
}

@article {pmid40304259,
year = {2025},
author = {Kim, J and Orozaliev, A and Sahloul, S and Van, AD and Dang, VT and Pham, VS and Oh, Y and Chehade, I and Al-Sayegh, M and Song, YA},
title = {Accelerating Cleavage Activity of CRISPR-Cas13 System on a Microfluidic Chip for Rapid Detection of RNA.},
journal = {Analytical chemistry},
volume = {97},
number = {18},
pages = {9858-9865},
doi = {10.1021/acs.analchem.5c00256},
pmid = {40304259},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; Humans ; *Lab-On-A-Chip Devices ; *MicroRNAs/analysis/genetics ; *Microfluidic Analytical Techniques ; SARS-CoV-2/genetics ; },
abstract = {It is extremely advantageous to detect nucleic acid levels in the early phases of disease management; such early detection facilitates timely treatment, and it can prevent altogether certain cancers and infectious diseases. A simple, rapid, and versatile detection platform without enzymatic amplification for both short and long sequences would be highly desirable in this regard. Our study addresses this need by introducing IMACC, an ICP-based Microfluidic Accelerator Combined with CRISPR, for amplification-free nucleic acid detection. It exploits electrokinetically induced ion concentration polarization (ICP) to concentrate target nucleic acids and CRISPR reagents near the depletion zone boundary within a microfluidic channel. This localized accumulation accelerates the CRISPR-guided promiscuous cleavage of reporter molecules while enhancing their fluorescence signals simultaneously. Simultaneous accumulation of RNA and ribonucleoproteins (RNP) in confined spaces was validated experimentally and numerically, showing overlapping regions. IMACC enabled detection of miRNA-21 (22 bp) down to 10 pM within 2 min of ICP. IMACC ensured CRISPR specificity (single mismatch (N = 1) sensitivity) during ICP, as shown by off-target and mismatch sequence experiments. IMACC was applied to long RNA samples (i.e., SARS-CoV-2), but it statistically remained challenging at this point due to nonlinear intensity trends with copy numbers and large deviations. IMACC enabled rapid detection of short RNAs such as microRNAs using only basic CRISPR reagents in a single microfluidic channel, eliminating the need for extra enzymes or buffer sets, streamlining workflow and reducing turnaround time. IMACC has the potential to advance CRISPR diagnostics and holds promise for improved detection and future prescreening applications.},
}

*CRISPR-Cas Systems
Humans
*Lab-On-A-Chip Devices
*MicroRNAs/analysis/genetics
*Microfluidic Analytical Techniques
SARS-CoV-2/genetics

@article {pmid40288511,
year = {2025},
author = {Sharma, N and Whinn, KS and Ghodke, H and van Oijen, AM and Lewis, JS and Spenkelink, LM},
title = {nCas9-based method for rolling-circle DNA substrate generation.},
journal = {Analytical biochemistry},
volume = {703},
number = {},
pages = {115883},
doi = {10.1016/j.ab.2025.115883},
pmid = {40288511},
issn = {1096-0309},
mesh = {*DNA, Circular/genetics/chemistry/metabolism ; *CRISPR-Associated Protein 9/metabolism ; *DNA Replication ; RNA, Guide, CRISPR-Cas Systems/genetics ; *DNA ; },
abstract = {Rolling-circle DNA replication is a DNA-duplication mechanism whereby circular DNA templates are continuously copied to produce long DNA products. It is widely used in molecular diagnostics, DNA sequencing, nanotechnology, and in vitro DNA replication studies. The efficiency of rolling-circle replication reaction heavily relies on the quality of the rolling-circle DNA template. Existing methods to create rolling-circle DNA substrates often rely on unique restriction sites and have limited control over replication fork topology and position. To address these limitations, we present a straightforward, customizable, and efficient strategy for producing rolling-circle DNA substrates with control over gap size and fork position. Our method relies on the use of nickase Cas9 (nCas9), which can be programmed to target specific DNA sequences using guide RNAs. In a one-pot reaction, we target nCas9 to four sites on an 18-kb plasmid to create 8-11-bp fragments. These fragments are removed and a flap oligo is ligated, to construct a fork with precisely controlled flap length and gap size. We demonstrate the application of this DNA substrate in an in vitro single-molecule rolling-circle DNA-replication assay. With our method, any plasmid DNA can be converted into a rolling-circle template, permitting generation of more physiologically-relevant DNA templates.},
}

*DNA, Circular/genetics/chemistry/metabolism
*CRISPR-Associated Protein 9/metabolism
*DNA Replication
RNA, Guide, CRISPR-Cas Systems/genetics
*DNA

@article {pmid40203601,
year = {2025},
author = {Li, X and Zhang, Y and He, M and Sun, J and Xiong, B and Wang, G},
title = {An ultrasensitive and specific fluorescence split-aptasensor for D-VP detection based on target-induced self-propelled 3D DNA walkers coupled with CRISPR-Cas12a.},
journal = {Talanta},
volume = {293},
number = {},
pages = {128102},
doi = {10.1016/j.talanta.2025.128102},
pmid = {40203601},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; *Aptamers, Nucleotide/chemistry/genetics/metabolism ; Humans ; *DNA/chemistry/genetics ; Fluorescence ; *Endodeoxyribonucleases/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism ; *Bacterial Proteins/metabolism ; DNA, Catalytic/chemistry/metabolism ; Spectrometry, Fluorescence ; },
abstract = {In this work, we present an ultrasensitive, specific, and high-signal-to-background ratio fluorescence split-aptasensor for D-vasopressin (D-VP) detection. This sensor is based on target-induced self-propelled 3D DNA walkers in conjunction with CRISPR-Cas12a technology. Two split probes (SDA 1 and SDA 2) were designed to undergo structural recombination and function as a walking chain (SDA) under the induction of D-VP. Simultaneously, an intact Mg[2+]-dependent DNAzyme domain was formed at the tail of SDA and subsequently activated. The activated Mg[2+]-dependent DNAzyme continuously propelled the 3D DNA walker, enabling the generation of signal strand DNA (activator DNA). The activator DNA can subsequently trigger the activation of the Cas12a protein, enabling it to cleave the FAM-ssDNA-BHQ1 substrate. This process leads to signal amplification and the specific detection of D-VP. Under optimal conditions, the designed split-aptasensor exhibits excellent linearity across a concentration range of 5 ng/mL to 1215 ng/mL, with a detection limit (LOD) as low as 0.22 ng/mL. This split-aptasensor were employed to identify D-VP in human serum and urine samples, yielding highly satisfactory results. This unique design acts as a proof of concept and illustrates considerable promise for the detection of a wide range of analytes.},
}

*CRISPR-Cas Systems
*Biosensing Techniques/methods
Limit of Detection
*Aptamers, Nucleotide/chemistry/genetics/metabolism
Humans
*DNA/chemistry/genetics
Fluorescence
*Endodeoxyribonucleases/metabolism/genetics
*CRISPR-Associated Proteins/metabolism
*Bacterial Proteins/metabolism
DNA, Catalytic/chemistry/metabolism
Spectrometry, Fluorescence

@article {pmid40194458,
year = {2025},
author = {Liu, S and Hu, J and Zhang, R and Tian, H and Wang, F and Chou, SH and He, J and Ma, L and Yin, W},
title = {Catalytic hairpin assembly assists CRISPR/Cas12a-mediated high-sensitivity detection of aflatoxin B1.},
journal = {Talanta},
volume = {293},
number = {},
pages = {128043},
doi = {10.1016/j.talanta.2025.128043},
pmid = {40194458},
issn = {1873-3573},
mesh = {*Aflatoxin B1/analysis ; *CRISPR-Cas Systems ; *Food Contamination/analysis ; Limit of Detection ; Aptamers, Nucleotide/chemistry ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/metabolism/genetics ; Biocatalysis ; *Inverted Repeat Sequences ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Aflatoxin B1 (AFB1) is recognized the most toxic and carcinogenic mycotoxin and is widely present in cereals and various foods. Therefore, its precise detection is crucial to safeguard food quality and human health. In this study, we proposed a highly sensitive detection system for AFB1 by combining the catalytic hairpin assembly (CHA) and CRISPR/Cas12a techniques. The Aptamer of Aptamer-Initiator interacts with AFB1 to release the blocked Antisense. As a result, the Initiator of the Aptamer-Initiator becomes free and can act as a toehold to bind with H1, which can initiate the CHA to generate a large amount of double-stranded DNA, which hybridized with the Cas12a-crRNA duplex to form the Cas12a-crRNA-DNA ternary complex, wherein Cas12a subsequently cleaves the FAM-ssDNA-BHQ1 probe in trans to generate fluorescence signals. After optimization, we observed a linear relationship between fluorescence intensity and the AFB1 concentration in the range of 50 pM to 1 nM, with a limit of detection (LOD) of 10 pM. Also, the system was robust and could operate with excellent reliability and accuracy even in complex samples. The recovery values in food samples ranged from 92.23 % to 111.72 %, with relative standard deviation (RSD) below 5.68 %. The system exhibited remarkable advantages, including high sensitivity, strong specificity, and rapid response, thereby showed great potential in the efficient detection of AFB1 contaminants in food.},
}

*Aflatoxin B1/analysis
*CRISPR-Cas Systems
*Food Contamination/analysis
Limit of Detection
Aptamers, Nucleotide/chemistry
*Biosensing Techniques/methods
*Endodeoxyribonucleases/metabolism/genetics
Biocatalysis
*Inverted Repeat Sequences
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid40187288,
year = {2025},
author = {Li, Z and Wang, J and Shen, K and Zhao, X and Lin, Z and Yi, H},
title = {Signal-on electrochemiluminescence resonance energy transfer biosensor for miRNA-543 based on CRISPR/Cas13a and magnetic separation.},
journal = {Talanta},
volume = {293},
number = {},
pages = {128085},
doi = {10.1016/j.talanta.2025.128085},
pmid = {40187288},
issn = {1873-3573},
mesh = {*MicroRNAs/analysis/genetics/blood ; *Biosensing Techniques/methods ; Humans ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; Silicon Dioxide/chemistry ; *Luminescent Measurements/methods ; Gold/chemistry ; Energy Transfer ; Female ; Limit of Detection ; Metal Nanoparticles/chemistry ; DNA, Single-Stranded/chemistry ; },
abstract = {In this study, an electrochemiluminescence resonance energy transfer (ECL-RET) biosensor with high sensitivity and strong resistance to interference was constructed based on the CRISPR/Cas13a system and magnetic separation for ovarian cancer biomarker miR-543 detection. Mesoporous silica nanoparticles embedded with Ru(bpy)3[2+] (Ru@SiO2) have high electrochemiluminescence (ECL) response was chosen as energy donor. Single-stranded DNA S1 containing "rUrU" motif was immobilized on AuNRs (AuNRs-S1), which hybridized with single-stranded DNA S2 modified SAMBs (SAMBs-S2) to form AuNRs-S1/S2-SAMBs complex, this has been used as energy acceptor. In the absence of the target, Cas13a remained inactive, preventing the cleavage of S1, thereby maintaining the association of AuNRs with SAMBs. Then they were added in Ru@SiO2 solution after magnetic separation. The electrostatic adsorption between the negatively charged AuNRs and the positively charged Ru@SiO2 cause the occurrence of ECL-RET and low ECL signal had been detected. When the target was added, Cas13a was activated and resulted in the non-specifically cleaving of S1, so AuNRs detached from SAMBs. After magnetic separation, fewer AuNRs participated in ECL-RET, leading to an enhanced ECL signal detected. The change in ECL intensity (ΔECL) exhibited a linear correlation with the logarithm of miR-543 concentration within the range of 10 fM to 10 nM, with a detection limit of 6.91 fM. The biosensor had been applied to detect miR-543 in clinical samples with high accuracy.},
}

*MicroRNAs/analysis/genetics/blood
*Biosensing Techniques/methods
Humans
*Electrochemical Techniques/methods
*CRISPR-Cas Systems
Silicon Dioxide/chemistry
*Luminescent Measurements/methods
Gold/chemistry
Energy Transfer
Female
Limit of Detection
Metal Nanoparticles/chemistry
DNA, Single-Stranded/chemistry

@article {pmid40157661,
year = {2025},
author = {Mukherjee, A and Jodder, J and Chowdhury, S and Das, H and Kundu, P},
title = {A novel stress-inducible dCas9 system for solanaceous plants.},
journal = {International journal of biological macromolecules},
volume = {308},
number = {Pt 3},
pages = {142462},
doi = {10.1016/j.ijbiomac.2025.142462},
pmid = {40157661},
issn = {1879-0003},
mesh = {*Solanum lycopersicum/genetics ; Gene Expression Regulation, Plant ; *CRISPR-Cas Systems/genetics ; *Stress, Physiological/genetics ; Plant Proteins/genetics ; Transcription Factors/genetics ; Plants, Genetically Modified/genetics ; Heat-Shock Response/genetics ; },
abstract = {Conditional manipulation of gene expression is essential in plant biology, yet a simple stimuli-based inducible system for regulating any plant gene is lacking. Here, we present an innovative stress-inducible CRISPR/dCas9-based gene-regulatory toolkit tailored for intentional gene regulation in solanaceous plants. We have translationally fused the transmembrane domain of a tomato membrane-bound NAC transcription factor with dCas9 to utilize the reversible-tethering-based activation mechanism. This system sequesters dCas9 to the plasma membrane under normal conditions and allows membrane detachment in response to heat induction and NLS-mediated nuclear transfer, enabling stress-inducible gene regulation. Transient assays with tomato codon-optimized dCas9-assisted inducible CRISPR activation and interference systems confirmed their superior ability on transcriptional control, rapid induction, and reversibility after stimulus withdrawal in solanaceous plants. The transformative potential of the toolkit was exemplified by enhancing tomato immunity against bacterial speck disease under elevated temperatures by precisely regulating crucial salicylic acid signalling components, SlCBP60g and SlSARD1. Additionally, it was instrumental in engineering heat-stress tolerance in tomato plants through multiplex activation of heat-responsive transcription factors, SlNAC2 and SlHSFA6b. These findings demonstrate the unprecedented temporal control offered by this novel stress-inducible toolkit over gene-expression dynamics, paving the way for favourable manipulation of complex traits in environmentally-challenged crops.},
}

*Solanum lycopersicum/genetics
Gene Expression Regulation, Plant
*CRISPR-Cas Systems/genetics
*Stress, Physiological/genetics
Plant Proteins/genetics
Transcription Factors/genetics
Plants, Genetically Modified/genetics
Heat-Shock Response/genetics

@article {pmid40082271,
year = {2025},
author = {Sharma, N and Thakur, K and Zinta, R and Mangal, V and Dalamu, and Tiwari, JK and Sood, S and Dutt, S and Kumar, V and Singh, B and Thakur, AK},
title = {Genome editing research initiatives and regulatory landscape of genome edited crops in India.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {13},
pmid = {40082271},
issn = {1573-9368},
support = {ICAR-Genome Editing Project, Cabin Project, AINP on Biotech Crops//Indian Council of Agricultural Research/ ; },
mesh = {*Crops, Agricultural/genetics/growth & development ; *Gene Editing/legislation & jurisprudence/methods ; India ; *Plants, Genetically Modified/genetics/growth & development ; *Genome, Plant/genetics ; Agriculture/legislation & jurisprudence ; Humans ; CRISPR-Cas Systems ; },
abstract = {Food and nutritional security are the top priorities in Indian agriculture. Exponential population growth coupled with climate change effects has become a serious challenge for sustainable agriculture. Genome editing has revolutionized the agricultural sector because of its ability to create precise, stable and predictable modifications in the genome and therefore, offers great opportunities for crop improvement in India. However, for harvesting the real benefits of this technology in agriculture sector, there is a strong need of creating awareness among the end users and development of suitable policies for regularization of genome edited products. Many regulatory agencies around the world have been modernizing their regulatory approaches to be more risk proportionate and to reflect a more science-based approach. In this article, recent research initiatives and developments undertaken by different Indian institutes/organizations for the genetic improvement of agricultural and horticultural crops via genome editing technologies are summarized. Furthermore, to benefit from this potential technology in our country, regulatory policies must be clear, science-based and proportionate. Therefore, in the present review, the regulatory policies related to the genome editing of crop products in India are discussed in detail. This review will sensitize researchers and stakeholders to the application of genome editing techniques in crop improvement and various biosafety committees involved in the development and regulation of genome edited crops.},
}

*Crops, Agricultural/genetics/growth & development
*Gene Editing/legislation & jurisprudence/methods
India
*Plants, Genetically Modified/genetics/growth & development
*Genome, Plant/genetics
Agriculture/legislation & jurisprudence
Humans
CRISPR-Cas Systems

@article {pmid40075351,
year = {2025},
author = {Huang, Z and Zhao, X and Jiang, Z and Qiu, X and Sun, X and Wang, D and Zhang, H and Chen, Q and Tan, R and Shen, Y},
title = {IP6K2 mutations as a novel mechanism of resistance to oncolytic virus therapy.},
journal = {Journal of translational medicine},
volume = {23},
number = {1},
pages = {311},
pmid = {40075351},
issn = {1479-5876},
support = {U22A20326//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Oncolytic Virotherapy ; Animals ; *Mutation/genetics ; *Oncolytic Viruses/physiology ; Apoptosis/genetics ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; Cell Proliferation ; Virus Replication ; Herpesvirus 1, Human/physiology ; CRISPR-Cas Systems/genetics ; Signal Transduction ; Mice ; Neoplasms/therapy/genetics ; },
abstract = {BACKGROUND: Oncolytic virus therapy (OVT) represents a promising frontier in cancer treatment. Despite its efficacy in clinical trials, variability in patient response, particularly resistance development, highlights the need for tailored therapeutic strategies.

METHODS: The Inositol Hexakisphosphate Kinase 2 (IP6K2) gene knock out was carried by CRISPR/Cas9 system. The evaluation of biomarkers of apoptosis and relevant pathways was conducted to be assessed. Attachment assay was conducted to verify the binding ability of virus to the host cells. Cell proliferation and apoptosis was assessed. Subcutaneous xenograft model was used to evaluate IP6K2 knock out influence in vivo. cBioPortal and TCGA database were applied to analyze genomic alterations in pan-cancer.

RESULTS: IP6K2 was essential for effective Herpes Simplex Virus Type1 (HSV-1) replication and subsequent cell apoptosis, acting through the tumor Protein p53 (p53) and Cyclin-Dependent Kinase Inhibitor 1 A (p21) signaling axis. The tumor model demonstrated that tumors lacking IP6K2 exhibited resistance to HSV-1 oncolysis, resulting in diminished therapeutic outcomes. Analysis of cBioPortal and TCGA databases corroborated the potential resistance stemming from IP6K2 mutations across various cancer types, underscoring the necessity for pre-treatment IP6K2 status assessment.

CONCLUSIONS: This study underscores the role of IP6K2 as potential markers of resistance, which opens avenues for precision medicine approaches in OVT.},
}

Humans
*Oncolytic Virotherapy
Animals
*Mutation/genetics
*Oncolytic Viruses/physiology
Apoptosis/genetics
Cell Line, Tumor
Xenograft Model Antitumor Assays
Cell Proliferation
Virus Replication
Herpesvirus 1, Human/physiology
CRISPR-Cas Systems/genetics
Signal Transduction
Mice
Neoplasms/therapy/genetics

@article {pmid39487259,
year = {2025},
author = {Tian, S and Qin, Y and Wu, Y and Dong, M},
title = {Design, performance, processing, and validation of a pooled CRISPR perturbation screen for bacterial toxins.},
journal = {Nature protocols},
volume = {20},
number = {5},
pages = {1158-1195},
pmid = {39487259},
issn = {1750-2799},
mesh = {*Bacterial Toxins/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; *CRISPR-Cas Systems ; Genomics/methods ; },
abstract = {Unbiased forward genetic screens have been extensively employed in biological research to elucidate functional genomics. In pooled clustered regularly interspaced short palindromic repeats (CRISPR) perturbation screens, various genetically encoded gain-of-function or loss-of-function mutations are introduced into a heterogeneous population of cells. Subsequently, these cells are screened for phenotypes, perturbation-associated genotypes are analyzed and a connection between genotype and phenotype is determined. CRISPR screening techniques enable the investigation of important biological questions, such as how bacterial toxins kill cells and cause disease. However, the broad spectrum of effects caused by diverse toxins presents a challenge when selecting appropriate screening strategies. Here, we provide a step-by-step protocol for a genome-wide pooled CRISPR perturbation screen to study bacterial toxins. We describe technical considerations, pilot experiments, library construction, screen execution, result analysis and validation of the top enriched hits. These screens are applicable for many different types of toxins and are anticipated to reveal a repertoire of host factors crucial in the intoxication pathway, such as receptors, trafficking/translocation factors and substrates. The entire protocol takes 21-27 weeks and does not require specialized knowledge beyond basic biology.},
}

*Bacterial Toxins/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Humans
*CRISPR-Cas Systems
Genomics/methods

@article {pmid40332465,
year = {2025},
author = {Zhang, RX and Zhang, YF and Yang, H and Zhang, XD and Yang, ZG and Li, BB and Sun, WH and Yang, Z and Liu, WT and Chen, KM},
title = {An Optimized Editing Approach for Wheat Genes by Improving sgRNA Design and Transformation Strategies.},
journal = {International journal of molecular sciences},
volume = {26},
number = {8},
pages = {},
pmid = {40332465},
issn = {1422-0067},
support = {32270372//National Natural Science Foundation of China/ ; },
mesh = {*Triticum/genetics ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; *Transformation, Genetic ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Biolistics ; Agrobacterium/genetics ; },
abstract = {Hexaploid wheat has a large genome, making it difficult for transgenes to produce phenotypes due to gene redundancy and tight linkage among genes. Multiple gene copies typically necessitate multiple targeting events during gene editing, followed by several generations of self-crossing to achieve homozygous genotypes. The high cost of transgenesis in wheat is another issue, which hinders the easy availability of gene-edited materials in wheat. In this study, we developed a comprehensive approach to improve wheat gene editing efficiency. First, we established a protoplast-based system to evaluate the relative efficiency of gene editing targets, which enabled the rapid and effective selection of optimal sgRNAs. We then compared two transformation strategies: biolistic bombardment and Agrobacterium-mediated transformation for generating edited wheat lines. Although biolistic bombardment showed higher initial editing efficiency, Agrobacterium-mediated transformation proved more effective for obtaining homozygous mutants. Notably, we discovered that deploying the same sgRNA through different vectors enhanced editing efficiency, whereas overlapping but distinct sgRNAs exhibited interference effects. Finally, we optimized the VITF-edit (virus-induced transgene free editing) technique using BSMV delivery to establish a relatively simple and easily applied wheat gene editing method for general laboratories.},
}

*Triticum/genetics
*Gene Editing/methods
Plants, Genetically Modified/genetics
*Transformation, Genetic
*RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems
Biolistics
Agrobacterium/genetics

@article {pmid40252733,
year = {2025},
author = {Barreto, JA and Lacôrte E Silva, MVM and Marin, DC and Brienzo, M and Jacobus, AP and Contiero, J and Gross, J},
title = {Engineering adaptive alleles for Escherichia coli growth on sucrose using the EasyGuide CRISPR system.},
journal = {Journal of biotechnology},
volume = {403},
number = {},
pages = {126-139},
doi = {10.1016/j.jbiotec.2025.04.016},
pmid = {40252733},
issn = {1873-4863},
mesh = {*Escherichia coli/genetics/growth & development/metabolism ; *Sucrose/metabolism ; Alleles ; Plasmids/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genetic Engineering/methods ; },
abstract = {Adaptive Laboratory Evolution (ALE) is a powerful approach for mining genetic data to engineer industrial microorganisms. This evolution-informed design requires robust genetic tools to incorporate the discovered alleles into target strains. Here, we introduce the EasyGuide CRISPR, a five-plasmid platform that exploits E. coli's natural recombination system to assemble gRNA plasmids from overlapping PCR fragments. The production of gRNAs and donor DNA is further facilitated by using recombination cassettes generated through PCR with 40-60-mer oligos. With the new CRISPR toolkit, we constructed 22 gene edits in E. coli DH5α, most of which corresponded to alleles mapped in E. coli DH5α and E2348/69 ALE populations selected for sucrose propagation. For DH5α ALE, sucrose consumption was supported by the cscBKA operon expression from a high-copy plasmid. During ALE, plasmid integration into the chromosome, or its copy number reduction due to the pcnB deletion, conferred a 30-35 % fitness gain, as demonstrated by CRISPR-engineered strains. A ∼5 % advantage was also associated with a ∼40.4 kb deletion involving fli operons for flagella assembly. In E2348/69 ALE, inactivation of the hfl system suggested selection pressures for maintaining λ-prophage dormancy (lysogeny). We further enhanced our CRISPR toolkit using yeast for in vivo assembly of donors and expression cassettes, enabling the establishment of polyhydroxybutyrate synthesis from sucrose. Overall, our study highlights the importance of combining ALE with streamlined CRISPR-mediated allele editing to advance microbial production using cost-effective carbon sources.},
}

*Escherichia coli/genetics/growth & development/metabolism
*Sucrose/metabolism
Alleles
Plasmids/genetics
*CRISPR-Cas Systems/genetics
Gene Editing/methods
Genetic Engineering/methods

@article {pmid40222553,
year = {2025},
author = {Sadler, RL and Greenman, AC and Methawasin, M and Fan, J and Harris, SP},
title = {The L348P point mutation in cardiac myosin binding protein-C alters transient responses to stretch, slows cardiac relaxation, and is embryonic lethal in homozygous CRISPR gene-edited mice.},
journal = {Journal of molecular and cellular cardiology},
volume = {203},
number = {},
pages = {35-46},
doi = {10.1016/j.yjmcc.2025.04.007},
pmid = {40222553},
issn = {1095-8584},
mesh = {Animals ; *Point Mutation/genetics ; *Carrier Proteins/genetics/metabolism ; Mice ; *Homozygote ; *Gene Editing ; Myocytes, Cardiac/metabolism ; Mice, Transgenic ; Gene Knock-In Techniques ; *Embryo Loss/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems ; Myocardial Contraction/genetics ; Amino Acid Substitution ; Disease Models, Animal ; Female ; },
abstract = {Mutations in cardiac myosin binding protein-C (cMyBP-C) are a common cause of hypertrophic cardiomyopathy (HCM), an inherited autosomal dominant disease affecting 1 in 250-500 people. We previously identified a single amino acid substitution (L348P) in the regulatory motif (M-domain) of cMyBP-C that slowed relaxation and caused diastolic dysfunction in transgenic mice. Here we attempted to increase expression of the mutant protein by creating a CRISPR gene-edited knock-in mouse model (L348P-CR) and breeding mice to homozygosity for the mutant allele. Results showed that L348P-CR homozygous mice died in utero, but that heterozygous knock-in mice developed contractile deficits and diastolic dysfunction comparable to transgenic mice. To overcome the lethal homozygous expression of the L348P mutation, we used our "cut-and-paste" approach to fully replace endogenous wild-type cMyBP-C with recombinant L348P cMyBP-C in permeabilized cardiomyocytes from SpyC3 mice. Results showed that replacement of wild-type cMyBP-C with recombinant L348P recapitulated mechanical effects seen in transgenic and L348P-CR mice, validating the utility of our cut-and-paste method for evaluating functional effects of cMyBP-C. We conclude that L348P-CR knock-in mice are a robust model of diastolic dysfunction due to a single point mutation in cMyBP-C and that the cut-and-paste approach offers a rapid and cost-effective approach for evaluating mutations in cMyBP-C, especially those that are lethal in traditional animal models.},
}

Animals
*Point Mutation/genetics
*Carrier Proteins/genetics/metabolism
Mice
*Homozygote
*Gene Editing
Myocytes, Cardiac/metabolism
Mice, Transgenic
Gene Knock-In Techniques
*Embryo Loss/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Cas Systems
Myocardial Contraction/genetics
Amino Acid Substitution
Disease Models, Animal
Female

@article {pmid40174865,
year = {2025},
author = {Bardani, E and Katsarou, K and Mitta, E and Andronis, C and Štefková, M and Wassenegger, M and Kalantidis, K},
title = {Broadening the Nicotiana benthamiana research toolbox through the generation of dicer-like mutants using CRISPR/Cas9 approaches.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {356},
number = {},
pages = {112490},
doi = {10.1016/j.plantsci.2025.112490},
pmid = {40174865},
issn = {1873-2259},
mesh = {*Nicotiana/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/physiology ; *Caspase 9/genetics/metabolism ; Streptococcus pyogenes/genetics/metabolism ; *RNA Interference ; Stress, Physiological ; Epigenomics ; Cell Cycle Proteins/genetics ; *CRISPR-Cas Systems/genetics ; Genes, Plant/physiology ; Plant Proteins/genetics/metabolism ; *RNA, Plant/genetics ; },
abstract = {RNA silencing in plants plays a pivotal role in various biological processes, including development, epigenetic modifications and stress response. Key components of this network are Dicer-like (DCL) proteins. Nicotiana benthamiana encodes four DCLs, each responsible for the generation of distinct small RNA (sRNA) populations, which regulate different functions. However, elucidating the precise role of each DCL has been proven challenging, as overlapping functions exist within DCLs. In our present study, we have successfully generated dcl2, dcl3 and dcl4 homozygous mutants, employing two different CRISPR/Cas9 approaches. The first approach is based on a transgene-mediated delivery of the single-guide RNA (sgRNA), while the second approach employs a viral vector for sgRNA delivery. By utilizing a suite of screening techniques, including polymerase chain reaction (PCR), T7 endonuclease I (T7E1) assay, high-resolution melt analysis (HRMA) and DNA sequencing, we successfully generated dcl2, dcl3 and dcl4 homozygous mutants harboring identical mutations in every allele. To evaluate these dcl mutants, we examined their sRNA profiles and phenotypes. We further have indications that homozygous mutations of a gene do not always lead to the desired loss-of-function, highlighting the importance of mutant evaluation. dcl mutants represent invaluable tools to explore how overlapping silencing pathways are connected to essential plant functions, including development, stress responses and pathogen defense. Additionally, they hold potential for biotechnological applications, such as crop improvement and gene silencing tools. We anticipate that our study will make significant contributions to enhance understanding of the role of DCLs in plants.},
}

*Nicotiana/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/physiology
*Caspase 9/genetics/metabolism
Streptococcus pyogenes/genetics/metabolism
*RNA Interference
Stress, Physiological
Epigenomics
Cell Cycle Proteins/genetics
*CRISPR-Cas Systems/genetics
Genes, Plant/physiology
Plant Proteins/genetics/metabolism
*RNA, Plant/genetics

@article {pmid40169039,
year = {2025},
author = {Dam, MI and Ding, BJ and Brauburger, K and Wang, HL and Powell, D and Groot, AT and Heckel, DG and Löfstedt, C},
title = {Sex pheromone biosynthesis in the Oriental fruit moth Grapholita molesta involves Δ8 desaturation.},
journal = {Insect biochemistry and molecular biology},
volume = {180},
number = {},
pages = {104307},
doi = {10.1016/j.ibmb.2025.104307},
pmid = {40169039},
issn = {1879-0240},
mesh = {Animals ; *Moths/metabolism/genetics/enzymology ; *Sex Attractants/biosynthesis ; *Fatty Acid Desaturases/metabolism/genetics ; *Insect Proteins/metabolism/genetics ; Female ; Male ; CRISPR-Cas Systems ; },
abstract = {The Oriental fruit moth Grapholita molesta is distributed throughout temperate regions and considered to be a pest in peach production and other high-value fruit crops in the rose family. Insecticide treatment has led to resistance development, but the use of sex pheromones in pest management has shown great promise. We investigated the pheromone biosynthesis pathway in G. molesta with the aim of elucidating pheromone evolution in the Olethreutinae subfamily of moths and harnessing pathway genes in biotechnological production of sex pheromone for use in pest management. In vivo labelling experiments suggested that an uncommon Δ8 fatty acyl desaturase is involved in sex pheromone biosynthesis. CRISPR/Cas9 knock-out of the highly expressed candidate desaturase gene Gmol_CPRQ almost completely blocked the production of Δ8 pheromone components in vivo. Heterologous expression of Gmol_CPRQ protein in yeast- or Sf9 insect cells, however, failed to demonstrate the expected Δ8 desaturase activity. Instead, Δ9 desaturase activity was observed. Co-expression in the yeast system of the electron donor, cytochrome b5, from G. molesta still produced only Δ9 desaturase activity. We suggest that Gmol_CPRQ is intimately involved in pheromone production in vivo, via an unknown reaction mechanism that may possibly involve another co-factor that is absent in the yeast and Sf9 expression systems, or depend on its subcellular site of activity. Solving this puzzle will shed further light on pheromone biosynthesis in the family Tortricidae and will be required for successful biotechnological production of fatty acids and pheromones requiring Δ8 desaturation.},
}

Animals
*Moths/metabolism/genetics/enzymology
*Sex Attractants/biosynthesis
*Fatty Acid Desaturases/metabolism/genetics
*Insect Proteins/metabolism/genetics
Female
Male
CRISPR-Cas Systems

@article {pmid40132674,
year = {2025},
author = {Li, Z and Yoon, JS and Zhong, Z and Ruan, Y and Yang, C and Zhou, X and Zhang, Y and Pan, H},
title = {HvStaufenC contributes to the high RNAi efficiency in the 28-spotted ladybeetle, Henosepilachna vigintioctopunctata.},
journal = {Insect biochemistry and molecular biology},
volume = {180},
number = {},
pages = {104304},
doi = {10.1016/j.ibmb.2025.104304},
pmid = {40132674},
issn = {1879-0240},
mesh = {Animals ; *Coleoptera/genetics/growth & development/metabolism ; *RNA Interference ; Larva/genetics/growth & development/metabolism ; *Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Female ; },
abstract = {RNA interference (RNAi) has been shown to be relatively effective in coleopteran insects, with limited exploration into the molecular mechanisms that underlie this effectiveness. This study specifically examines the 28-spotted ladybeetle, Henosepilachna vigintioctopunctata (Hvig), known for its high RNAi efficiency. Here, we utilized RNAi and CRISPR/Cas9 techniques to identify and validate the genes involved in the RNAi pathway that enhance RNAi efficacy in Hvig. We identified a total of 15 potential genes within the RNAi pathway that may impact RNAi efficiency. The bioassay results showed that only knockdown of HvStaufenC in the 3rd instar larvae could block the abnormal body color phenotype and lethality induced by the subsequent silencing of the two marker genes, HvTH (tyrosine hydroxylase) and HvABCH1 (ATP-binding cassette H transporter gene), respectively. Additionally, successful CRISPR/Cas9-mediated knockout of HvStaufenC led to the generation of stable, heritable mutants that exhibited insensitivity to RNAi, displaying no response to RNAi targeting HvTH and HvABCH1. Compared to the wild-type strain, the HvStaufenC knockout (HvStaufenCKO) mutant females demonstrated a 42 % decrease in oviposition rate and a 41.3 % reduction in egg hatchability. This study demonstrates that HvStaufenC gene is crucial for the RNAi efficiency of Hvig and offers new evidence into the RNAi mechanisms in coleopteran species.},
}

Animals
*Coleoptera/genetics/growth & development/metabolism
*RNA Interference
Larva/genetics/growth & development/metabolism
*Insect Proteins/genetics/metabolism
CRISPR-Cas Systems
Female

@article {pmid40064881,
year = {2025},
author = {Fu, Y and He, X and Ma, L and Gao, XD and Liu, P and Shi, H and Chai, P and Ge, S and Jia, R and Liu, DR and Fan, X and Yang, Z},
title = {In vivo prime editing rescues photoreceptor degeneration in nonsense mutant retinitis pigmentosa.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {2394},
pmid = {40064881},
issn = {2041-1723},
support = {82200961//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Retinitis Pigmentosa/genetics/therapy/pathology ; *Gene Editing/methods ; *Cyclic Nucleotide Phosphodiesterases, Type 6/genetics/metabolism ; Mice ; Disease Models, Animal ; *Codon, Nonsense/genetics ; Retinal Rod Photoreceptor Cells/metabolism/pathology ; Genetic Therapy/methods ; Dependovirus/genetics ; *Retinal Degeneration/genetics/therapy ; Humans ; Female ; CRISPR-Cas Systems ; Male ; Mice, Inbred C57BL ; },
abstract = {The next-generation gene editing tool, prime editing (PE), is adept at correcting point mutations precisely with high editing efficiency and rare off-target events and shows promising therapeutic value in treating hereditary diseases. Retinitis pigmentosa (RP) is the most common type of inherited retinal dystrophy and is characterized by progressive degeneration of retinal photoreceptors and, consequently, visual decline. To date, effective treatments for RP are lacking. Herein, a PE system is designed to target the PDE6B Y347X mutation in the rd1 mouse strain, a preclinical RP model. We screen and develop the PE system with epegRNA and RT[ΔRnH], which is delivered via dual-AAV in vivo with an editing efficiency of 26.47 ± 13.35%, with negligible off-target effects confirmed by AID-Seq and PE-tag. Treatment with the PE system in vivo greatly restores PDE6B protein expression and protects rod cells from degeneration. Mouse behavioural experiments also show that compared with no treatment, prime editing inhibits vision deterioration in littermate rd1 mice. This study provides a therapeutic opportunity for the use of PE to correct mutated RPs at the genomic level.},
}

Animals
*Retinitis Pigmentosa/genetics/therapy/pathology
*Gene Editing/methods
*Cyclic Nucleotide Phosphodiesterases, Type 6/genetics/metabolism
Mice
Disease Models, Animal
*Codon, Nonsense/genetics
Retinal Rod Photoreceptor Cells/metabolism/pathology
Genetic Therapy/methods
Dependovirus/genetics
*Retinal Degeneration/genetics/therapy
Humans
Female
CRISPR-Cas Systems
Male
Mice, Inbred C57BL

@article {pmid40064677,
year = {2025},
author = {Parsons, VA and Vadlamudi, S and Voos, KM and Rohy, AE and Moxley, AH and Cannon, ME and Rosen, JD and Mills, CA and Herring, LE and Broadaway, KA and Lorenzo, DN and Mohlke, KL},
title = {TBC1D30 regulates proinsulin and insulin secretion and is the target of a genomic association signal for proinsulin.},
journal = {Diabetologia},
volume = {68},
number = {6},
pages = {1169-1183},
pmid = {40064677},
issn = {1432-0428},
support = {F31DK132982/NH/NIH HHS/United States ; P30AR069619/NH/NIH HHS/United States ; P30CA016086/NH/NIH HHS/United States ; R01DK072193/NH/NIH HHS/United States ; T32GM135128/NH/NIH HHS/United States ; T32HL129982/NH/NIH HHS/United States ; UM1DK126185/NH/NIH HHS/United States ; 1-19-JDF-081//American Diabetes Association/ ; F31DK132982/NH/NIH HHS/United States ; P30AR069619/NH/NIH HHS/United States ; P30CA016086/NH/NIH HHS/United States ; R01DK072193/NH/NIH HHS/United States ; T32GM135128/NH/NIH HHS/United States ; T32HL129982/NH/NIH HHS/United States ; UM1DK126185/NH/NIH HHS/United States ; },
mesh = {Humans ; *Proinsulin/metabolism/genetics ; *Insulin Secretion/genetics/physiology ; Genome-Wide Association Study ; *GTPase-Activating Proteins/genetics/metabolism ; *Insulin/metabolism ; Animals ; Cell Line, Tumor ; Insulin-Secreting Cells/metabolism ; Rats ; CRISPR-Cas Systems ; Polymorphism, Single Nucleotide ; },
abstract = {AIMS/HYPOTHESIS: Components of the insulin processing and secretion pathways remain incompletely understood. Here, we examined a genome-wide association study (GWAS) signal for plasma proinsulin levels. Lead GWAS variant rs150781447-T encodes an Arg279Cys substitution in TBC1 domain family member 30 (TBC1D30), but no role for this protein in insulin processing or secretion has been established previously. This study aimed to evaluate whether TBC1D30 drives the GWAS association signal by determining whether TBC1D30 is involved in proinsulin secretion and, if so, to examine the effects of variant alleles and potential mechanisms.

METHODS: Using CRISPR/Cas9 genome editing to create double-strand breaks and prime editing to install substitutions in INS1 832/13 insulinoma cells, we generated clonal cell lines with altered TBC1D30, as well as homozygous and heterozygous lines carrying the lead GWAS variant. We characterised lines by Sanger sequencing, quantitative PCR and ELISAs to measure glucose-stimulated proinsulin and insulin secretion. We also tested the effects of TBC1D30 knockdown on proinsulin and insulin secretion in human islets. We further assessed TBC1D30's contribution to secretory pathways by examining the effects of altered gene function on intracellular proinsulin and insulin content and insulin localisation, and by identifying potential proteins that interact with TBC1D30 using affinity purification mass spectrometry.

RESULTS: Compared with mock-edited cells, cell lines with reduced TBC1D30 expression or altered Rab GTPase-activating protein (RabGAP) domain had significantly more secreted proinsulin, 1.8- and 2.6-fold more than controls, respectively. Similarly, cells expressing the variant substitution demonstrated increased proinsulin secretion. Cell lines with a partial deletion of a critical functional domain showed 1.8-fold higher expression of Tbc1d30 and at least 2.0-fold less secreted proinsulin. Cells with altered RabGAP domain sequence also demonstrated, to a lesser extent, changes in secreted insulin levels. TBC1D30 knockdown in human islets resulted in increased insulin secretion with no significant effect on proinsulin secretion. The effects of altered TBC1D30 on mislocalisation of insulin, intracellular proinsulin and insulin content and the identities of interacting proteins are consistent with a role for TBC1D30 in proinsulin and insulin secretion.

CONCLUSIONS/INTERPRETATION: These findings suggest that effects on TBC1D30 are responsible for the GWAS signal and that TBC1D30 plays a critical role in the secretion of mature insulin.},
}

Humans
*Proinsulin/metabolism/genetics
*Insulin Secretion/genetics/physiology
Genome-Wide Association Study
*GTPase-Activating Proteins/genetics/metabolism
*Insulin/metabolism
Animals
Cell Line, Tumor
Insulin-Secreting Cells/metabolism
Rats
CRISPR-Cas Systems
Polymorphism, Single Nucleotide

@article {pmid40050992,
year = {2025},
author = {Li, X and Zhang, W and Fang, Y and Sun, T and Chen, J and Tian, R},
title = {Large-scale CRISPRi screens link metabolic stress to glioblastoma chemoresistance.},
journal = {Journal of translational medicine},
volume = {23},
number = {1},
pages = {289},
pmid = {40050992},
issn = {1479-5876},
support = {32100766//National Natural Science Foundation of China/ ; 82171416//National Natural Science Foundation of China/ ; 2023B1515020075//Basic and Applied Basic Research Foundation of Guangdong Province/ ; JCYJ20220530112602006//Shenzhen Fundamental Research Program/ ; RCYX20221008092845052//Shenzhen Fundamental Research Program/ ; A2303039//Shenzhen Medical Research Fund/ ; },
mesh = {*Glioblastoma/genetics/drug therapy/metabolism/pathology ; Humans ; *Drug Resistance, Neoplasm/genetics/drug effects ; Cell Line, Tumor ; Temozolomide/pharmacology/therapeutic use ; *Stress, Physiological/genetics/drug effects ; Phosphoglycerate Kinase/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Animals ; *Brain Neoplasms/genetics/drug therapy/pathology/metabolism ; Gene Expression Regulation, Neoplastic/drug effects ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism ; Signal Transduction/drug effects ; Mice ; },
abstract = {BACKGROUND: Glioblastoma (GBM) patients frequently develop resistance to temozolomide (TMZ), the standard chemotherapy. While targeting cancer metabolism shows promise, the relationship between metabolic perturbation and drug resistance remains poorly understood.

METHODS: We performed high-throughput CRISPR interference screens in GBM cells to identify genes modulating TMZ sensitivity. Findings were validated using multiple GBM cell lines, patient-derived glioma stem cells, and clinical data. Molecular mechanisms were investigated through transcriptome analysis, metabolic profiling, and functional assays.

RESULTS: We identified phosphoglycerate kinase 1 (PGK1) as a key determinant of TMZ sensitivity. Paradoxically, while PGK1 inhibition suppressed tumor growth, it enhanced TMZ resistance by inducing metabolic stress. This activated AMPK and HIF-1α pathways, leading to enhanced DNA damage repair through 53BP1. PGK1 expression levels correlated with TMZ sensitivity across multiple GBM models and patient samples.

CONCLUSIONS: Our study reveals an unexpected link between metabolic stress and chemoresistance, demonstrating how metabolic adaptation can promote therapeutic resistance. These findings caution against single-agent metabolic targeting and suggest PGK1 as a potential biomarker for TMZ response in GBM.},
}

*Glioblastoma/genetics/drug therapy/metabolism/pathology
Humans
*Drug Resistance, Neoplasm/genetics/drug effects
Cell Line, Tumor
Temozolomide/pharmacology/therapeutic use
*Stress, Physiological/genetics/drug effects
Phosphoglycerate Kinase/metabolism/genetics
*CRISPR-Cas Systems/genetics
Animals
*Brain Neoplasms/genetics/drug therapy/pathology/metabolism
Gene Expression Regulation, Neoplastic/drug effects
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Hypoxia-Inducible Factor 1, alpha Subunit/metabolism
Signal Transduction/drug effects
Mice

@article {pmid39422616,
year = {2025},
author = {Sikiru, AB and Truong, MN and Zohdy, W},
title = {Future prospects for the advancement of treatment of men with NOA: focus on gene editing, artificial sperm, stem cells, and use of imaging.},
journal = {Asian journal of andrology},
volume = {27},
number = {3},
pages = {433-439},
doi = {10.4103/aja202486},
pmid = {39422616},
issn = {1745-7262},
mesh = {Humans ; Male ; *Gene Editing/methods ; *Azoospermia/therapy/genetics ; Sperm Retrieval ; Sperm Injections, Intracytoplasmic ; CRISPR-Cas Systems ; Spermatozoa ; Stem Cells ; },
abstract = {Nonobstructive azoospermia (NOA) affects about 60% of men with azoospermia, representing a severe form of male infertility. The current approach to manage NOA primarily involves testicular sperm retrieval methods such as conventional testicular sperm extraction (c-TESE) and microdissection testicular sperm extraction (micro-TESE). While combining testicular sperm retrieval with intracytoplasmic sperm injection (ICSI) offers hope for patients, the overall sperm retrieval rate (SRR) stands at around 50%. In cases where micro-TESE fails to retrieve sperm, limited options, like donor sperm or adoption, can be problematic in certain cultural contexts. This paper delves into prospective treatments for NOA management. Gene editing technologies, particularly clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein 9 (CRISPR/Cas9), hold potential for correcting genetic mutations underlying testicular dysfunction. However, these technologies face challenges due to their complexity, potential off-target effects, ethical concerns, and affordability. This calls for research to address key challenges associated with NOA management within the clinical settings. This also necessitate ongoing research essential for developing more sensitive diagnostic tests, validating novel treatments, and customizing current treatment strategies for individual patients. This review concluded that the future of NOA management may entail a combination of these treatment options, tailored to each patient's unique circumstances, providing a comprehensive approach to address NOA challenges.},
}

Humans
Male
*Gene Editing/methods
*Azoospermia/therapy/genetics
Sperm Retrieval
Sperm Injections, Intracytoplasmic
CRISPR-Cas Systems
Spermatozoa
Stem Cells

@article {pmid40081542,
year = {2025},
author = {Salvesen, HA and Dearden, PK},
title = {Genome editing in hymenoptera.},
journal = {Insect biochemistry and molecular biology},
volume = {180},
number = {},
pages = {104300},
doi = {10.1016/j.ibmb.2025.104300},
pmid = {40081542},
issn = {1879-0240},
mesh = {Animals ; *Gene Editing/methods ; *Hymenoptera/genetics ; *Genome, Insect ; CRISPR-Cas Systems ; },
abstract = {The application of genome editing tools in Hymenoptera has transformative potential for functional genetics and understanding their unique biology. Hymenoptera comprise one of the most diverse Orders of animals, and the development of methods for efficiently creating precise genome modifications could have applications in conservation, pest management and agriculture. To date, sex determination, DNA methylation, taste and smell sensory systems as well as phenotypic markers have been selected for gene editing investigations. From these data, insights into eusociality, the nature of haplodiploidy and the complex communication systems that Hymenoptera possess have provided an understanding of their evolutionary history that has led them to become so diverse and successful. Insights from these functional genetics analyses have been supported by the ever-improving suite of CRIPSR tools and further expansion will allow more specific biological hypotheses to be tested and applications beyond the lab. Looking ahead, genome editing tools have potential for Hymenopteran applications in modifying biocontrol agents of agricultural pests and for use in managing invasive species through the development of technologies such as gene drives. This review provides accessibility to information regarding the status of Hymenopteran genome editing, intending to support the considered development of CRISPR tools in novel species as well as innovation and refinement of methods in species in which it has already been achieved.},
}

Animals
*Gene Editing/methods
*Hymenoptera/genetics
*Genome, Insect
CRISPR-Cas Systems

@article {pmid40044744,
year = {2025},
author = {Yamada, H and Odagiri, M and Yamakita, K and Chiba, A and Ukai, A and Yasui, M and Honma, M and Sugiyama, KI and Ura, K and Sassa, A},
title = {Dual-directional epi-genotoxicity assay for assessing chemically induced epigenetic effects utilizing the housekeeping TK gene.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {7780},
pmid = {40044744},
issn = {2045-2322},
support = {21KA1001//Ministry of Health, Labor and Welfare/ ; 24KA1008//Consumer Affairs Agency/ ; 22H03748//JSPS/ ; },
mesh = {Humans ; *Epigenesis, Genetic/drug effects ; *Thymidine Kinase/genetics ; Mutagenicity Tests/methods ; Cell Line ; *Genes, Essential ; DNA Methylation/drug effects ; Tetradecanoylphorbol Acetate/toxicity ; Promoter Regions, Genetic ; Carcinogens/toxicity ; Mutation ; CRISPR-Cas Systems ; Histones/metabolism ; },
abstract = {Numerous chemicals are associated with carcinogenesis through epigenetic alterations in cells. To detect global epigenetic changes induced by carcinogens, the housekeeping gene can serve as a reporter locus, offering a baseline for identifying shifts in epigenetic marks. To investigate this potential, we developed a simple, cost-effective, and quantitative reporter system to assess chemically induced epigenetic effects, utilizing the thymidine kinase (TK) gene mutation assay as a foundation. Using a standard genotoxicity test cell line, human lymphoblast TK6, we edited the CpG promoter loci of the endogenous TK gene using the CRISPR/dCas9-SunTag-DNMT3A system. This epi-genotoxicity assay, employing modified mTK6 cells, provides a simple method for quantifying chemically induced epigenetic effects. The assay successfully detects both increased TK reversion rates induced by DNMT inhibitors, such as 5-Aza-2'-deoxycytidine and GSK-3484862, and, for the first time, a significant reduction in TK revertant frequency caused by the non-genotoxic carcinogen 12-O-tetradecanoylphorbol-13-acetate (TPA). Chromatin immunoprecipitation and western blotting analyses revealed that TPA treatment led to a global decrease in H3K27Ac levels, likely driven by TPA-mediated inflammation. These results demonstrate the utility of the epi-genotoxicity assay as a valuable tool for evaluating dual-directional epigenetic changes triggered by chemical exposure.},
}

Humans
*Epigenesis, Genetic/drug effects
*Thymidine Kinase/genetics
Mutagenicity Tests/methods
Cell Line
*Genes, Essential
DNA Methylation/drug effects
Tetradecanoylphorbol Acetate/toxicity
Promoter Regions, Genetic
Carcinogens/toxicity
Mutation
CRISPR-Cas Systems
Histones/metabolism

@article {pmid40025280,
year = {2025},
author = {Ha, TY and Chan, SW and Wang, Z and Law, PWN and Miu, KK and Lu, G and Chan, WY},
title = {SOX9 haploinsufficiency reveals SOX9-Noggin interaction in BMP-SMAD signaling pathway in chondrogenesis.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {82},
number = {1},
pages = {99},
pmid = {40025280},
issn = {1420-9071},
support = {Acc 8601011//CUHK VC Discretionary Fund/ ; YFJGJS1.0//CUHK Laboratory Support Special Fund for Key Laboratory for Regenerative Medicine, Ministry of Education, China/ ; N_CUHK 428/22//NSFC/RGC Joint Grant/ ; N_CUHK 434/24//NSFC/RGC Joint Grant/ ; MHP/005/23//Innovation and Technology Commission grant (Mainland-Hong Kong Joint Funding Scheme, MHKJFS)/ ; JLFS/M-403/24//Co-funding Mechaism on Joint Laboratories with the CAS/ ; },
mesh = {Humans ; *SOX9 Transcription Factor/genetics/metabolism ; *Chondrogenesis/genetics ; *Haploinsufficiency/genetics ; *Signal Transduction ; *Bone Morphogenetic Proteins/metabolism ; *Carrier Proteins/metabolism/genetics ; Male ; Chondrocytes/metabolism/cytology ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Smad Proteins/metabolism ; Campomelic Dysplasia/genetics/pathology/metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; },
abstract = {Campomelic Dysplasia (CD) is a rare congenital disease caused by haploinsufficiency (HI) in SOX9. Patients with CD typically present with skeletal abnormalities and 75% of them have sex reversal. In this study, we use CRISPR/Cas9 to generate a human induced pluripotent stem cell (hiPSC) model from a heathy male donor, based on a previously reported SOX9 splice site mutation in a CD patients. This hiPSCs-derived chondrocytes from heterozygotes (HT) and homozygotes (HM) SOX9 mutation carriers showed significant defects in chondrogenesis. Bulk RNA profiling revealed that the BMP-SMAD signaling pathway, ribosome-related, and chromosome segregation-related gene sets were altered in the HT chondrocytes. The profile also showed significant noggin upregulation in CD chondrocytes, with ChIP-qPCR confirming that SOX9 binds to the distal regulatory element of noggin. This suggests SOX9 plays a feedback role in the BMP signaling pathway by modulating noggin expression rather than acting solely as a downstream regulator. This provides further insights into its dosage sensitivity in chondrogenesis. Overexpression of SOX9 showed promising results with improved sulfated glycosaminoglycans (GAGs) aggregation and COL2A1 expression following differentiation. We hope this finding could provide a better understanding of the dosage-dependent role of SOX9 in chondrogenesis and contribute to the development of improved therapeutic targets for CD patients.},
}

Humans
*SOX9 Transcription Factor/genetics/metabolism
*Chondrogenesis/genetics
*Haploinsufficiency/genetics
*Signal Transduction
*Bone Morphogenetic Proteins/metabolism
*Carrier Proteins/metabolism/genetics
Male
Chondrocytes/metabolism/cytology
Induced Pluripotent Stem Cells/metabolism/cytology
*Smad Proteins/metabolism
Campomelic Dysplasia/genetics/pathology/metabolism
CRISPR-Cas Systems
Cell Differentiation

@article {pmid40328729,
year = {2025},
author = {Hermann, J and Borteçen, T and Kalis, R and Kowar, A and Pechincha, C and Vogt, V and Schneider, M and Helm, D and Krijgsveld, J and Loayza-Puch, F and Zuber, J and Palm, W},
title = {mTORC1 cooperates with tRNA wobble modification to sustain the protein synthesis machinery.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4201},
pmid = {40328729},
issn = {2041-1723},
support = {Projektnummer 2024.086.1//Wilhelm Sander-Stiftung (Wilhelm Sander Foundation)/ ; ERC StG No. 759579//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; },
mesh = {*RNA, Transfer/metabolism ; Animals ; Humans ; Mice ; Cell Line, Tumor ; Gene Knockout Techniques ; *Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors/metabolism ; *Protein Biosynthesis ; CRISPR-Cas Systems ; Codon/metabolism ; Uridine/metabolism ; Ribosomes/metabolism ; },
abstract = {Synthesizing the cellular proteome is a demanding process that is regulated by numerous signaling pathways and RNA modifications. How precisely these mechanisms control the protein synthesis machinery to generate specific proteome subsets remains unclear. Here, through genome-wide CRISPR screens we identify genes that enable mammalian cells to adapt to inactivation of the kinase mechanistic target of rapamycin complex 1 (mTORC1), the central driver of protein synthesis. When mTORC1 is inactive, enzymes that modify tRNAs at wobble uridines (U34-enzymes), Elongator and Ctu1/2, become critically essential for cell growth in vitro and in tumors. By integrating quantitative nascent proteomics, steady-state proteomics and ribosome profiling, we demonstrate that the loss of U34-enzymes particularly impairs the synthesis of ribosomal proteins. However, when mTORC1 is active, this biosynthetic defect only mildly affects steady-state protein abundance. By contrast, simultaneous suppression of mTORC1 and U34-enzymes depletes cells of ribosomal proteins, globally inhibiting translation. Thus, mTORC1 cooperates with tRNA U34-enzymes to sustain the protein synthesis machinery and support the high translational requirements of cell growth.},
}

*RNA, Transfer/metabolism
Animals
Humans
Mice
Cell Line, Tumor
Gene Knockout Techniques
*Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors/metabolism
*Protein Biosynthesis
CRISPR-Cas Systems
Codon/metabolism
Uridine/metabolism
Ribosomes/metabolism

@article {pmid40286769,
year = {2025},
author = {Yang, X and Gao, S},
title = {Competitive rDNA binding by dCas9 induces outside-in disassembly of the nucleolus.},
journal = {Biochemical and biophysical research communications},
volume = {766},
number = {},
pages = {151883},
doi = {10.1016/j.bbrc.2025.151883},
pmid = {40286769},
issn = {1090-2104},
mesh = {*Cell Nucleolus/metabolism/genetics ; Nucleophosmin ; *DNA, Ribosomal/metabolism/genetics ; Humans ; Nuclear Proteins/metabolism/genetics ; HeLa Cells ; Promoter Regions, Genetic ; *CRISPR-Cas Systems ; Transcription, Genetic ; Pol1 Transcription Initiation Complex Proteins/metabolism ; },
abstract = {The inside-out assembly and outside-in disassembly of the nucleolus are well-accepted models, yet direct in-cell evidence remains elusive. Here, we employed a dCas9-based competitive binding system to specifically target the rDNA promoter within the nucleolus, effectively inhibiting rDNA transcription. This transcriptional blockade induced a stepwise, outside-in disassembly of the nucleolus. NPM1 was the first to disappear from the nucleolus, followed by a progressive reduction in the fluorescence intensities of FBL and UBF. Additionally, UBF relocated from the nucleolar core to the periphery. These findings provide the first direct evidence in cells supporting the outside-in disassembly of the nucleolus. Furthermore, our results suggest that the dynamic inside-out assembly and outside-in disassembly of the nucleolus.},
}

*Cell Nucleolus/metabolism/genetics
Nucleophosmin
*DNA, Ribosomal/metabolism/genetics
Humans
Nuclear Proteins/metabolism/genetics
HeLa Cells
Promoter Regions, Genetic
*CRISPR-Cas Systems
Transcription, Genetic
Pol1 Transcription Initiation Complex Proteins/metabolism

@article {pmid40252377,
year = {2025},
author = {Zhou, T and Shen, G and Zhong, L and Chen, G and Meng, L and He, W and Liu, J and Yang, S and Luo, Y and Wang, X},
title = {crRNA array-mediated CRISPR/Cas12a coupling with dual RPA for highly sensitive detection of Streptomyces aureofaciens Tü117 from hypertension with multi-signal output.},
journal = {Biosensors & bioelectronics},
volume = {282},
number = {},
pages = {117493},
doi = {10.1016/j.bios.2025.117493},
pmid = {40252377},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Animals ; *Streptomyces/isolation & purification/genetics ; Humans ; Nucleic Acid Amplification Techniques/methods ; Mice ; *Hypertension/microbiology/diagnosis ; Limit of Detection ; },
abstract = {Accurate and sensitive detection of Streptomyces aureofaciens Tü117 is crucial for hypertension classification and early warning. To achieve this, a dual recombinase polymerase amplification coupled with a crRNA array-mediated CRISPR/Cas12a assay (DR-CAMCas) was developed, enabling multi-signal output for precise identification and detection of S. aureofaciens Tü117. The 16S rDNA and LipReg4 genes of S. aureofaciens Tü117 are amplified simultaneously via one-step dual RPA, activating the crRNA array-mediated CRISPR/Cas12a system to cleave exogenous FQ-reporters, releasing fluorescent signals. DR-CAMCas offers high amplification efficiency, multi-site recognition through crRNA array signal superposition, and the programmability of CRISPR/Cas12a, achieving ultrasensitive detection with a linear range of 10 to 10[8] cfu/mL and a limit of detection of approximately 3 cfu/mL. DR-CAMCas successfully detected S. aureofaciens Tü117 in fecal samples from high-salt diet-induced hypertensive mice and hypertensive patients, matching qPCR results and demonstrating high reliability and practicality. Additionally, target-induced cleavage of a DNA linker by DR-CAMCas dispersed AuNPs-DNA probes, enabling colorimetric detection. Integrated onto lateral flow sensors, DR-CAMCas allows point-of-care testing via simple visual strip analysis. Its triple signal output meets diverse detection needs, offering a promising tool for diagnosing salt-sensitive hypertension.},
}

*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
Animals
*Streptomyces/isolation & purification/genetics
Humans
Nucleic Acid Amplification Techniques/methods
Mice
*Hypertension/microbiology/diagnosis
Limit of Detection

@article {pmid40216108,
year = {2025},
author = {Yi, JY and Park, S and Kim, M and Jeong, Y and Shin, H and Cho, Y and Jeon, M and Oh, MK and Sung, C},
title = {Emerging wound-healing injectable polydeoxyribonucleotide: potential as a prohibited doping method and its simple detection via CRISPR/Cas12a system.},
journal = {International journal of biological macromolecules},
volume = {309},
number = {Pt 3},
pages = {142999},
doi = {10.1016/j.ijbiomac.2025.142999},
pmid = {40216108},
issn = {1879-0003},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Polydeoxyribonucleotides/administration & dosage/pharmacology/urine/genetics ; *Doping in Sports/prevention & control ; Animals ; *Wound Healing/drug effects ; },
abstract = {Polydeoxyribonucleotide (PDRN), derived from chum salmon (Oncorhynchus keta), is a mixture of hydrolyzed DNA fragments used in various clinical applications. Its therapeutic value stems from its ability to promote wound healing by upregulating growth factors like VEGF, FGF, and HIF-1. However, PDRN's regenerative properties raise concerns about its potential misuse in sports. Studies suggest it may enhance athletic performance by stimulating muscle growth, recovery, and endurance through mechanisms such as satellite cell activation, angiogenesis, and anti-inflammatory effects. These potential performance-enhancing effects could be considered gene or cell doping, prohibited by the World Anti-Doping Agency (WADA). To address this concern, we developed a sensitive and specific detection method for PDRN misuse based on the CRISPR-Cas12a system. This method targets conserved 12S and 16S rDNA sequences unique to salmonids. A direct PCR method was optimized to amplify these target sequences from human plasma and urine without prior DNA extraction. The amplified DNA was then subjected to Cas12a-mediated detection, resulting in a fluorescent signal upon successful target recognition. This method demonstrated high sensitivity, detecting as little as 0.8 pg(0.3 genome copies) of O. keta DNA in 10 μL of biological samples within 90 min, surpassing the detection limits of many current doping agents.},
}

*CRISPR-Cas Systems/genetics
Humans
*Polydeoxyribonucleotides/administration & dosage/pharmacology/urine/genetics
*Doping in Sports/prevention & control
Animals
*Wound Healing/drug effects

@article {pmid40118423,
year = {2025},
author = {Lee, MH and Thomas, JL and Lin, YL and Lin, HY},
title = {In vitro activation of anti-cancer gene expression by delivery of CRISPR/dCas9 ribonucleoproteins to suppress glioblastoma.},
journal = {International journal of biological macromolecules},
volume = {308},
number = {Pt 1},
pages = {142289},
doi = {10.1016/j.ijbiomac.2025.142289},
pmid = {40118423},
issn = {1879-0003},
mesh = {Humans ; *Glioblastoma/genetics/therapy/pathology ; *Ribonucleoproteins/genetics ; Cell Line, Tumor ; *CRISPR-Cas Systems/genetics ; *Gene Expression Regulation, Neoplastic ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics ; Astrocytes/metabolism ; Apoptosis/genetics ; Tumor Suppressor Protein p53/genetics ; },
abstract = {Cancer has been a leading cause of death for decades. While many anti-cancer drugs exist, precisely targeting malignant cells is crucial for successful tumor treatment. This targeting can be achieved by activating anti-cancer genes, which specifically destroy malignant cells. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) therapeutics provide a promising approach for gene activation. The technology involves utilizing the denatured Cas9 (CRISPR-associated) protein conjugated with a protein activator to deliver a ribonucleoprotein (RNP) complex including guide RNA into cells for the overexpression of specific proteins. In this study, several guide RNAs targeting cancer suppressor genes were employed. These genes included tumor protein p53 (TP53), human alpha-lactalbumin made lethal to tumor cells (HAMLET), melanoma differentiation-associated gene-7 (MDA7, IL24), phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1, NOXA), pro-apoptotic WT1 regulator (PAWR, PAR4), and TNF superfamily member 10 (TNFSF10, TRAIL). The dCas9/guide RNA complexes were then adsorbed onto magnetic epitope-imprinted nanoparticles. Uppsala 87 malignant glioma (U87MG) cells and induced astrocytes (noncancerous cells) were then treated with the RNP / nanoparticles. The overexpression of MDA7 and NOXA was monitored for at least 30 days using enzyme-linked immunosorbent assay (ELISA) kits. Finally, the induced astrocytes, first activated with these anti-cancer genes, were co-cultured with U87MG cells. This resulted in a "bystander" effect: the malignant U87MG cells underwent apoptosis, while the astrocytes survived.},
}

Humans
*Glioblastoma/genetics/therapy/pathology
*Ribonucleoproteins/genetics
Cell Line, Tumor
*CRISPR-Cas Systems/genetics
*Gene Expression Regulation, Neoplastic
RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Associated Protein 9/genetics
Astrocytes/metabolism
Apoptosis/genetics
Tumor Suppressor Protein p53/genetics

@article {pmid40118400,
year = {2025},
author = {Kong, F and Peng, S and Zhang, Y and Zhang, H and Wang, J and Wang, D},
title = {Spatial double-layer hydrogels enabled visual detection of Cladobotryum mycophilum based on recombinase-aided amplification - CRISPR/Cas12a.},
journal = {International journal of biological macromolecules},
volume = {308},
number = {Pt 1},
pages = {142304},
doi = {10.1016/j.ijbiomac.2025.142304},
pmid = {40118400},
issn = {1879-0003},
mesh = {*CRISPR-Cas Systems ; *Hydrogels/chemistry ; *Ascomycota/isolation & purification/genetics ; *Recombinases/metabolism ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Cladobotryum mycophilum, a pathogen responsible for cobweb disease, caused significant and irreversible losses in the mushroom industry. Effective monitoring and early prevention rely on the development of advanced diagnosis methods. This study introduced a novel hydrogel-based C. mycophilum detection method that integrates recombinase-aided amplification (RAA) with the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) system (CRISPR/Cas12a), referred as RCCH. The RAA reaction occurs within cross-linked PEG hydrogel, which is subsequently overlaid with a CRISPR/Cas12a-functionalized hydrogel. The porous network of the PEG hydrogel traps essential enzymes, facilitating spatial co-localization of target DNA and the CRISPR/Cas12a-crRNA complex. Upon activation of Cas12a's trans-cleavage activity, clear and countable fluorescent spots are generated for visual detection. RCCH demonstrates a limit of detection as low as 1 fg/μL, and exceptional selectivity against common fungi Trichoderma viride and T. harzianum and the host mushroom Lentinula edodes. The entire process is completed in under 40 min, indicating RCCH's potential as a rapid, accurate, and practical detection method for monitoring mushroom diseases. This innovative approach offers significant support for enhancing safety in the mushroom industry.},
}

*CRISPR-Cas Systems
*Hydrogels/chemistry
*Ascomycota/isolation & purification/genetics
*Recombinases/metabolism
*Nucleic Acid Amplification Techniques/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid39988603,
year = {2025},
author = {Cocito, C and Xiang, C and Huang, M and Gongora, T and Surana, P and Davuluri, R and Dahmane, N and Greenfield, JP},
title = {Immunoglobulin superfamily 3 (Igsf3) function is dispensable for brain development.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {6526},
pmid = {39988603},
issn = {2045-2322},
support = {R01 NS111997/NS/NINDS NIH HHS/United States ; 5RO1NS111997//NIH-NINDS/ ; },
mesh = {Animals ; Mice ; Mice, Knockout ; *Brain/metabolism/growth & development/embryology ; Neurons/metabolism/cytology ; Cell Movement ; Neurogenesis ; *Membrane Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; },
abstract = {The Immunoglobulin superfamily (IgSF) is a heterogeneous and conserved family of adhesion proteins crucial during the development of the central nervous system including neuronal migration and synaptogenesis. The Immunoglobulin superfamily member 3 (IGSF3) is expressed in the developing brain and has been suggested to play a role during morphological development of the granule cells neurites in the cerebellum. In addition, a role for IGSF3 in supporting glioma progression has been recently demonstrated. Remaining unexplored is the physiological role of IGSF3 in regulating brain development, including neocortical development. We generated an Igsf3 knockout (KO) mouse using a CRISPR/Cas9-based approach and explored the function of Igsf3 in regulating cortical development. We found that Igsf3 largely co-localizes with other IgSF proteins during cortical development and despite its expression being developmentally regulated in neuronal progenitors and in postmitotic neurons, Igsf3 is not essential for brain development, neuronal migration, or neuronal maturation.},
}

Animals
Mice
Mice, Knockout
*Brain/metabolism/growth & development/embryology
Neurons/metabolism/cytology
Cell Movement
Neurogenesis
*Membrane Proteins/metabolism/genetics
CRISPR-Cas Systems
Gene Expression Regulation, Developmental

@article {pmid39954947,
year = {2025},
author = {Souza, HCA and Panzenhagen, P and Dos Santos, AMP and Portes, AB and Fidelis, J and Conte-Junior, CA},
title = {Unravelling the advances of CRISPR-Cas9 as a precise antimicrobial therapy: A systematic review.},
journal = {Journal of global antimicrobial resistance},
volume = {42},
number = {},
pages = {51-60},
doi = {10.1016/j.jgar.2025.02.002},
pmid = {39954947},
issn = {2213-7173},
mesh = {*CRISPR-Cas Systems ; Humans ; *Anti-Bacterial Agents/pharmacology ; *Drug Resistance, Bacterial/genetics ; Plasmids/genetics ; Escherichia coli/genetics/drug effects ; Gene Editing ; },
abstract = {UNLABELLED: Antimicrobial resistance is a critical public health threat, compromising treatment effectiveness. The spread of resistant pathogens, facilitated by genetic variability and horizontal gene transfer, primarily through plasmids, poses significant challenges to health systems.

OBJECTIVE: This review explores the potential of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology and Cas9 nucleases in combating antimicrobial resistance.

METHODS: The literature review followed the PRISMA guidelines using PubMed, Embase, and Scopus databases until July 2023.

RESULTS: The Enterobacterales family, particularly Escherichia coli, was the main focus. The resistance genes targeted were mainly associated with β-lactam antibiotics, specifically bla genes, and colistin resistance linked to the mcr-1 gene. Plasmid vectors have been the primary delivery method for the CRISPR-Cas9 system, with conjugative plasmids resensitizing bacterial strains to various antimicrobials. Other delivery methods included electroporation, phage-mediated delivery, and nanoparticles. The efficacy of the CRISPR-Cas9 system in resensitizing bacterial strains ranged from 4.7% to 100%.

CONCLUSIONS: Despite challenges in delivery strategies and clinical application, studies integrating nanotechnology present promising approaches to overcome these limitations. This review highlights new perspectives for the clinical use of CRISPR-Cas9 as a specific and efficient antimicrobial agent, potentially replacing traditional broad-spectrum antimicrobials in the future.},
}

*CRISPR-Cas Systems
Humans
*Anti-Bacterial Agents/pharmacology
*Drug Resistance, Bacterial/genetics
Plasmids/genetics
Escherichia coli/genetics/drug effects
Gene Editing

@article {pmid40262200,
year = {2025},
author = {Tian, SC and Song, XH and Feng, KK and Li, CL and Tu, YF and Hu, YS and Shao, JW},
title = {Self-oxygenating nanoplatform integrating CRISPR/Cas9 gene editing and immune activation for highly efficient photodynamic therapy.},
journal = {Journal of colloid and interface science},
volume = {693},
number = {},
pages = {137632},
doi = {10.1016/j.jcis.2025.137632},
pmid = {40262200},
issn = {1095-7103},
mesh = {*CRISPR-Cas Systems ; *Photochemotherapy ; *Gene Editing ; Animals ; Humans ; Mice ; *Manganese Compounds/chemistry/pharmacology ; *Oxides/chemistry/pharmacology ; *Nanoparticles/chemistry ; Reactive Oxygen Species/metabolism ; *Photosensitizing Agents/pharmacology/chemistry ; Tumor Microenvironment/drug effects ; Particle Size ; *Oxygen/chemistry/metabolism ; Phosphoric Monoester Hydrolases/genetics ; Surface Properties ; *Antineoplastic Agents/pharmacology/chemistry ; },
abstract = {Photodynamic therapy (PDT) has arisen as a promising method due to its spatiotemporal precision and minimal invasiveness. It encounters significant obstacles in solid tumors due to hypoxia-induced therapeutic resistance and the self-protective mechanisms of cancer cells facilitated by MutT homolog 1 (MTH1), an enzyme involved in oxidative damage repair. Herein, we fabricate a tumor-microenvironment responsive CRISPR nanoplatform based on hollow mesoporous manganese dioxide (H-MnO2) for PDT. This platform utilizes H-MnO2 to produce oxygen (O2) through the decomposition of hydrogen peroxide (H2O2) in TME, thereby mitigating hypoxia and enhancing reactive oxygen species (ROS) generation. The high concentration of glutathione (GSH) and hyaluronidase (HAase) in TME induces the release of CRISPR/Cas9 ribonucleoproteins (RNP) to target the MTH1 gene, thereby impairs oxidative damage repair pathways and amplifys ROS-mediated cytotoxicity. The released Mn[2+] ions function as immunomodulatory agents, activate innate immune responses via stimulating STING signal pathway. In vitro, IHMRH NPs markedly increased intracellular O2 levels, ROS production, lipid peroxidation and DNA damage, leading to tumor cell death, immune activation, and effective gene editing. In vivo, the nanoplatform suppressed tumor growth, diminished MTH1 gene expression, stimulated dendritic cell (DC) maturation through immunogenic cell death (ICD). This multimodal nanosystem may amplifies oxidative stress, collaborates with innate and adaptive immune activation to surpass the constraints of traditional PDT. The research presents a novel framework for cancer combination therapy by systematically integrating nanotechnology with precision gene editing.},
}

*CRISPR-Cas Systems
*Photochemotherapy
*Gene Editing
Animals
Humans
Mice
*Manganese Compounds/chemistry/pharmacology
*Oxides/chemistry/pharmacology
*Nanoparticles/chemistry
Reactive Oxygen Species/metabolism
*Photosensitizing Agents/pharmacology/chemistry
Tumor Microenvironment/drug effects
Particle Size
*Oxygen/chemistry/metabolism
Phosphoric Monoester Hydrolases/genetics
Surface Properties
*Antineoplastic Agents/pharmacology/chemistry

@article {pmid40245636,
year = {2025},
author = {Pu, Q and Ren, H and Ou, Q and Yang, X and Wei, T and Zhao, L and Han, Y and Lou, Y and Kashyap, S and Liu, S},
title = {SHMT, SHMTML and PRPS1 synergize to regulate blood digestion and nutrient metabolism in Aedes aegypti mosquitoes.},
journal = {International journal of biological macromolecules},
volume = {309},
number = {Pt 4},
pages = {143243},
doi = {10.1016/j.ijbiomac.2025.143243},
pmid = {40245636},
issn = {1879-0003},
mesh = {Animals ; *Aedes/metabolism/genetics ; *Glycine Hydroxymethyltransferase/metabolism/genetics/chemistry ; Female ; *Nutrients/metabolism ; *Insect Proteins/metabolism/genetics ; Digestion ; CRISPR-Cas Systems ; },
abstract = {Mosquitoes rely on vertebrate blood for nutrients vital for ovarian development. The enzyme serine hydroxymethyltransferase (SHMT) is crucial for amino acid and one‑carbon metabolism, playing a significant role in blood digestion and nutrient use in mosquitoes, though its functional mechanism remains further investigation. Using CRISPR/Cas9 to knock out the SHMT gene, we observed impaired blood digestion, delayed ovarian development, and inability to fly in mosquitoes. Multi-omics analysis revealed that SHMT deletion affected genes and metabolites related to amino acid metabolism. Knocking down SHMT-responsive genes mitochondrial-like serine hydroxymethyltransferase (SHMTML) and ribose-phosphate pyrophosphokinase 1 (PRPS1) also hindered blood digestion and ovarian development, mirroring SHMT-deficient mosquitoes. The interaction between SHMT, SHMTML, and PRPS1 was confirmed through various experiments, including Co-IP, GST pull-down, immunofluorescence colocalization, BiFC, molecular docking, and functional studies. Further research reveals that missing any of these proteins in mosquitoes results in ammonia and reactive oxygen species buildup, leading to mitochondrial problems, midgut cell damage, and abnormal enzyme expression. This study highlights a new molecular mechanism of SHMT and emphasizes its crucial interaction with SHMTML and PRPS1 in blood digestion and nutrient metabolism in vector mosquitoes. These findings may offer a strategic foundation for the development of innovative mosquito control measures.},
}

Animals
*Aedes/metabolism/genetics
*Glycine Hydroxymethyltransferase/metabolism/genetics/chemistry
Female
*Nutrients/metabolism
*Insect Proteins/metabolism/genetics
Digestion
CRISPR-Cas Systems

@article {pmid40228775,
year = {2025},
author = {Qiao, J and Sun, W and Yin, W and Hu, L and Wang, X and Liu, Y},
title = {Direct preparation of Cas9 ribonucleoproteins with an extended 'gRNA-shRNA' construct in Escherichia coli for precise genome manipulation.},
journal = {International journal of biological macromolecules},
volume = {309},
number = {Pt 4},
pages = {143121},
doi = {10.1016/j.ijbiomac.2025.143121},
pmid = {40228775},
issn = {1879-0003},
mesh = {*Escherichia coli/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Ribonucleoproteins/genetics/metabolism ; Humans ; *CRISPR-Cas Systems ; *RNA, Small Interfering/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; DNA Ligase ATP/genetics ; },
abstract = {Gene perturbation approaches have emerged as powerful tools for elucidating gene function and treating hereditary disorders. Previously, we developed a method for streamlined production of ready-to-use Cas9 ribonucleoproteins (RNPs) in Escherichia coli BL21(DE3). In this study, we present an improved approach by assembling Cas9 RNPs with an extended 'gRNA-shRNA' construct in the RNase III deficient strain HT115(DE3). Transfection of these engineered Cas9 RNPs into mammalian cells enables multidimensional genome manipulation, including simultaneous knockdown and knockout of target genes. Furthermore, the design of shRNA specifically targeting human DNA ligase IV (LIG4) significantly enhances efficiency in homology-directed repair genome editing. Collectively, our findings establish a user-friendly CRISPR/Cas9 RNP tool with immense potential for precise genome editing, gene function analysis, and gene therapy.},
}

*Escherichia coli/genetics/metabolism
*RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing/methods
*Ribonucleoproteins/genetics/metabolism
Humans
*CRISPR-Cas Systems
*RNA, Small Interfering/genetics
*CRISPR-Associated Protein 9/genetics/metabolism
DNA Ligase ATP/genetics

@article {pmid40222509,
year = {2025},
author = {Xiao, G and Shi, H and Liu, M and Huang, M and Li, S and Zhou, X and Li, H and Zhang, G},
title = {Trans-cleavage activity of Cas12a effectors can be unleashed by both double-stranded DNA and single-stranded RNA targeting in absence of PAM.},
journal = {International journal of biological macromolecules},
volume = {309},
number = {Pt 4},
pages = {142992},
doi = {10.1016/j.ijbiomac.2025.142992},
pmid = {40222509},
issn = {1879-0003},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *DNA/metabolism/genetics/chemistry ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; DNA, Single-Stranded/genetics ; *Bacterial Proteins/metabolism/chemistry/genetics ; Humans ; },
abstract = {CRISPR-Cas12a is a powerful tool in nucleic acid detection, but the relationship between its trans-cleavage activity and protospacer adjacent motif (PAM) sequences remains incompletely understood. In this study, we synthesized diverse PAM-sequence substrates and conducted systematic cis-cleavage and trans-cleavage experiments with three Cas12a orthologs. We found that double-stranded DNA (dsDNA) can activate Cas12a's trans-cleavage activity even without PAM and this activation occurring independently of cis-cleavage. Notably, our results also revealed that single-stranded RNA (ssRNA) can directly initiate the trans-cleavage activity of Cas12a.We also experimentally validated the feasibility of CRISPR-Cas12a in detecting target dsDNA lacking PAM sequences, including identifying mutated sites in clinical samples. Structural prediction using AlphaFold 3 revealed the potential mechanism of Cas12a's PAM-independent trans-cleavage. Our research expands the understanding of Cas12a's trans-cleavage mechanism and demonstrates its potential for nucleic acid detection beyond PAM-dependent targets. This discovery broadens the application scope of Cas12a, providing new opportunities for developing highly sensitive and versatile diagnostic platforms.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism/chemistry/genetics
*DNA/metabolism/genetics/chemistry
*Endodeoxyribonucleases/metabolism/chemistry/genetics
DNA, Single-Stranded/genetics
*Bacterial Proteins/metabolism/chemistry/genetics
Humans

@article {pmid40210053,
year = {2025},
author = {Bai, J and Lu, H and Xue, Y and Li, M and Fang, S and Cui, L and He, Y and Wang, Y and Li, H and He, F and Zhao, B and Ai, L and Wang, S},
title = {Deletion of the DEAD-box helicase CshA in Lacticaseibacillus paracasei elicits heightened low-temperature sensitivity and reduced post-acidification capacity.},
journal = {International journal of biological macromolecules},
volume = {309},
number = {Pt 4},
pages = {142970},
doi = {10.1016/j.ijbiomac.2025.142970},
pmid = {40210053},
issn = {1879-0003},
mesh = {*Cold Temperature ; *DEAD-box RNA Helicases/genetics/metabolism ; *Gene Deletion ; *Bacterial Proteins/genetics/metabolism ; Hydrogen-Ion Concentration ; Fermentation ; CRISPR-Cas Systems ; Animals ; },
abstract = {While cold chain logistics remains the primary strategy to mitigate post acidification in fermented dairy products, maintaining reliable refrigeration systems presents substantial challenges, especially in resource-limited settings. Biological approaches targeting strain low-temperature sensitivity modulation for post-acidification control are increasingly recognized as promising alternatives. This study utilized CRISPR/Cas9-mediated knockout of DEAD-box helicase genes in Lacticaseibacillus paracasei, successfully identifying a low-temperature-sensitive strain, ∆AF91_12,710. Under optimal conditions, ∆AF91_12,710 showed comparable growth patterns and pH profiles to the wild-type strain. However, under low-temperature conditions, ∆AF91_12,710 demonstrated significantly impaired growth capacity and distinct pH regulation patterns. Metabolomic analysis of post-acidification revealed 39 unique differential metabolites in ∆AF91_12,710 fermented milk over 21 days, featuring decreased ATP, γ-linolenic acid, and dGMP concentrations, alongside elevated glucose-6-phosphate, cAMP, and D-galactose levels. These metabolic alterations indicate impaired glycolysis, likely resulting from DEAD-box helicase gene deletion-induced RNA instability and reduced metabolic activity in ∆AF91_12,710 under cold stress. The AF91_12,710 deletion significantly enhanced low-temperature sensitivity while reducing post-acidification capability in fermented milk. This research provides valuable insights into post-acidification attenuation in low-temperature-sensitive strains and demonstrates the potential for developing robust starter cultures in dairy applications.},
}

*Cold Temperature
*DEAD-box RNA Helicases/genetics/metabolism
*Gene Deletion
*Bacterial Proteins/genetics/metabolism
Hydrogen-Ion Concentration
Fermentation
CRISPR-Cas Systems
Animals

@article {pmid40187665,
year = {2025},
author = {Pablo-Marcos, D and Fernández-Diego, L and Rodríguez-Grande, J and Fraile-Valcárcel, N and Ortiz-Cartagena, C and Pacios, O and García-García, S and García-Fernández, S and Blasco, L and Ocampo-Sosa, A and Calvo-Montes, J and Tomás, M},
title = {An accurate amplification-free CRISPR/Cas12a-based assay for GES β-lactamase detection.},
journal = {International journal of antimicrobial agents},
volume = {66},
number = {1},
pages = {107506},
doi = {10.1016/j.ijantimicag.2025.107506},
pmid = {40187665},
issn = {1872-7913},
mesh = {*beta-Lactamases/genetics/analysis ; *Pseudomonas aeruginosa/genetics/enzymology/isolation & purification ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; Humans ; *Molecular Diagnostic Techniques/methods ; *Enterobacteriaceae/genetics/enzymology/isolation & purification ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {OBJECTIVE: Guiana-Extended-Spectrum (GES) β-lactamases belong to the minor class A β-lactamases and are probably underdiagnosed due to a lack of specific diagnostic tests. There is therefore an urgent need to develop new molecular diagnostic tools that will be able to fill the gap in the detection of rare β-lactamases. Here, we propose an optimized, amplification-free CRISPR/Cas12a-based assay for the accurate detection of GES β-lactamases and we validate its application with clinical isolates (Graphic abstract). Based on the results of examination of 79 standard collection, the proposed assay exhibited 100% sensitivity and specificity, as well as 100% positive and negative predictive values in less than 1.5 hours.

METHODS: We optimized the CRISPR/Cas12a method by harnessing a multiplex crRNA strategy, a highly efficient DNA reporter (TTATT-5C) and the Murine RNase Inhibitor to prevent crRNA degradation.

RESULTS: Our yielded limits of detection of 1 ng/µL and 3 ng/µL in Enterobacterales and Pseudomonas aeruginosa, respectively. The observed difference is due to the location of the blaGES gene. The gene occurs in a chromosomal integron present only in one to three copies in P. aeruginosa, whereas it occurs in plasmids present in multiple copies in Enterobacterales.

CONCLUSIONS: The proposed method could be established as a routine diagnostic tool in clinical microbiology laboratories to fill the gap in availability of commercial diagnostic tests for GES β-lactamases.},
}

*beta-Lactamases/genetics/analysis
*Pseudomonas aeruginosa/genetics/enzymology/isolation & purification
Sensitivity and Specificity
*CRISPR-Cas Systems
Humans
*Molecular Diagnostic Techniques/methods
*Enterobacteriaceae/genetics/enzymology/isolation & purification
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40122420,
year = {2025},
author = {Chen, Y and Lv, B and Wang, W and Wu, Y and Li, D},
title = {Structure-switching G-quadruplex: An efficient CRISPR/Cas12a signal reporter for label-free colorimetric biosensing.},
journal = {International journal of biological macromolecules},
volume = {307},
number = {Pt 4},
pages = {142410},
doi = {10.1016/j.ijbiomac.2025.142410},
pmid = {40122420},
issn = {1879-0003},
mesh = {*G-Quadruplexes ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Colorimetry/methods ; MicroRNAs/analysis/genetics ; DNA, Catalytic/chemistry ; Adenosine Triphosphate/analysis ; Humans ; Hemin/chemistry ; DNA/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {G-quadruplex is widely used as a signal reporter for colorimetric biosensor construction. However, the effectiveness of CRISPR/Cas12a in trans-cleaving G-quadruplexes is significantly influenced by their resistance to nuclease, resulting in a weak colorimetric signal response. Herein, a structure-switching G-quadruplex regulated by transducer DNA is used as a signal reporter to construct CRISPR/Cas12a-based biosensors. The transducer DNA lacks a stable secondary structure, enabling efficient cleavage by CRISPR/Cas12a, which subsequently affects the catalytic activity of the G-quadruplex/hemin DNAzyme. We used microRNAs (miRNAs) and ATP as model targets to develop a label-free colorimetric detection platform. By optimizing the DNA sequences and reaction conditions, the biosensors exhibit excellent detection selectivity and sensitivity. The reliability of the proposed method was validated by its consistency with RT-qPCR for miRNAs detection and a commercial chemiluminescence kit for ATP assay, demonstrating its potential in clinical diagnosis and bioanalytical studies. The assay is concise and cost-effective because it does not require DNA labeling, magnetic separation, or enzymatic DNA amplification. Our design strategy avoids the use of G-quadruplex as a cleavage substrate for CRISPR/Cas12a while ensuring an efficient response of the G-quadruplex/hemin DNAzyme to CRISPR/Cas12a system, addressing the issue of G-quadruplex resistance to CRISPR/Cas12a nuclease activity.},
}

*G-Quadruplexes
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
*Colorimetry/methods
MicroRNAs/analysis/genetics
DNA, Catalytic/chemistry
Adenosine Triphosphate/analysis
Humans
Hemin/chemistry
DNA/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40107560,
year = {2025},
author = {Xiong, SS and Wang, XR and Han, KK and Tang, JQ and Lu, WT and Kong, XY and Fan, R and Sun, XL and Ji, YL and Wu, K and Guo, DD and Wan, Z and Xun, JN and Jiao, S and Zhai, H},
title = {Creation of dual-purpose soybean germplasm for grain and forage by CRISPR/Cas9-mediated targeting mutation of GmFT2a and GmFT5a.},
journal = {International journal of biological macromolecules},
volume = {307},
number = {Pt 4},
pages = {142234},
doi = {10.1016/j.ijbiomac.2025.142234},
pmid = {40107560},
issn = {1879-0003},
mesh = {*Glycine max/genetics/growth & development ; *CRISPR-Cas Systems/genetics ; *Mutation ; *Plant Proteins/genetics ; *Seeds/genetics/growth & development ; *Edible Grain/genetics ; Gene Editing ; Gene Expression Regulation, Plant ; Plants, Genetically Modified/genetics ; },
abstract = {Soybean [Glycine max (L.) Merr.] seeds are a rich source of high-quality protein and edible oil, and their foliage supports the rearing of the economically significant insect, 'Doudan'. This study utilized CRISPR/Cas9 to edit the GmFT2a and GmFT5a genes, resulting in soybean germplasm with enhanced grain and forage traits. Under short-day conditions, the double mutant ft2a ft5a showed significant increases in grain yield and yield-related traits, including main stem nodes, branching, and pod count per plant. Under long-day conditions, the double mutant exhibited a substantial increase in total vegetative biomass, with prolonged vegetative growth, larger leaves, and increased branching and nodes. The soluble protein and soluble sugar contents remained unchanged, while phenylalanine levels increased and tannin content decreased, which is beneficial for Doudan feeding. The ft2a ft5a double mutant is suitable for both grain and forage soybeans. We further revealed the molecular basis for the influence of GmFT2a and GmFT5a on soybean architecture. GmFT2a and GmFT5a proteins interact with each other and with Dt2 and SOC1a critical genes for branching and stem growth in soybeans. This interaction forms a complex that potently activates the downstream GmAp1s gene expression. The simultaneous mutation of GmFT2a and GmFT5a significantly downregulates GmAp1s expression, impacting soybean plant architecture. Overall, this study not only identifies the dual role of GmFT2a and GmFT5a in soybean yield and biomass but also uncovers their molecular interactions with Dt2 and SOC1a, providing a foundation for the genetic enhancement of soybean varieties tailored for both grain and forage production, underscoring the potential for improving agricultural sustainability and economic value.},
}

*Glycine max/genetics/growth & development
*CRISPR-Cas Systems/genetics
*Mutation
*Plant Proteins/genetics
*Seeds/genetics/growth & development
*Edible Grain/genetics
Gene Editing
Gene Expression Regulation, Plant
Plants, Genetically Modified/genetics

@article {pmid40049443,
year = {2025},
author = {Svoboda, T and Niederdöckl-Loibl, D and Schüller, A and Hummel, K and Schlosser, S and Razzazi-Fazeli, E and Strauss, J},
title = {Locus-specific chromatin proteomics using dCas-guided proximity labelling in Aspergillus nidulans.},
journal = {Fungal genetics and biology : FG & B},
volume = {178},
number = {},
pages = {103973},
doi = {10.1016/j.fgb.2025.103973},
pmid = {40049443},
issn = {1096-0937},
mesh = {*Chromatin/genetics/metabolism ; *Aspergillus nidulans/genetics/metabolism ; *Proteomics/methods ; Fungal Proteins/genetics/metabolism ; Biotinylation ; Transcription Factors/genetics ; CRISPR-Cas Systems ; Chromatin Immunoprecipitation ; Carbon-Nitrogen Ligases/genetics ; },
abstract = {Proximity labelling that uses promiscuous biotin ligases (BirA) fused to a bait protein is a powerful tool to identify protein interaction partners in vivo under different metabolic or developmental conditions. BirA can also be used to determine protein composition and interaction partners at specific chromatin locations when it is fused with enzymatically-disabled Cas9 (dCas9) and then guided to the location of interest by sgRNAs. We adapted this method (called CasID) for fungal cells using the nitrate assimilation gene cluster of A. nidulans as a model locus and estrogen-inducible expression of the dCas9-BirA fusion to improve condition-specific labelling. For method establishment, we first verified the presence of dCas-BirA and a known transcription factor at the nitrate locus by chromatin immunoprecipitation (ChIP). Results show that both dCas-BirA and the AreA transcription factor are present at the locus of interest under the conditions used for biotinylation. We then optimized the CasID procedure for efficient labelling and background reduction using the CasID-sgRNA strain and two control strains, one lacking the sgRNA and another one lacking the whole CasID system. Here we provide proof-of-concept for the suitability of the method by showing that biotinylated proteins are enriched in the CasID strains in comparison to the controls. After background reduction, 32 proteins remained in two independent experiments exclusively enriched in the Cas-ID-sgRNA strain. Among these proteins was NmrA, an AreA-interacting regulator, and we also found several chromatin-associated proteins. Overall, our results demonstrate that CasID is suitable for locus-specific labelling and identification of chromatin-associated proteins and transcription factors in A. nidulans. However, the high background of proteins that are biotinylated out of chromatin context or unspecifically attach to the affinity purification matrix needs to be addressed by implementing a set of rigorous controls. In summary, we herewith provide a detailed protocol for application of the method that proved to be useful for the identification of novel chromatin-associated proteins and their interaction partners at a specific genomic locus in divers metabolic and developmental conditions. AUTHOR SUMMARY: This study demonstrates that locus-specific proteomics can be carried out by dCas-BirA guided proximity labelling in Aspergillus nidulans. For establishment, we targeted the well-described bidirectional promoter region between niaD, a nitrate reductase, and niiA, a nitrite reductase. At this locus we could test by chromatin immunoprecipitation (ChIP) in combination with qPCR if both, the dCas9-BirA fusion as well as a central transcription factor are at the locus under the conditions of our CasID experiment. After this first control step, we considered that unspecific labelling by dCas-BirA during the time from translation to landing at the targeted chromatin locus may be one of the most relevant drawbacks of the method. Therefore, we developed a number of control strains that would allow us to clearly discriminate between background and sgRNA-dependent specific labelling at the locus. Our protein MS results validated these estimates and only considering the results of these controls enabled us to distinguish the set of locus-specific proteins from a very high general background. Finally, enrichment of biotinylated proteins through affinity purification with streptavidin resin and subsequent LC-MS/MS analysis showed that more than 800 proteins were detected in each sample, emphasizing the high background of the purification method. After background reduction of the control samples, we were able to identify 32 proteins which were exclusively detected in the test strain in two independent measurements, including several chromatin-associated proteins and NmrA, a negative regulator of the nitrate locus transcription factor AreA.},
}

*Chromatin/genetics/metabolism
*Aspergillus nidulans/genetics/metabolism
*Proteomics/methods
Fungal Proteins/genetics/metabolism
Biotinylation
Transcription Factors/genetics
CRISPR-Cas Systems
Chromatin Immunoprecipitation
Carbon-Nitrogen Ligases/genetics

@article {pmid39981976,
year = {2025},
author = {He, X and Mauki, DH and Zhao, X and Dai, S and Yang, H and Zheng, Y and Xia, Q and Wang, R and Wang, T},
title = {Targeting LncRNA-Vof16: A Novel Therapeutic Strategy for Neuropathic Pain Relief.},
journal = {CNS neuroscience & therapeutics},
volume = {31},
number = {2},
pages = {e70241},
pmid = {39981976},
issn = {1755-5949},
support = {2023NSFSC1567//Natural Science Foundation of Sichuan Province of China/ ; 2023SCUH0033//Sichuan University Innovation Research Project/ ; 81471268//National Natural Science Foundation of China/ ; 2020YFS0043//Key Research and Development Program of Sichuan Science and Technology Agency/ ; },
mesh = {Animals ; *Neuralgia/metabolism/therapy/genetics ; Rats ; *RNA, Long Noncoding/genetics/metabolism ; Rats, Sprague-Dawley ; Male ; Peripheral Nerve Injuries/complications/metabolism ; Hyperalgesia ; CRISPR-Cas Systems ; Disease Models, Animal ; Spinal Cord Dorsal Horn/metabolism ; },
abstract = {AIMS: Neuropathic pain (NP) is a debilitating condition characterized by chronic pain resulting from nerve damage or lesion. Despite the ongoing efforts of clinically defining NP, its distinctive mechanisms that lead to various NP phenotypes remain unresolved.

METHODS: Using a spared nerve injury (SNI) model, we investigated the mechanisms underlying the development of NP caused by injury in the peripheral nerves. With CRISPR-Cas9-mediated knockout and virus-mediated overexpression strategies, we investigated the role of LncRNA Vof16 (abbreviated as Vof16) during SNI-induced NP.

RESULTS: Our results revealed that SNI led to the downregulation of Vof16 expression in spinal dorsal horn (SDH) of lumbar enlargement. This was evidently confirmed when we disrupted the expression of Vof16 in SNI rats of which we observed exacerbation of hyperalgesia; while overexpressing it alleviated the pain.

CONCLUSION: Our findings suggest that Vof16 plays a crucial role in maintaining normal sensory function in healthy states and a protective shield against NP following peripheral nerve injury. We therefore propose Vof16 as a new therapeutic target for alleviating NP.},
}

Animals
*Neuralgia/metabolism/therapy/genetics
Rats
*RNA, Long Noncoding/genetics/metabolism
Rats, Sprague-Dawley
Male
Peripheral Nerve Injuries/complications/metabolism
Hyperalgesia
CRISPR-Cas Systems
Disease Models, Animal
Spinal Cord Dorsal Horn/metabolism

@article {pmid39955231,
year = {2025},
author = {Oliveira, DM and Saleme, MLS and Smith, RA and Vangeel, T and Lima, MF and Chanoca, AA and Mota, TR and Vanhevel, Y and Coussens, G and Van Aelst, K and Geerts, J and Cornet, I and Vaneechoutte, D and Vandepoele, K and Pauwels, L and Goeminne, G and Morreel, K and Sels, BF and Ralph, J and Vanholme, R and Boerjan, W},
title = {CRISPR/Cas9 editing of p-COUMAROYL-CoA:MONOLIGNOL TRANSFERASE 1 in maize alters phenolic metabolism, lignin structure, and lignin-first biomass processing.},
journal = {Trends in biotechnology},
volume = {43},
number = {5},
pages = {1166-1195},
doi = {10.1016/j.tibtech.2025.01.006},
pmid = {39955231},
issn = {1879-3096},
mesh = {*Lignin/metabolism/chemistry ; *Zea mays/genetics/metabolism/enzymology ; *CRISPR-Cas Systems/genetics ; Biomass ; *Gene Editing/methods ; *Phenols/metabolism ; Plants, Genetically Modified ; *Plant Proteins/genetics/metabolism ; },
abstract = {Valorization of lignocellulosic biomass for sustainable production of high-value chemicals is challenged by the complexity of lignin, a phenolic biopolymer. Beyond the classical lignin monomers derived from p-coumaryl, coniferyl, and sinapyl alcohol, grass lignins incorporate substantial amounts of monolignol p-coumarates that are produced by p-COUMAROYL-CoA:MONOLIGNOL TRANSFERASE (PMT). Here, the CRISPR/Cas9-mediated mutation of ZmPMT1 in maize enabled the design of biomass depleted in p-coumaroylated lignin and enriched in guaiacyl lignin. Lignin-first biorefining of stem biomass from zmpmt1 mutants by reductive catalytic fractionation (RCF) generated a lignin oil depleted in carboxylates and enriched in guaiacyl-derived alcohols, which are desirable substrates for bio-based polyurethane synthesis. The reported lignin engineering in maize is a promising strategy for designing a dual-purpose crop, providing both food and feed, along with a renewable feedstock for the production of plant-based chemicals.},
}

*Lignin/metabolism/chemistry
*Zea mays/genetics/metabolism/enzymology
*CRISPR-Cas Systems/genetics
Biomass
*Gene Editing/methods
*Phenols/metabolism
Plants, Genetically Modified
*Plant Proteins/genetics/metabolism

@article {pmid39873911,
year = {2025},
author = {Wang, W and Li, S and Yang, J and Li, J and Yan, L and Zhang, C and He, Y and Xia, L},
title = {Exploiting the efficient Exo:Cas12i3-5M fusions for robust single and multiplex gene editing in rice.},
journal = {Journal of integrative plant biology},
volume = {67},
number = {5},
pages = {1246-1253},
pmid = {39873911},
issn = {1744-7909},
support = {2023ZD04074//Biological Breeding - Major Projects/ ; 32188102//National Natural Science Foundation of China/ ; 2021YFF1000204//National Key Research and Development Program of China/ ; B23CJ0208//Hainan Seed Industry Lab/ ; //National Engineering Research Centre of Crop Molecular Breeding/ ; 32100328//National Natural Science Foundation of China/ ; },
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; },
abstract = {The development of a single and multiplex gene editing system is highly desirable for either functional genomics or pyramiding beneficial alleles in crop improvement. CRISPR/Cas12i3, which belongs to the Class II Type V-I Cas system, has attracted extensive attention recently due to its smaller protein size and less restricted canonical "TTN" protospacer adjacent motif (PAM). However, due to its relatively lower editing efficiency, Cas12i3-mediated multiplex gene editing has not yet been documented in plants. Here, we fused four 5' exonucleases (Exo) including T5E, UL12, PapE, ME15 to the N terminal of an optimized Cas12i3 variant (Cas12i3-5M), respectively, and systematically evaluated the editing activities of these Exo:Cas12i3-5M fusions across six endogenous targets in rice stable lines. We demonstrated that the Exo:Cas12i3-5M fusions increased the gene editing efficiencies by up to 12.46-fold and 1.25-fold compared with Cas12i3 and Cas12i3-5M, respectively. Notably, the UL12:Cas12i3-5M fusion enabled robust single gene editing with editing efficiencies of up to 90.42%-98.61% across the six tested endogenous genes. We further demonstrated that, although all the Exo:Cas12i5-5M fusions were capable of multiplex gene editing, UL12:Cas12i3-5M exhibited a superior performance in the simultaneous editing of three, four, five or six genes with efficiencies of 82.76%, 61.36%, 52.94%, and 51.06% in rice stable lines, respectively. Together, we evaluated different Exo:Cas12i3-5M fusions systemically and established UL12:Cas12i3-5M as the more robust system for single and multiplex gene editing in rice. The development of an alternative robust single and multiplex gene editing system will enrich plant genome editing toolkits and facilitate pyramiding of agronomically important traits for crop improvement.},
}

*Oryza/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Plants, Genetically Modified

@article {pmid39873367,
year = {2025},
author = {Zhang, Y and Liu, J and Li, Y and Ma, H and Ji, J and Wang, Y and Zhuang, M and Yang, L and Fang, Z and Li, J and Zhang, C and Liu, L and Lebedeva, M and Taranov, V and Zhang, Y and Lv, H},
title = {Generation of novel bpm6 and dmr6 mutants with broad-spectrum resistance using a modified CRISPR/Cas9 system in Brassica oleracea.},
journal = {Journal of integrative plant biology},
volume = {67},
number = {5},
pages = {1214-1216},
doi = {10.1111/jipb.13842},
pmid = {39873367},
issn = {1744-7909},
support = {2023YFE0111400//National Key Research and Development Program of China/ ; },
mesh = {*Brassica/genetics/microbiology ; *CRISPR-Cas Systems/genetics ; *Disease Resistance/genetics ; *Mutation/genetics ; *Plant Proteins/genetics/metabolism ; Plant Diseases/microbiology/genetics ; Plants, Genetically Modified ; Genes, Plant ; },
abstract = {Using an optimized CRISPR/Cas9 system to knock out the BTB-POZ and MATH domain gene BoBPM6 and the DOWNY MILDEW RESISTANCE 6 gene in Brassica oleracea resulted in new lines with broad-spectrum disease resistance.},
}

*Brassica/genetics/microbiology
*CRISPR-Cas Systems/genetics
*Disease Resistance/genetics
*Mutation/genetics
*Plant Proteins/genetics/metabolism
Plant Diseases/microbiology/genetics
Plants, Genetically Modified
Genes, Plant

@article {pmid39424445,
year = {2025},
author = {Liberty, JT and Poudel, B and Ihedioha, O and Lin, H and Habanabakize, E and Gao, Z and Adesoji, AT and Liberty, SJ},
title = {Gene editing technology: shaping international standards for health and food safety assurance.},
journal = {Trends in biotechnology},
volume = {43},
number = {5},
pages = {985-988},
doi = {10.1016/j.tibtech.2024.09.019},
pmid = {39424445},
issn = {1879-3096},
mesh = {*Gene Editing/standards/ethics/methods ; *Food Safety ; Humans ; CRISPR-Cas Systems ; International Cooperation ; },
abstract = {The emergence of gene editing technologies like CRISPR-Cas9 has revolutionized health and food safety, necessitating robust international standards. This Science & Society examines how these advances have shaped global regulatory frameworks, ethical standards, and international collaborations, emphasizing the need for cohesive and ethical applications across various sectors.},
}

*Gene Editing/standards/ethics/methods
*Food Safety
Humans
CRISPR-Cas Systems
International Cooperation

@article {pmid38511958,
year = {2024},
author = {Gager, C and Flores-Mireles, AL},
title = {Blunted blades: new CRISPR-derived technologies to dissect microbial multi-drug resistance and biofilm formation.},
journal = {mSphere},
volume = {9},
number = {4},
pages = {e0064223},
pmid = {38511958},
issn = {2379-5042},
support = {R01 DK128805/DK/NIDDK NIH HHS/United States ; },
mesh = {*Biofilms/growth & development ; *Drug Resistance, Multiple, Bacterial/genetics ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Bacteria/drug effects/genetics ; Humans ; Anti-Bacterial Agents/pharmacology ; },
abstract = {The spread of multi-drug-resistant (MDR) pathogens has rapidly outpaced the development of effective treatments. Diverse resistance mechanisms further limit the effectiveness of our best treatments, including multi-drug regimens and last line-of-defense antimicrobials. Biofilm formation is a powerful component of microbial pathogenesis, providing a scaffold for efficient colonization and shielding against anti-microbials, which further complicates drug resistance studies. Early genetic knockout tools didn't allow the study of essential genes, but clustered regularly interspaced palindromic repeat inference (CRISPRi) technologies have overcome this challenge via genetic silencing. These tools rapidly evolved to meet new demands and exploit native CRISPR systems. Modern tools range from the creation of massive CRISPRi libraries to tunable modulation of gene expression with CRISPR activation (CRISPRa). This review discusses the rapid expansion of CRISPRi/a-based technologies, their use in investigating MDR and biofilm formation, and how this drives further development of a potent tool to comprehensively examine multi-drug resistance.},
}

*Biofilms/growth & development
*Drug Resistance, Multiple, Bacterial/genetics
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
*Bacteria/drug effects/genetics
Humans
Anti-Bacterial Agents/pharmacology

@article {pmid38499776,
year = {2024},
author = {Han, F and Zhang, X and Liu, Y and Liu, Y and Zhao, H and Li, Z},
title = {One-step creation of CMS lines using a BoCENH3-based haploid induction system in Brassica crop.},
journal = {Nature plants},
volume = {10},
number = {4},
pages = {581-586},
pmid = {38499776},
issn = {2055-0278},
mesh = {*Haploidy ; *Brassica/genetics/physiology ; *Plant Infertility/genetics ; *Plant Breeding/methods ; Crops, Agricultural/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {Heterosis utilization in a large proportion of crops depends on the use of cytoplasmic male sterility (CMS) tools, requiring the development of homozygous fertile lines and CMS lines[1]. Although doubled haploid (DH) technology has been developed for several crops to rapidly generate fertile lines[2,3], CMS lines are generally created by multiple rounds of backcrossing, which is time consuming and expensive[4]. Here we describe a method for generating both homozygous fertile and CMS lines through in vivo paternal haploid induction (HI). We generated in-frame deletion and restored frameshift mutants of BoCENH3 in Brassica oleracea using the CRISPR/Cas9 system. The mutants induced paternal haploids by outcrossing. We subsequently generated HI lines with CMS cytoplasm, which enabled the generation of homozygous CMS lines in one step. The BoCENH3-based HI system provides a new DH technology to accelerate breeding in Brassica and other crops.},
}

*Haploidy
*Brassica/genetics/physiology
*Plant Infertility/genetics
*Plant Breeding/methods
Crops, Agricultural/genetics
CRISPR-Cas Systems
Plants, Genetically Modified

@article {pmid38454137,
year = {2024},
author = {Arriaga, JM and Ronaldson-Bouchard, K and Picech, F and Nunes de Almeida, F and Afari, S and Chhouri, H and Vunjak-Novakovic, G and Abate-Shen, C},
title = {In vivo genome-wide CRISPR screening identifies CITED2 as a driver of prostate cancer bone metastasis.},
journal = {Oncogene},
volume = {43},
number = {17},
pages = {1303-1315},
pmid = {38454137},
issn = {1476-5594},
support = {K22 CA258806/CA/NCI NIH HHS/United States ; R01 CA173481/CA/NCI NIH HHS/United States ; P01 CA265768/CA/NCI NIH HHS/United States ; P30 CA013696/CA/NCI NIH HHS/United States ; R01 CA193442/CA/NCI NIH HHS/United States ; P41 EB027062/EB/NIBIB NIH HHS/United States ; R01 CA249799/CA/NCI NIH HHS/United States ; UH3 EB025765/EB/NIBIB NIH HHS/United States ; U54 CA274506/CA/NCI NIH HHS/United States ; UL1 TR001873/TR/NCATS NIH HHS/United States ; P01 CA221757/CA/NCI NIH HHS/United States ; R01 CA183929/CA/NCI NIH HHS/United States ; },
mesh = {Male ; *Bone Neoplasms/secondary/genetics ; Humans ; *Prostatic Neoplasms/genetics/pathology ; *Trans-Activators/genetics/metabolism ; *Repressor Proteins/genetics/metabolism ; Animals ; Mice ; Cell Line, Tumor ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Most cancer deaths are due to metastatic dissemination to distant organs. Bone is the most frequently affected organ in metastatic prostate cancer and a major cause of prostate cancer deaths. Yet, our partial understanding of the molecular factors that drive bone metastasis has been a limiting factor for developing preventative and therapeutic strategies to improve patient survival and well-being. Although recent studies have uncovered molecular alterations that occur in prostate cancer metastasis, their functional relevance for bone metastasis is not well understood. Using genome-wide CRISPR activation and inhibition screens we have identified multiple drivers and suppressors of prostate cancer metastasis. Through functional validation, including an innovative organ-on-a-chip invasion platform for studying bone tropism, our study identifies the transcriptional modulator CITED2 as a novel driver of prostate cancer bone metastasis and uncovers multiple new potential molecular targets for bone metastatic disease.},
}

Male
*Bone Neoplasms/secondary/genetics
Humans
*Prostatic Neoplasms/genetics/pathology
*Trans-Activators/genetics/metabolism
*Repressor Proteins/genetics/metabolism
Animals
Mice
Cell Line, Tumor
CRISPR-Cas Systems
Gene Expression Regulation, Neoplastic
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid40332425,
year = {2025},
author = {Guo, X and Wang, X and Wang, J and Ma, M and Ren, Q},
title = {Current Development of iPSC-Based Modeling in Neurodegenerative Diseases.},
journal = {International journal of molecular sciences},
volume = {26},
number = {8},
pages = {},
doi = {10.3390/ijms26083774},
pmid = {40332425},
issn = {1422-0067},
support = {81901088//National Natural Science Foundation of China/ ; E2019050019//Hundred Talents Program of Hebei Province/ ; H2022206562//Hebei Provincial Natural Science Foundation Precision Medicine Joint Fund Cultivation Project/ ; NV20230011//Key laboratory of Neural and Vascular Biology, Ministry of Education of China/ ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Neurodegenerative Diseases/pathology/therapy/metabolism/genetics ; Animals ; Gene Editing ; Organoids/metabolism ; CRISPR-Cas Systems ; Cell Differentiation ; *Models, Biological ; Neurons/metabolism ; Huntington Disease/pathology ; },
abstract = {Over the past two decades, significant advancements have been made in the induced pluripotent stem cell (iPSC) technology. These developments have enabled the broader application of iPSCs in neuroscience, improved our understanding of disease pathogenesis, and advanced the investigation of therapeutic targets and methods. Specifically, optimizations in reprogramming protocols, coupled with improved neuronal differentiation and maturation techniques, have greatly facilitated the generation of iPSC-derived neural cells. The integration of the cerebral organoid technology and CRISPR/Cas9 genome editing has further propelled the application of iPSCs in neurodegenerative diseases to a new stage. Patient-derived or CRISPR-edited cerebral neurons and organoids now serve as ideal disease models, contributing to our understanding of disease pathophysiology and identifying novel therapeutic targets and candidates. In this review, we examine the development of iPSC-based models in neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Neurodegenerative Diseases/pathology/therapy/metabolism/genetics
Animals
Gene Editing
Organoids/metabolism
CRISPR-Cas Systems
Cell Differentiation
*Models, Biological
Neurons/metabolism
Huntington Disease/pathology

@article {pmid40332149,
year = {2025},
author = {Navarro, C and Díaz, MP and Duran, P and Castro, A and Díaz, A and Cano, C and Carbonell-Zabaleta, AK and Solano-Jimenez, DS and Rivera-Porras, D and Contreras-Velásquez, JC and Bermúdez, V},
title = {CRISPR-Cas Systems: A Functional Perspective and Innovations.},
journal = {International journal of molecular sciences},
volume = {26},
number = {8},
pages = {},
doi = {10.3390/ijms26083645},
pmid = {40332149},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Bacteria/genetics/virology ; Archaea/genetics ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Adaptation is a fundamental tenet of evolutionary biology and is essential for the survival of all organisms, including prokaryotes. The evolution of clustered regularity exemplifies this principle of interspaced short palindromic repeats (CRISPR) and associated proteins (Cas), an adaptive immune system that confers resistance to viral infections. By integrating short segments of viral genomes into their own, bacteria and archaea develop a molecular memory that enables them to mount a rapid and targeted response upon subsequent viral challenges. The fortuitous discovery of this immune mechanism prompted many studies and introduced researchers to novel tools that could potentially be developed from CRISPR-Cas and become clinically relevant as biotechnology rapidly advances in this area. Thus, a deeper understanding of the underpinnings of CRISPR-Cas and its possible therapeutic applications is required. This review analyses the mechanism of action of the CRISPR-Cas systems in detail and summarises the advances in developing biotechnological tools based on CRISPR, opening the field for further research.},
}

*CRISPR-Cas Systems/genetics
Gene Editing/methods
Bacteria/genetics/virology
Archaea/genetics
Humans
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40331919,
year = {2025},
author = {Xu, C and Zhang, Y and Zhu, X and Hua, D and Yang, L and Huang, X and Gao, H and Luo, A and Deng, R and Xia, X},
title = {Preamplification-Free Detection of Viable Microorganisms in Fermentation Using Tandem CRISPR Nuclease Probe.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c01068},
pmid = {40331919},
issn = {1520-5118},
abstract = {Accurate detection of viable bacteria is crucial for evaluating and monitoring the fermentation process. However, the complexity of fermentation samples presents challenges to developing precise and rapid detection tools. Here, we present a Cas13a-Csm6 tandem nuclease probe capable of the one-pot detection of viable microorganisms during fermentation, eliminating the need for nucleic acid preamplification. The RNA-activated CRISPR-Cas13a generates cleavage substrates that serve as activators for the CRISPR/Cas III-A Csm6 system. Leveraging the high specificity and efficient amplification capacity of the CRISPR cascade, this nuclease probe can detect 1% of viable Lactobacillus and Bacillus, facilitating the monitoring of bacterial populations throughout fermentation. This approach completes detection within 30 min and improves sensitivity for bacterial profiling by 16-fold compared with using Cas13 alone. The Cas13a-Csm6 tandem nuclease probe offers a precise and rapid analytical tool for the on-site quality monitoring of fermented foods.},
}

@article {pmid40331862,
year = {2025},
author = {Anglada, T and Rodriguez-Muñoz, M and Pulido-Artola, N and Genescà, A},
title = {Engineering Chromosome Bridges Through CRISPR/Cas9 to Decipher the Impact of Intercentromeric Distance on Resolution Dynamics.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {39},
number = {9},
pages = {e70599},
doi = {10.1096/fj.202402258RR},
pmid = {40331862},
issn = {1530-6860},
support = {//Fundació la Marató de TV3 (Fundació la Marató)/ ; //Generalitat de Catalunya (Government of Catalonia)/ ; //Consejo de Seguridad Nuclear (CSN)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Mitosis/genetics ; *Chromatin/genetics/metabolism ; Cell Line ; Kinetochores/metabolism ; *Centromere/genetics ; },
abstract = {Resolution of chromosome bridges during mitosis is a critical yet incompletely understood process with implications for genomic stability and cancer development. In this study, we investigated the impact of the bridging chromatin length on the timing and mechanism of chromosome bridge resolution. Using CRISPR/Cas9 technology, we engineered chromosome bridges with precisely defined intercentromeric distances in human RPE-1 cells. Our study revealed a decline in the frequency of chromosome bridges as cells progressed from early anaphase to late telophase, indicating resolution during mitosis. Moreover, the longer the bridging chromatin length, the higher the frequency of chromosome bridges observed at the mitotic exit, demonstrating that the size of the bridge influences its resolution during mitosis. Additionally, the separation between the bridge kinetochores needed for bridge breakage was strongly dependent on the megabase length of the bridging chromatin, with longer chromosome bridges requiring greater separation for their resolution. Given that chromosome bridge resolution occurs in a concerted manner with spindle elongation and is influenced by the length of the bridging chromatin, we posit that the traction forces generated by microtubules attaching to dicentric chromosomes play a significant role in resolving chromosome bridges during mitosis. Our study underscores the intricate interplay between chromosome bridge geometry and mechanical forces in mitotic chromosome bridge resolution. Our model offers a valuable framework for future investigations into the molecular mechanisms underlying chromosome bridge resolution, with potential implications for cancer biology and genomic stability maintenance.},
}

Humans
*CRISPR-Cas Systems/genetics
*Mitosis/genetics
*Chromatin/genetics/metabolism
Cell Line
Kinetochores/metabolism
*Centromere/genetics

@article {pmid40330464,
year = {2025},
author = {Li, TF and Rothhaar, P and Lang, A and Grünvogel, O and Colasanti, O and Ugarte, SMO and Traut, J and Piras, A and Acosta-Rivero, N and Gonçalves Magalhães, V and Springer, E and Betz, A and Huang, HE and Park, J and Qiu, R and Gnouamozi, GE and Mehnert, AK and Thi, VLD and Urban, S and Muckenthaler, M and Schlesner, M and Wohlleber, D and Binder, M and Bartenschlager, R and Pichlmair, A and Lohmann, V},
title = {RBM39 shapes innate immunity by controlling the expression of key factors of the interferon response.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1568056},
pmid = {40330464},
issn = {1664-3224},
mesh = {*RNA-Binding Proteins/genetics/metabolism/immunology ; *Immunity, Innate/genetics ; Humans ; Toll-Like Receptor 3/metabolism/genetics/immunology ; Hepatocytes/immunology/metabolism ; Signal Transduction ; *Interferons/immunology/metabolism ; *Gene Expression Regulation ; CRISPR-Cas Systems ; Cell Line ; },
abstract = {BACKGROUND AND AIMS: The contribution of innate immunity to clearance of viral infections of the liver, in particular sensing via Toll-like receptor 3 (TLR3), is incompletely understood. We aimed to identify the factors contributing to the TLR3 response in hepatocytes via CRISPR/Cas9 screening.

METHODS: A genome-wide CRISPR/Cas9 screen on the TLR3 pathway was performed in two liver-derived cell lines, followed by siRNA knockdown validation. SiRNA knockdown and indisulam treatment were used to study the role of RNA-binding motif protein 39 (RBM39) in innate immunity upon poly(I:C) or cytokine treatment and viral infections. Transcriptome, proteome, and alternative splicing were studied via RNA sequencing and mass spectrometry upon depletion of RBM39.

RESULTS: Our CRISPR/Cas9 screen identified RBM39, which is highly expressed in hepatocytes, as an important regulator of the TLR3 pathway. Knockdown of RBM39 or treatment with indisulam, an aryl sulfonamide drug targeting RBM39 for proteasomal degradation, strongly reduced the induction of interferon-stimulated genes (ISGs) in response to double-stranded RNA (dsRNA) or viral infections. RNA sequencing (seq) and mass spectrometry identified that transcription and/or splicing of the key pathway components IRF3, RIG-I, and MDA5 were affected by RBM39 depletion, along with multiple other cellular processes identified previously. RBM39 knockdown further restrained type I and type III IFN pathways by reducing the expression of individual receptor subunits and STAT1/2. The function of RBM39 was furthermore not restricted to hepatocytes.

CONCLUSION: We identified RBM39 as a regulatory factor of cell intrinsic innate immune signaling. Depletion of RBM39 impaired TLR3, RIG-I/MDA5, and IFN responses by affecting the basal expression of key pathway components.},
}

*RNA-Binding Proteins/genetics/metabolism/immunology
*Immunity, Innate/genetics
Humans
Toll-Like Receptor 3/metabolism/genetics/immunology
Hepatocytes/immunology/metabolism
Signal Transduction
*Interferons/immunology/metabolism
*Gene Expression Regulation
CRISPR-Cas Systems
Cell Line

@article {pmid40329691,
year = {2025},
author = {Su, R and Shen, G and Xiao, X and Zheng, Y and Liu, F and Chen, D},
title = {Generation of a Novel Inducible and Dermal Papilla-Specific Wif1-CreER Knock-In Mouse Line for Hair Follicle Research.},
journal = {Experimental dermatology},
volume = {34},
number = {5},
pages = {e70109},
doi = {10.1111/exd.70109},
pmid = {40329691},
issn = {1600-0625},
support = {//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Hair Follicle/cytology/metabolism ; Mice ; Gene Knock-In Techniques ; Integrases/genetics/metabolism ; *Adaptor Proteins, Signal Transducing/genetics ; Mice, Transgenic ; *Dermis/cytology/metabolism ; CRISPR-Cas Systems ; Tamoxifen/pharmacology ; },
abstract = {Dermal papilla (DP) cells are essential niche cells that regulate hair follicle development, cycling and regeneration. Despite the establishment of several DP cell mouse lines in prior research, these tools are limited by incomplete specificity and spatiotemporal control. The Wnt inhibitory factor 1 (Wif1) has been identified as a DP signature gene. To address the need for precise labelling and manipulation of DP cells, we developed a novel genetic tool-Wif1-CreER knock-in mice. Using CRISPR/Cas9-mediated homologous recombination, the CreERT2 sequences were inserted into the endogenous Wif1 locus, under the control of the native promoter. PCR and sequencing analysis confirmed the accurate insertion of the CreERT2 sequence. Crossing Wif1-CreER mice with a reporter line demonstrated efficient and specific Cre recombinase activity in DP cells during anagen, catagen and telogen upon tamoxifen treatment across hair types. Importantly, DP-restricted labelling was confirmed by immunofluorescence and colocalised with Crabp1 and alkaline phosphatase (AP)-staining activity, exhibiting minimal to negligible expression in other tissues. This innovative mouse model overcomes the limitations of current tools and provides a valuable resource for advancing our understanding of hair biology and developing targeted therapies for hair-related disorders, offering unprecedented precision in the manipulation of dermal papilla cells.},
}

Animals
*Hair Follicle/cytology/metabolism
Mice
Gene Knock-In Techniques
Integrases/genetics/metabolism
*Adaptor Proteins, Signal Transducing/genetics
Mice, Transgenic
*Dermis/cytology/metabolism
CRISPR-Cas Systems
Tamoxifen/pharmacology

@article {pmid40329173,
year = {2025},
author = {Piché, LC and Bories, S and Liato, V and Paquet, VE and Saucier, L and Létourneau-Montminy, MP and Charette, SJ and Dubar, R and Labrie, SJ and Lagüe, P and Vincent, AT},
title = {Evolutionary responses of Escherichia coli to phage pressure: insights into mucoidy and colanic acid overexpression.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {448},
pmid = {40329173},
issn = {1471-2164},
support = {IA120638//MAPAQ (Innov'Action)/ ; IA120638//MAPAQ (Innov'Action)/ ; RGPIN-2022-03321//Natural Sciences and Engineering Research Council of Canada/ ; },
mesh = {*Escherichia coli/virology/genetics/metabolism ; *Evolution, Molecular ; *Bacteriophages/physiology ; Escherichia coli Proteins/genetics/metabolism/chemistry ; *Coliphages/physiology ; Mutation ; Polysaccharides ; },
abstract = {BACKGROUND: Antibiotic resistance is a major issue affecting all spheres of human activity, including agriculture. One significant example is the Avian Pathogenic Escherichia coli (APEC), a bacterium that infects poultry and leads to substantial economic losses in the farming industry. As antibiotics lose efficacity, bacteriophages (phages) -viruses that specifically target bacteria-are emerging as a promising alternative to antibiotics for treating and preventing bacterial infections. However, bacteria can develop resistance to phages through various mechanisms. Studying the coevolution between a phage and its host bacterium is important to gain insight into the phage's potential as a therapeutic agent. This study investigates the evolutionary responses of an APEC strain and a laboratory E. coli strain to a commercial phage originally isolated from APEC.

RESULTS: In most cases, phage resistance resulted in a significant increase in mucoidy. Genomic analysis revealed that this resistance consistently correlated with amino acid changes, particularly in proteins involved in colanic acid production, such as YrfF. Further investigation of a mutation found in the YrfF protein demonstrated that this mutation altered the protein's structure and its interaction with the membrane. Transcriptomic analysis confirmed that the genes involved in colanic acid production were significantly overexpressed. Although the strains possessed a CRISPR-Cas system, it did not contribute to phage resistance.

CONCLUSIONS: This study suggests that specific amino acid changes in key proteins may be a mechanism employed by E. coli, including APEC, to defend against phage infections.},
}

*Escherichia coli/virology/genetics/metabolism
*Evolution, Molecular
*Bacteriophages/physiology
Escherichia coli Proteins/genetics/metabolism/chemistry
*Coliphages/physiology
Mutation
Polysaccharides

@article {pmid40328743,
year = {2025},
author = {Huang, F and Wang, Y and Zhang, X and Gao, W and Li, J and Yang, Y and Mo, H and Prince, E and Long, Y and Hu, J and Jiang, C and Kang, Y and Chen, Z and Hu, YC and Zeng, C and Yang, L and Chen, CW and Chen, J and Huang, H and Weng, H},
title = {m[6]A/IGF2BP3-driven serine biosynthesis fuels AML stemness and metabolic vulnerability.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4214},
pmid = {40328743},
issn = {2041-1723},
support = {82270168//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Leukemia, Myeloid, Acute/metabolism/genetics/pathology ; Humans ; *Serine/biosynthesis/metabolism ; *RNA-Binding Proteins/metabolism/genetics ; Animals ; *Neoplastic Stem Cells/metabolism/pathology ; *Adenosine/analogs & derivatives/metabolism ; Mice ; Cell Line, Tumor ; Glycine/metabolism ; Methyltransferases/metabolism/genetics ; CRISPR-Cas Systems ; RNA, Messenger/metabolism/genetics ; Gene Expression Regulation, Leukemic ; },
abstract = {Metabolic reprogramming of amino acids represents a vulnerability in cancer cells, yet the mechanisms underlying serine metabolism in acute myeloid leukemia (AML) and leukemia stem/initiating cells (LSCs/LICs) remain unclear. Here, we identify RNA N[6]-methyladenosine (m[6]A) modification as a key regulator of serine biosynthesis in AML. Using a CRISPR/Cas9 screen, we find that depletion of m[6]A regulators IGF2BP3 or METTL14 sensitizes AML cells to serine and glycine (SG) deprivation. IGF2BP3 recognizies m[6]A on mRNAs of key serine synthesis pathway (SSP) genes (e.g., ATF4, PHGDH, PSAT1), stabilizing these transcripts and sustaining serine production to meet the high metabolic demand of AML cells and LSCs/LICs. IGF2BP3 silencing combined with dietary SG restriction potently inhibits AML in vitro and in vivo, while its deletion spares normal hematopoiesis. Our findings reveal the critical role of m[6]A modification in the serine metabolic vulnerability of AML and highlight the IGF2BP3/m[6]A/SSP axis as a promising therapeutic target.},
}

*Leukemia, Myeloid, Acute/metabolism/genetics/pathology
Humans
*Serine/biosynthesis/metabolism
*RNA-Binding Proteins/metabolism/genetics
Animals
*Neoplastic Stem Cells/metabolism/pathology
*Adenosine/analogs & derivatives/metabolism
Mice
Cell Line, Tumor
Glycine/metabolism
Methyltransferases/metabolism/genetics
CRISPR-Cas Systems
RNA, Messenger/metabolism/genetics
Gene Expression Regulation, Leukemic

@article {pmid40328723,
year = {2025},
author = {Yang, X and Shi, J and Wang, H and Wang, L and Su, H and Chen, C and Zhao, C},
title = {[Construction of mouse podocyte clone-5 cell lines with Smad3 knockout by CRISPR/Cas9].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {4},
pages = {1658-1670},
doi = {10.13345/j.cjb.240803},
pmid = {40328723},
issn = {1872-2075},
mesh = {Animals ; *Smad3 Protein/genetics ; *CRISPR-Cas Systems/genetics ; Mice ; *Podocytes/cytology/metabolism ; Transforming Growth Factor beta1/pharmacology ; Cell Line ; *Gene Knockout Techniques ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {This study established the mouse podocyte clone-5 (MPC5) with Smad3 knockout and studied the effect of transforming growth factor-beta 1 (TGF-β1) on the dedifferentiation of the MPC5 cells with Smad3 knockout, aiming to provide a cell tool for studying the role of Smad3 in mouse podocytes. The single-guide RNA (sgRNA) sequence targeting Smad3 was designed according to the principles of CRISPR/Cas9 design. The pX458-Smad3 vector was constructed and introduced into competent cells, and then the vector was extracted and used to transfect MPC5 cells. The successfully transfected cells were sorted by a flow cytometer. After single-cell clone expansion, PCR amplification of sequences adjacent to the edition site of Smad3 and sequencing were performed to identify potential cells with gene knockout. Western blotting was employed to verify the knockout efficiency of Smad3. Finally, the effect of Smad3 knockout on TGF-β1-induced dedifferentiation of MPC5 cells was analyzed by reverse transcription-polymerase chain reacting (RT-PCR), Western blotting, and the immunofluorescence method. The sgRNA was designed to target the fifth exon of Smad3. EGFP expression was observed 24 h after transfection of the pX458-Smad3 plasmid into MPC5 cells, with the transfection efficiency of 0.1% as determined by flow cytometry. From the transfected cells, 21 cell clones were obtained through flow cytometric sorting and single-cell clone expansion. PCR amplification and sequencing of the region around the sgRNA target site in Smad3 identified two cell clones with biallelic frameshift mutations. Western blotting results confirmed the absence of Smad3 expression in these clones, indicating successful establishment of the MPC5 cell line with Smad3 knockout. In normal MPC5 cells, TGF-β1 stimulation promoted the expression of fibrosis-related genes fibronectin and Col1a1 (collagen I) and inhibited the expression of the podocyte marker proteins synaptopodin and podocin, which suggested epithelial-mesenchymal transition and podocyte injury. However, in the two MPC5 cell lines with Smad3 knockout, TGF-β1-induced expression of epithelial-mesenchymal transition markers was significantly suppressed. The MPC5 cell lines with Smad3 knockout that were constructed by CRISPR/Cas9 provide a valuable cell model for functional studies of Smad3 protein and highlight the critical role of Smad3 in cell dedifferentiation.},
}

Animals
*Smad3 Protein/genetics
*CRISPR-Cas Systems/genetics
Mice
*Podocytes/cytology/metabolism
Transforming Growth Factor beta1/pharmacology
Cell Line
*Gene Knockout Techniques
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40328722,
year = {2025},
author = {Cai, Q and Wang, M and Zhu, J and Wu, J},
title = {[A universal counter-selection strategy based on replacement of sgRNA expression cassettes targeting multi-copy genes].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {4},
pages = {1649-1657},
doi = {10.13345/j.cjb.240505},
pmid = {40328722},
issn = {1872-2075},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Plasmids/genetics ; },
abstract = {Selection markers are essential tools in gene editing, the utility of such systems is inherently constrained by species-specific limitations, governed by divergent host genetic backgrounds and metabolic compatibility. To address this limitation, we leveraged the CRISPR/Cas9 system to develop a universal counter-selection tool. We designed and introduced an sgRNA expression cassettes as counter-selection markers, which directs the Cas9 protein to target and cleave genomic DNA, allowing for the selection of the strains where the sgRNA expression cassette has been replaced. Optimized to target multiple copy sites with sgRNA, this system significantly enhances cell lethality, boosting counter-selection efficiency to over 85.00%. This counter-selection tool is not limited to single strains and is suitable for various scenarios, including multi-copy plasmid assembly and plasmid editing, demonstrating broad application potential.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
Plasmids/genetics

@article {pmid40327602,
year = {2025},
author = {Sharma, I and Hall, K and Moonah, S},
title = {CRISPR genome editing using a combined positive and negative selection system.},
journal = {PloS one},
volume = {20},
number = {5},
pages = {e0321881},
pmid = {40327602},
issn = {1932-6203},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Polymorphism, Single Nucleotide ; *Selection, Genetic ; beta-Globins/genetics ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system is a powerful genome editing tool that has revolutionized research. Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation in humans. Only a subset of these SNPs has been shown to be linked to genetic diseases, while the biological relevance of the majority remains unclear. Investigating these variants of unknown significance could provide valuable insights into their roles in biological processes, disease susceptibility, and treatment responses. While CRISPR/Cas has emerged as a transformative technology, its ability to make single nucleotide substitutions remains a significant limitation. Other techniques in single nucleotide editing, such as base editing and prime editing, offer promising possibilities to complement CRISPR/Cas systems, though they also have their own limitations. Hence, alternative approaches are necessary to overcome the limitations of CRISPR. Here, to improve the feasibility of generating single base edits in the genome, we provide a protocol that introduces a multiple expression and dual selection (MEDS) system, which, alongside CRISPR, utilizes the opposing roles of cytosine deaminase/uracil phosphoribosyltransferase (CD/UPRT) for negative selection and neomycin phosphotransferase II (NPT II) for positive selection. As a proof of concept and to demonstrate feasibility of the method, we used MEDS, along with traditional CRISPR-Cas9, to generate sickle hemoglobin by introducing a point mutation (A → T) in the sixth codon of the hemoglobin beta gene.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Polymorphism, Single Nucleotide
*Selection, Genetic
beta-Globins/genetics

@article {pmid40327171,
year = {2025},
author = {Yang, J and Li, W and Hu, Y and Han, Y and Lei, C and Wang, H},
title = {Establishment of a rapid RAA-CRISPR/Cas12a system targeting the recN gene for on-site detection of Streptococcus suis in livestock and fresh pork meat.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {99},
pmid = {40327171},
issn = {1438-7948},
support = {2025ZNSFSC0209//Natural Science Foundation of Sichuan Province/ ; U21A20257//National Natural Science Foundation of China/ ; 2021ZDZX0010//Sichuan Science and Technology Programs/ ; },
mesh = {*Streptococcus suis/genetics/isolation & purification ; Animals ; *CRISPR-Cas Systems ; Swine ; *Livestock/microbiology ; *Bacterial Proteins/genetics ; *Pork Meat/microbiology ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {Streptococcus suis is a major bacterial pathogen in the swine industry, causing meningitis, arthritis, and other diseases in infected pigs. It also poses significant public health risks due to its zoonotic potential, particularly in individuals with skin lesions. Current detection methods, including traditional culture-based techniques and PCR assays, are time-consuming, labor-intensive, and lack sufficient accuracy. To address these limitations, this study aimed to develop a rapid and precise detection method for S. suis. By leveraging whole-genome sequencing (WGS) and multiple sequence alignment, the recN gene was identified as a highly specific molecular target. A novel isothermal detection method, integrating recombinase-aided amplification (RAA) with CRISPR/Cas12a, was subsequently established. This RAA-CRISPR/Cas12a-based system demonstrated superior sensitivity compared to conventional PCR (targeting the gdh gene), achieving detection within 30 min without requiring specialized equipment. This method achieves 2.44 × 10[1] copies/µL and 2.1 × 10[1] CFU sensitivity and 100% specificity within 30 min, outperforming conventional PCR in speed and reliability while eliminating dependency on specialized equipment. Designed for field applications, it offers a cost-effective (US$1/test), user-friendly solution for on-site S. suis detection in swine farms and fresh pork meat, enhancing outbreak control and preventive healthcare in the livestock industry.},
}

*Streptococcus suis/genetics/isolation & purification
Animals
*CRISPR-Cas Systems
Swine
*Livestock/microbiology
*Bacterial Proteins/genetics
*Pork Meat/microbiology
*Nucleic Acid Amplification Techniques/methods

@article {pmid40326084,
year = {2025},
author = {Barman, K and Goswami, P},
title = {Recent Advances in Diagnostics and Therapeutic Interventions for Drug-Resistant Malaria.},
journal = {ACS infectious diseases},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsinfecdis.4c00962},
pmid = {40326084},
issn = {2373-8227},
abstract = {The emergence of drug-resistant malarial parasites has been a growing challenge to medical science to safeguard public health in the malaria-endemic regions of the globe. With time, the parasite develops newer resistance mechanisms to defunct the drug's action one after another. Genetic mutation is the prime weapon parasites rely upon to initiate the resistance mechanism in a case-specific manner, following various strategies such as structural changes in the target protein, metabolic alterations, and tweaking the drug-transported channels. In order to combat these resistances, different approaches have evolved among these developing inhibitors against critical parasite enzymes and metabolic pathways, combinatorial/hybrid drug therapies, exploring new drug targets and analogues of existing drugs, use of resistance-reversal agents, drug-repurposing, gene blocking/altering using RNA interference and CRISPR/Cas systems are prominent. However, the effectiveness of these approaches needs to be earnestly monitored for better management of the disease, which demands the development of a reliable diagnosis technique. Several methodologies have been investigated in search of a suitable diagnosis technique, such as in vivo, in vitro, ex vivo drug efficacy studies, and molecular techniques. A parallel effort to transform the efficient method into an inexpensive and portable diagnosis tool for rapid screening of drug resistance malaria among masses in the societal landscape is advocated. This review gives an insight into the historical perspectives of drug-resistant malaria and the recent developments in malaria diagnosis and antimalarial drug discovery. Efforts have been made to update recent strategies formulated to combat and diagnose drug-resistant malaria. Finally, a concluding remark with a future perspective on the subject has been forwarded.},
}

@article {pmid40294287,
year = {2025},
author = {Huang, P and Liu, Y and Zhao, C and Wang, C and Wang, L and Luo, M and Wang, W and Shan, W and Liu, X and Li, B and Wang, Z and Deng, H and Chen, X},
title = {Permanent Efferocytosis Prevention by Terminating MerTK Recycle on Tumor-Associated Macrophages for Cancer Immunotherapy.},
journal = {Journal of the American Chemical Society},
volume = {147},
number = {18},
pages = {15901-15914},
doi = {10.1021/jacs.5c05640},
pmid = {40294287},
issn = {1520-5126},
mesh = {*c-Mer Tyrosine Kinase/metabolism/genetics/immunology/antagonists & inhibitors ; Animals ; Proto-Oncogene Mas ; Mice ; *Immunotherapy ; *Tumor-Associated Macrophages/metabolism/immunology ; Humans ; Phagocytosis ; Mice, Inbred C57BL ; Cell Line, Tumor ; Phosphatidylserines/metabolism ; *Neoplasms/therapy/immunology ; CRISPR-Cas Systems ; Efferocytosis ; },
abstract = {Efferocytosis of apoptotic tumor cells by tumor-associated macrophages mediated through the phosphatidylserine (PtdSer)/MER proto-oncogene tyrosine kinase (MerTK) axis can exacerbate tumor immunosuppression, and conversely, prevention of efferocytosis via blocking PtdSer-MerTK association using prevalent antibodies represents a promising strategy for reversing tumor immunosuppression and boosting antitumor immunity. However, it remains unclear whether the antibody blockade can induce durable efferocytosis prevention and achieve sustained tumor growth inhibition. Here, we have shown that utilizing PtdSer and MerTK antibodies induced only a transient rather than a persistent efferocytosis prevention effect, and little enhancement was observed even after improving antibody enrichment in tumor sites. Further mechanistic studies suggested that degradation of anti-MerTK antibody and recycling of the MerTK receptor to the cell membrane would compromise the therapeutic benefits of antibody blockade. Based on these findings, we developed a CRISPR/Cas9 gene editing system deployed using Cas9 mRNA and MerTK sgRNA to permanently knock out MerTK, which achieved durable efferocytosis prevention, elicited persistent in situ vaccination immune responses via enhancing X-ray irradiation-induced immunogenic cell death, and led to sustained tumor suppression effects together with anti-PtdSer antibody and X-ray irradiation treatment in multiple B16 melanoma tumor models. Our findings provide a reliable gene-editing-mediated strategy for long-term modulating MerTK homeostasis and overcoming MerTK-dependent cancer immune evasion, generating adaptive antitumor immune responses for sustained cancer immunotherapy.},
}

*c-Mer Tyrosine Kinase/metabolism/genetics/immunology/antagonists & inhibitors
Animals
Proto-Oncogene Mas
Mice
*Immunotherapy
*Tumor-Associated Macrophages/metabolism/immunology
Humans
Phagocytosis
Mice, Inbred C57BL
Cell Line, Tumor
Phosphatidylserines/metabolism
*Neoplasms/therapy/immunology
CRISPR-Cas Systems
Efferocytosis

@article {pmid39951551,
year = {2025},
author = {Zhang, B and Zhang, P and Wang, H and Wang, X and Hu, Z and Wang, F and Li, Z},
title = {Dual Protein Corona-Mediated Target Recognition System for Visual Detection and Single-Molecule Counting of Nucleic Acids.},
journal = {ACS nano},
volume = {19},
number = {7},
pages = {6929-6941},
doi = {10.1021/acsnano.4c13924},
pmid = {39951551},
issn = {1936-086X},
mesh = {Gold/chemistry ; Metal Nanoparticles/chemistry ; Surface Plasmon Resonance/methods ; *Protein Corona/chemistry ; Humans ; *Nucleic Acids/analysis ; CRISPR-Cas Systems ; *Single Molecule Imaging/methods ; Biosensing Techniques/methods ; Point-of-Care Systems ; },
abstract = {Rapid, highly sensitive, and specific nucleic acid detection plays a crucial role in advancing point-of-care (POC) diagnostics for pathogens and viruses, cancer monitoring, and optimizing clinical treatments. Herein, leveraging the precise recognition ability of CRISPR/dCas9 and the powerful localized surface plasmon resonance (LSPR) of gold nanoparticles (AuNPs), we report the design of a dual protein corona-mediated detection platform to simultaneously fulfill rapid POC testing and single-molecule counting of nucleic acids in a one-pot and one-step manner. This system uses guide RNA as a molecular bridge to anchor dCas9 protein onto AuNPs, forming artificial protein coronas. Upon recognizing a target, the interaction between the two protein coronas on the same nucleic acid molecule triggers cross-linked aggregation of AuNPs. Then, a target as low as 100 aM can be visually detected within 30 min, making the platform particularly well-suited for rapid POC application and the screening of emerging epidemics. Additionally, the superior LSPR properties of AuNPs increase the light-scattering signal generated during target-induced aggregation, enabling the visualization of the aggregated AuNPs as diffraction-limited spots under confocal microscopy. By counting these spots, the platform achieves unprecedented detection sensitivity, identifying a target as low as 1 aM, which is equivalent to just 6 molecules in a 10 μL system, demonstrating single-molecule detection capability. This dual protein corona-mediated detection system offers exceptional promise for large-scale screening of pathogenic viruses and the early detection of cancer, particularly in applications requiring ultrahigh sensitivity at the single-molecule level.},
}

Gold/chemistry
Metal Nanoparticles/chemistry
Surface Plasmon Resonance/methods
*Protein Corona/chemistry
Humans
*Nucleic Acids/analysis
CRISPR-Cas Systems
*Single Molecule Imaging/methods
Biosensing Techniques/methods
Point-of-Care Systems

@article {pmid39941065,
year = {2025},
author = {Xia, X and Song, W and Zhang, F and Fan, Y and Zhang, B and Chen, X},
title = {ctdsp2 Knockout Induces Zebrafish Craniofacial Dysplasia via p53 Signaling Activation.},
journal = {International journal of molecular sciences},
volume = {26},
number = {3},
pages = {},
pmid = {39941065},
issn = {1422-0067},
support = {82271186 and 81974143//the General Programs of the National Natural Science Foundation of China/ ; 2022-PUMCH-C-029//the National High Level Hospital Clinical Research Funding/ ; 7232122//the Natural Science Foundation of Beijing, China/ ; },
mesh = {Animals ; *Zebrafish/genetics/metabolism/embryology ; *Tumor Suppressor Protein p53/metabolism/genetics ; *Zebrafish Proteins/genetics/metabolism ; *Signal Transduction ; *Craniofacial Abnormalities/genetics/metabolism/pathology ; Apoptosis/genetics ; Chondrocytes/metabolism/pathology ; Gene Knockout Techniques ; Neural Crest/metabolism ; Cell Proliferation ; Gene Expression Regulation, Developmental ; Cell Differentiation ; Cartilage/metabolism ; CRISPR-Cas Systems ; Disease Models, Animal ; },
abstract = {Hemifacial microsomia (HFM) is a rare congenital craniofacial deformity that significantly impacts the appearance and hearing. The genetic etiology of HFM remains largely unknown, although genetic factors are considered to be primary contributors. We previously identified CTDSP2 as a potential causative gene in HFM cases. Utilizing CRISPR/Cas9, we knocked out ctdsp2 in zebrafish and analyzed the spatiotemporal expression of ctdsp2 and neural crest cell (NCC) markers through in situ hybridization (ISH). Craniofacial cartilage and chondrocyte phenotypes were visualized using Alcian blue and wheat germ agglutinin (WGA) staining. Cell proliferation and apoptosis were assessed via immunofluorescence with PH3 and TUNEL. RNA sequencing was performed on ctdsp2[-/-] embryos and control siblings, followed by rescue experiments. Knockout of ctdsp2 in zebrafish resulted in craniofacial defects characteristic of HFM. We observed abnormalities in NCC apoptosis and proliferation in the pharyngeal arches, as well as impaired differentiation of chondrocytes in ctdsp2[-/-] embryos. RNA-Seq analysis revealed significantly higher expression of genes in the p53 signaling pathway in mutants. Furthermore, ctdsp2 mRNA injection and tp53 knockout significantly rescued pharyngeal arch cartilage dysplasia. Our findings suggest that ctdsp2 knockout induces zebrafish craniofacial dysplasia, primarily by disrupting pharyngeal chondrocyte differentiation and inhibiting NCC proliferation through p53 signaling pathway activation.},
}

Animals
*Zebrafish/genetics/metabolism/embryology
*Tumor Suppressor Protein p53/metabolism/genetics
*Zebrafish Proteins/genetics/metabolism
*Signal Transduction
*Craniofacial Abnormalities/genetics/metabolism/pathology
Apoptosis/genetics
Chondrocytes/metabolism/pathology
Gene Knockout Techniques
Neural Crest/metabolism
Cell Proliferation
Gene Expression Regulation, Developmental
Cell Differentiation
Cartilage/metabolism
CRISPR-Cas Systems
Disease Models, Animal

@article {pmid39939726,
year = {2025},
author = {Stewart, J and Krastev, DB and Brough, R and Zatreanu, D and Song, F and Baxter, JS and Sridhar, S and Frankum, J and Konde, A and Yang, W and Haider, S and Alexander, J and Betteridge, K and Gulati, A and Attygalle, AD and Vroobel, K and Natrajan, R and Khalique, S and Roumeliotis, TI and Choudhary, JS and Yeung, J and Wicks, AJ and Marlow, R and Banerjee, S and Pettitt, SJ and Tutt, ANJ and Lord, CJ},
title = {PPP2R1A mutations cause ATR inhibitor sensitivity in ovarian clear cell carcinoma.},
journal = {Oncogene},
volume = {44},
number = {9},
pages = {618-629},
pmid = {39939726},
issn = {1476-5594},
support = {DRCRPG-Nov21\100001//Cancer Research UK (CRUK)/ ; },
mesh = {Female ; Humans ; *Protein Phosphatase 2/genetics ; *Adenocarcinoma, Clear Cell/genetics/drug therapy/pathology ; *Ovarian Neoplasms/genetics/drug therapy/pathology ; Animals ; *Mutation ; Mice ; Cell Line, Tumor ; *Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors/genetics ; Transcription Factors/genetics ; DNA-Binding Proteins/genetics ; Drug Resistance, Neoplasm/genetics ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; },
abstract = {Identification of ARID1A/ATR synthetic lethality led to ATR inhibitor phase II trials in ovarian clear cell carcinoma (OCCC), a cancer of unmet need. Using multiple CRISPR-Cas9 mutagenesis and interference screens, we show that inactivation of protein phosphatase 2A (PP2A) subunits, including PPP2R1A, enhance ATRi sensitivity in ARID1A mutant OCCC. Analysis of a new OCCC cohort indicates that 52% possess oncogenic PPP2R1A p.R183 mutations and of these, one half possessed both ARID1A as well as PPP2R1A mutations. Using CRISPR-prime editing to generate new isogenic models of PPP2R1A mutant OCCC, we found that PPP2R1A p.R183W and p.R183P mutations cause ATRi-induced S phase stress, premature mitotic entry, genomic instability and ATRi sensitivity in OCCC tumour cells. p.R183 mutation also enhanced both in vitro and in vivo ATRi sensitivity in preclinical models of ARID1A mutant OCCC. These results argue for the assessment of PPP2R1A mutations as a biomarker of ATRi sensitivity.},
}

Female
Humans
*Protein Phosphatase 2/genetics
*Adenocarcinoma, Clear Cell/genetics/drug therapy/pathology
*Ovarian Neoplasms/genetics/drug therapy/pathology
Animals
*Mutation
Mice
Cell Line, Tumor
*Ataxia Telangiectasia Mutated Proteins/antagonists & inhibitors/genetics
Transcription Factors/genetics
DNA-Binding Proteins/genetics
Drug Resistance, Neoplasm/genetics
Xenograft Model Antitumor Assays
CRISPR-Cas Systems

@article {pmid39933924,
year = {2025},
author = {Lebon, S and Bruneel, A and Drunat, S and Albert, A and Csaba, Z and Elmaleh, M and Ntorkou, A and Ténier, Y and Fenaille, F and Gressens, P and Passemard, S and Boespflug-Tanguy, O and Dorboz, I and El Ghouzzi, V},
title = {A biallelic variant in GORASP1 causes a novel Golgipathy with glycosylation and mitotic defects.},
journal = {Life science alliance},
volume = {8},
number = {4},
pages = {},
pmid = {39933924},
issn = {2575-1077},
mesh = {Humans ; Glycosylation ; *Golgi Apparatus/metabolism/pathology/genetics ; *Golgi Matrix Proteins/genetics/metabolism ; *Mitosis/genetics ; Male ; Female ; Mutation ; Alleles ; Phenotype ; CRISPR-Cas Systems ; *Membrane Proteins/genetics ; },
abstract = {GRASP65 is a Golgi-associated peripheral protein encoded by the GORASP1 gene and required for Golgi cisternal stacking in vitro. A key role of GRASP65 in the regulation of cell division has also been suggested. However, depletion of GRASP65 in mice has little effect on the Golgi structure and the gene has not been associated with any human phenotype to date. Here, we report the identification of the first human pathogenic variant of GORASP1 (c.1170_1171del; p.Asp390Glufs*18) in a patient combining a neurodevelopmental disorder with neurosensory, neuromuscular, and skeletal abnormalities. Functional analysis revealed that the variant leads to a total absence of GRASP65. The structure of the Golgi apparatus did not show fragmentation, but glycosylation anomalies such as hyposialylation were detected. Mitosis analyses revealed an excess of prometaphases and metaphases with polar chromosomes, suggesting a delay in the cell cycle. These phenotypes were recapitulated in RPE cells in which a similar mutation was introduced by CRISPR/Cas9. These results indicate that loss of GRASP65 in humans causes a novel Golgipathy associated with defects in glycosylation and mitotic progression.},
}

Humans
Glycosylation
*Golgi Apparatus/metabolism/pathology/genetics
*Golgi Matrix Proteins/genetics/metabolism
*Mitosis/genetics
Male
Female
Mutation
Alleles
Phenotype
CRISPR-Cas Systems
*Membrane Proteins/genetics

@article {pmid39930358,
year = {2025},
author = {Zhang, Y and Newstead, S and Sarkies, P},
title = {Predicting substrates for orphan solute carrier proteins using multi-omics datasets.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {130},
pmid = {39930358},
issn = {1471-2164},
mesh = {Humans ; *Solute Carrier Proteins/metabolism/genetics ; Algorithms ; *Computational Biology/methods ; *Genomics/methods ; Substrate Specificity ; CRISPR-Cas Systems ; Multiomics ; },
abstract = {Solute carriers (SLC) are integral membrane proteins responsible for transporting a wide variety of metabolites, signaling molecules and drugs across cellular membranes. Despite key roles in metabolism, signaling and pharmacology, around one third of SLC proteins are 'orphans' whose substrates are unknown. Experimental determination of SLC substrates is technically challenging, given the wide range of possible physiological candidates. Here, we develop a predictive algorithm to identify correlations between SLC expression levels and intracellular metabolite concentrations by leveraging existing cancer multi-omics datasets. Our predictions recovered known SLC-substrate pairs with high sensitivity and specificity compared to simulated random pairs. CRISPR-Cas9 dependency screen data and metabolic pathway adjacency data further improved the performance of our algorithm. In parallel, we combined drug sensitivity data with SLC expression profiles to predict new SLC-drug interactions. Together, we provide a novel bioinformatic pipeline to predict new substrate predictions for SLCs, offering new opportunities to de-orphanise SLCs with important implications for understanding their roles in health and disease.},
}

Humans
*Solute Carrier Proteins/metabolism/genetics
Algorithms
*Computational Biology/methods
*Genomics/methods
Substrate Specificity
CRISPR-Cas Systems
Multiomics

@article {pmid40325455,
year = {2025},
author = {Gao, Y and Zou, Y and Wu, C and Tao, J and Nie, Z and Yan, J and Wang, P and Huang, X},
title = {Comparative evaluation of immunomodulatory cytokines for oncolytic therapy based on a high-efficient platform for oHSV1 reconstruction.},
journal = {Virology journal},
volume = {22},
number = {1},
pages = {133},
pmid = {40325455},
issn = {1743-422X},
support = {Kunming Medical University graduate Student Innovation Fund(2024S086)//Yufang Zou/ ; National Natural Science Foundation of China (82260486)//Xinwei Huang/ ; },
mesh = {Animals ; *Oncolytic Virotherapy/methods ; *Oncolytic Viruses/genetics/immunology ; *Cytokines/genetics/immunology ; Mice ; *Herpesvirus 1, Human/genetics/immunology ; Humans ; Female ; Cell Line, Tumor ; CRISPR-Cas Systems ; *Triple Negative Breast Neoplasms/therapy/immunology ; Mice, Inbred BALB C ; Tumor Microenvironment ; *Immunologic Factors/genetics ; },
abstract = {BACKGROUND: Triple-negative breast cancer (TNBC) presents significant therapeutic challenges due to its immunosuppressive tumor microenvironment (TME). Oncolytic herpes simplex virus type 1 (oHSV1) offers dual mechanisms of tumor lysis and immune activation, yet the optimal cytokine payloads for TNBC remain undefined.

METHODS: We developed a CRISPR/Cas9-mediated platform for high-efficiency oHSV1 engineering, replacing the ICP47 locus with murine IFN-γ, GM-CSF, or IL-15Rα/IL-15 fusion protein (IL15Fu). Constructs were validated for cytokine secretion, MHC modulation, and cytotoxicity in 4T1 TNBC and a panel of human cancer cell lines. Antitumor efficacy and immune remodeling were evaluated in a syngeneic 4T1 model using RNA sequencing and flow cytometry.

RESULTS: The CRISPR platform achieved 62.5-71.4% homologous recombination efficiency, enabling rapid virus construction. In vitro, OV-IFNG exhibited upregulated MHC I/II expression and potent cytotoxicity, while OV-GMCSF attenuated oncolysis in subsets of breast cancer cell lines. In the 4T1 model, OV-IL15Fu modestly improved tumor control and extended survival without apparent toxicity, while OV-IFNG induced early mortality associated with systemic toxicity. Transcriptomic profiling revealed divergent immune modulation: OV-IL15Fu enriched T cell/NK cytotoxicity pathways, OV-IFNG amplified cytokine/chemokine signaling, and OV-GMCSF paradoxically enhanced myeloid recruitment while inhibiting MHC-II pathways. Flow cytometry confirmed functional differences in immune activation: OV-IL15Fu expanding cytotoxic lymphocytes (CD8⁺ T/NK cells), OV-IFNG preferentially promote Th1 polarization and innate immune activation, and OV-GMCSF failed to activate T cells despite myeloid infiltration.

CONCLUSIONS: Our findings underscore the need for rational cytokine selection in oHSV1-based immunotherapy. While IFN-γ increased immunogenic markers, its systemic toxicity and myeloid effects may limit benefit. GM-CSF exacerbated immune suppression in this context, whereas IL15Fu showed favorable immunostimulatory properties without detectable toxicity. These data support IL15Fu as a contextually promising payload for further evaluation in TNBC-targeted oncolytic virotherapy.},
}

Animals
*Oncolytic Virotherapy/methods
*Oncolytic Viruses/genetics/immunology
*Cytokines/genetics/immunology
Mice
*Herpesvirus 1, Human/genetics/immunology
Humans
Female
Cell Line, Tumor
CRISPR-Cas Systems
*Triple Negative Breast Neoplasms/therapy/immunology
Mice, Inbred BALB C
Tumor Microenvironment
*Immunologic Factors/genetics

@article {pmid40324959,
year = {2025},
author = {Namata, MJ and Xu, J and Habyarimana, E and Palakolanu, SR and Wang, L and Li, J},
title = {Genome editing in maize and sorghum: A comprehensive review of CRISPR/Cas9 and emerging technologies.},
journal = {The plant genome},
volume = {18},
number = {2},
pages = {e70038},
doi = {10.1002/tpg2.70038},
pmid = {40324959},
issn = {1940-3372},
support = {32372134//National Natural Science Foundation of China/ ; S202310879188//Student Innovation and Entrepreneurship Project of Anhui Science and Technology University/ ; No.XK-XJGF001//Key Discipline Construction Funds of Anhui Sciences and Technology University/ ; 2023cxcysj179//Chuzhou "Star of Innovation and Entrepreneurship" Industrial Innovation Team, Postgraduate Innovation and Entrepreneurship Practice Project of Anhui Province/ ; },
mesh = {*Sorghum/genetics ; *Zea mays/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Plant Breeding/methods ; *Genome, Plant ; Plants, Genetically Modified ; Crops, Agricultural/genetics ; },
abstract = {The increasing changes in the climate patterns across the globe have deeply affected food systems where unparalleled and unmatched challenges are created. This jeopardizes food security due to an ever-increasing population. The extreme efficiency of C4 crops as compared to C3 crops makes them incredibly significant in securing food safety. C4 crops, maize (Zea mays L.) and sorghum (Sorghum bicolor L. Moench) in particular, have the ability to withstand osmotic stress induced by oxidative stress. Osmotic stress causes a series of physical changes in a plant thus facilitating reduced water uptake and photosynthesis inhibition, such as membrane tension, cell wall stiffness, and turgor changes. There has been a great advancement in plant breeding brought by introduction of clustered regularly interspaced short palindromic repeats (CRISPR) gene editing technology. This technology offers precise alterations to an organism's DNA through targeting specific genes for desired traits in a wide number of crop species. Despite its immense opportunities in plant breeding, it faces limitations such as effective delivery systems, editing efficiency, regulatory concerns, and off-target effects. Future prospects lie in optimizing next-generation techniques, such as prime editing, and developing novel genotype-independent delivery methods. Overall, the transformative role of CRISPR/Cas9 in sorghum and maize breeding underscores the need for responsible and sustainable utilization to address global food security challenges.},
}

*Sorghum/genetics
*Zea mays/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
Plant Breeding/methods
*Genome, Plant
Plants, Genetically Modified
Crops, Agricultural/genetics

@article {pmid40324075,
year = {2025},
author = {Engel, NW and Steinfeld, I and Ryan, D and Anupindi, K and Kim, S and Wellhausen, N and Chen, L and Wilkins, K and Baker, DJ and Rommel, PC and Jarocha, D and Gohil, M and Zhang, Q and Milone, MC and Fraietta, JA and Davis, M and Young, RM and June, CH},
title = {Quadruple adenine base-edited allogeneic CAR T cells outperform CRISPR/Cas9 nuclease-engineered T cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {20},
pages = {e2427216122},
doi = {10.1073/pnas.2427216122},
pmid = {40324075},
issn = {1091-6490},
support = {1-U54 CA244711//US NIH, National Cancer Institute/ ; Thought Leader Award 2020//Agilent Foundation (Agilent Technologies Foundation)/ ; Sponsored Research//Gilead | Kite Pharma/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *T-Lymphocytes/immunology/metabolism ; Animals ; Humans ; Mice ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; *Adenine/metabolism ; Immunotherapy, Adoptive/methods ; Graft vs Host Disease ; },
abstract = {Genome-editing technologies have enabled the clinical development of allogeneic cellular therapies, yet the optimal gene-editing modality for multiplex editing of therapeutic T cell product manufacturing remains elusive. In this study, we conducted a comprehensive comparison of CRISPR/Cas9 nuclease and adenine base editor (ABE) technologies in generating allogeneic chimeric antigen receptor (CAR) T cells, utilizing extensive in vitro and in vivo analyses. Both methods achieved high editing efficiencies across four target genes, critical for mitigating graft-versus-host disease and allograft rejection: TRAC or CD3E, B2M, CIITA, and PVR. Notably, ABE demonstrated higher manufacturing yields and distinct off-target profiles compared to Cas9, with translocations observed exclusively in Cas9-edited products. Functionally, ABE-edited CAR T cells exhibited superior in vitro effector functions under continuous antigen stimulation, including enhanced proliferative capacity and increased surface CAR expression. Transcriptomic analysis revealed that ABE editing resulted in reduced activation of p53 and DNA damage response pathways at baseline, along with sustained activation of metabolic pathways during antigen stress. Consistently, Assay for Transposase-Accessible Chromatin using sequencing data indicated that Cas9-edited, but not ABE-edited, CAR T cells showed enrichment of chromatin accessibility peaks associated with double-strand break repair and DNA damage response pathways. In a preclinical leukemia model, ABE-edited CAR T cells demonstrated improved tumor control and extended overall survival compared to their Cas9-edited counterparts. Collectively, these findings position ABE as superior to Cas9 nucleases for multiplex gene editing of therapeutic T cells.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*T-Lymphocytes/immunology/metabolism
Animals
Humans
Mice
*Receptors, Chimeric Antigen/genetics/immunology/metabolism
*Adenine/metabolism
Immunotherapy, Adoptive/methods
Graft vs Host Disease

@article {pmid40270429,
year = {2025},
author = {Shi, K and Luo, W and Cheng, Y and Li, H and Peng, L and Luo, X and Hu, Y and Zhang, J and Chen, J},
title = {Phosphorothioate-Modified Hairpin G-Triplex Reporter-Assisted Split CRISPR/Cas12a-Powered Biosensor for "Turn-On" Fluorescent Detection of Nucleic Acid and Non-Nucleic Acid Targets.},
journal = {Analytical chemistry},
volume = {97},
number = {17},
pages = {9361-9366},
doi = {10.1021/acs.analchem.5c00140},
pmid = {40270429},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; G-Quadruplexes ; *CRISPR-Cas Systems ; *MicroRNAs/analysis ; *DNA/analysis/chemistry ; Benzothiazoles/chemistry ; *Phosphorothioate Oligonucleotides/chemistry ; Fluorescent Dyes/chemistry ; Limit of Detection ; Humans ; Spectrometry, Fluorescence ; },
abstract = {CRISPR/Cas12a-powered biosensors with guanine (G)-rich sequence reporters (e.g., G-quadruplex and G-triplex) are widely used in detection applications due to their simplicity and sensitivity. However, when these biosensors are employed for molecular detection in complex samples, they may encounter difficulties such as high background signal and susceptibility to interference because of the "turn-off" signal output. Herein, we explore, for the first time, a set of phosphorothioate (ps)-modified G-quadruplex (G4) and G-triplex (G3) sequences that can bind with thioflavin T (ThT) in an active split CRISPR/Cas12a system (SCas12a) to generate a "turn-on" fluorescent signal. To apply this new phenomenon, we develop a universal SCas12a-powered biosensor for "turn-on" fluorescent detection of nucleic acid (miRNA-21) and non-nucleic acid (kanamycin) targets by using ps-modified hairpin G3 as a reporter (SCas12a/psHG3). Target recognition activates SCas12a's trans-cleavage activity, leading to cleavage at the loop region of the psHG3 reporter. The released prelocked psG3 DNA binds ThT to produce a strong fluorescence signal. Without preamplification, this strategy can detect miRNA-21 with a detection limit of 100 fM. Moreover, the SCas12a/psHG3 system was further utilized for detecting kanamycin by incorporating its aptamers, enabling the detection of kanamycin at concentrations as low as 100 pM. This work is the first to develop a "turn-on" SCas12a/psHG3 system, showcasing its improved performance and wide range of applications in synthetic biology-based sensing technology.},
}

*Biosensing Techniques/methods
G-Quadruplexes
*CRISPR-Cas Systems
*MicroRNAs/analysis
*DNA/analysis/chemistry
Benzothiazoles/chemistry
*Phosphorothioate Oligonucleotides/chemistry
Fluorescent Dyes/chemistry
Limit of Detection
Humans
Spectrometry, Fluorescence

@article {pmid40263956,
year = {2025},
author = {Yan, H and Qi, M and Li, H},
title = {Characterization and Full Sequencing of 100 Nt sgRNA and Large RNA Using Site-Directed Cleavage and Liquid Chromatography Tandem Mass Spectrometry.},
journal = {Analytical chemistry},
volume = {97},
number = {17},
pages = {9228-9238},
doi = {10.1021/acs.analchem.4c06606},
pmid = {40263956},
issn = {1520-6882},
mesh = {Tandem Mass Spectrometry/methods ; Ribonuclease H/metabolism ; Chromatography, Liquid ; DNA, Catalytic/metabolism/chemistry ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; *RNA/genetics/chemistry ; *Sequence Analysis, RNA/methods ; CRISPR-Cas Systems ; },
abstract = {CRISPR/Cas9 is widely recognized as the most effective, efficient, and precise genome editing tool, inspiring numerous applications in basic science, medicine, and biotechnology. In the CRISPR/Cas9 system, single guide RNA (sgRNA) and Cas9 enzyme form a ribonucleoprotein complex that specifically and effectively cleaves target DNA. Accurate sequencing of sgRNA, particularly identifying the target sequence within the first 20 nucleotides (nt) at the 5'-end, is crucial for quality assurance and regulatory compliance. In this study, we used site-directed cleavage using ribonuclease H (RNase H) and DNAzyme for the first time to digest 100 nt sgRNA, achieving full sequencing with 100% coverage by analyzing the two cleaved fragments separately via LC MS/MS. We evaluated four different DNA-RNA chimeras as capture probes for the RNase H site-directed cleavage approach, finding that the chimera with four deoxynucleotides provided the most specific cleavage. Compared to RNase H, the DNAzyme demonstrated higher specificity and stability for 100 nt sgRNA digestion, successfully identifying up to 200 nucleotides of large RNA with 100% sequence coverage by fully sequencing the four short cleaved fragments. Due to the high specificity of DNAzyme cleavage, we used this method to study the designed 5'-end N-X truncated impurities of 100 nt sgRNA, demonstrating accurate identification and relative quantification. For 100 nt sgRNA, the limited available cleavage site was set on the scaffold sequence for both site-directed cleavage approaches, and the captured probes designed for RNase H and DNAzyme can be universally applied to sequence all 100 nt sgRNAs because of the conserved scaffold sequence.},
}

Tandem Mass Spectrometry/methods
Ribonuclease H/metabolism
Chromatography, Liquid
DNA, Catalytic/metabolism/chemistry
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry
*RNA/genetics/chemistry
*Sequence Analysis, RNA/methods
CRISPR-Cas Systems

@article {pmid40215333,
year = {2025},
author = {Macias, LA and Lowther, J and Tillotson, EL and Rohde, E and Madsen, JA},
title = {Ion Mobility Gas-Phase Separation Enhances Top-Down Mass Spectrometry of Heavily Modified Guide RNA.},
journal = {Analytical chemistry},
volume = {97},
number = {17},
pages = {9430-9437},
doi = {10.1021/acs.analchem.5c00705},
pmid = {40215333},
issn = {1520-6882},
mesh = {*Mass Spectrometry/methods ; *RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/analysis ; Ion Mobility Spectrometry ; Gases/chemistry ; Phase Separation ; },
abstract = {As gene editing technologies enter the clinic, state-of-the-art characterization methods have been developed in parallel to assess the components of these paradigm-shifting medicines. One such component, the guide RNA (gRNA) element of CRISPR-based drugs, is a large synthetic heavily modified oligonucleotide that programs for the desired gene edit. Conventional oligonucleotide sequencing technologies can inform gRNA composition, but these methods may not completely capture the chemical modifications that are introduced during synthesis. Circumventing these challenges, mass spectrometry has demonstrated use in oligonucleotide analyses and has been combined here with ion mobility to deepen its characterization power. The use of ion mobility enabled us to perform gas-phase separation of the fragment ions produced by top-down mass spectrometry, yielding a significant increase in fragment identifications for a highly modified 100-mer gRNA by uncovering high-confidence assignments for heavily modified regions and for the important spacer region. Furthermore, the high-confidence fragment assignments empowered simultaneous de novo sequencing and chemical modification localization for the 5'-end spacer region as well as for 15 nucleotides on the heavily modified 3'-end. Overall, a total sequence coverage of 95% was achieved for the heavily modified 100-mer, ushering near complete sequence and chemical modification confirmation by top-down mass spectrometry.},
}

*Mass Spectrometry/methods
*RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/analysis
Ion Mobility Spectrometry
Gases/chemistry
Phase Separation

@article {pmid40020602,
year = {2025},
author = {Patra, N and Barker, GC and Maiti, MK},
title = {Knockout of fatty acid elongase1 homeoalleles in amphidiploid Brassica juncea leads to undetectable erucic acid in seed oil.},
journal = {Plant physiology and biochemistry : PPB},
volume = {222},
number = {},
pages = {109679},
doi = {10.1016/j.plaphy.2025.109679},
pmid = {40020602},
issn = {1873-2690},
mesh = {*Mustard Plant/genetics/metabolism/enzymology ; *Erucic Acids/metabolism ; *Fatty Acid Elongases/genetics/metabolism ; *Seeds/metabolism/genetics ; *Plant Oils/metabolism ; Gene Knockout Techniques ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {Indian mustard (Brassica juncea L.) is a major oilseed crop with considerable economic and nutritional importance globally. While its seed oil offers valuable dietary benefits due to a balanced ratio of human essential fatty acids, the traditional high oil-yielding varieties contain an elevated level of erucic acid (EA, C22:1) associated with adverse health effects. Therefore, developing low erucic acid (LEA) mustard cultivars is crucial for broader utilization and consumer safety. In this study, CRISPR/Cas9 genome editing tool was employed to disrupt the fatty acid elongase1 (FAE1) gene that encodes a key enzyme in EA biosynthesis in two high erucic acid (HEA) B. juncea cultivars, PCR7 (∼39% EA) and JD6 (∼45% EA). Targeted knockout (KO) of BjFAE1 homeoalleles (BjFAE1.1 and BjFAE1.2) in this amphidiploid plant species using CRISPR/Cas9 constructs, each carrying two guide RNAs led to generation of single (either fae1.1 or fae1.2) and double (fae1.1fae1.2) mutants. Best performing homozygous fae1.1fae1.2 KO lines showed a near-complete elimination of EA in both the cultivars (<0.5% in PCR7, undetectable in JD6) with a marked increase in nutritionally beneficial oleic acid (from ∼18% to ∼32% in PCR7, from ∼9% to ∼38% in JD6). Moreover, the content of essential fatty acids also increased substantially [linoleic acid (C18:2) 1.9-fold in PCR7 and 2.1-fold in JD6; linolenic acid (C18:3) 2.5-fold in PCR7 and 1.4-fold in JD6], suggesting rerouting of carbon flux from EA biosynthesis. Importantly, these LEA lines retained key agronomic traits like plant seed yield and oil content, matching the productivity of the unedited control elite cultivars. Our findings underscore the effectiveness of CRISPR/Cas9 technology for editing B. juncea genome, developing plant lines producing LEA seed oil with improved nutritional quality and broadening the utility of this important oilseed crop for food and non-food applications.},
}

*Mustard Plant/genetics/metabolism/enzymology
*Erucic Acids/metabolism
*Fatty Acid Elongases/genetics/metabolism
*Seeds/metabolism/genetics
*Plant Oils/metabolism
Gene Knockout Techniques
*Plant Proteins/genetics/metabolism
CRISPR-Cas Systems
Gene Editing

@article {pmid39916610,
year = {2025},
author = {Uusi-Mäkelä, M and Harjula, SE and Junno, M and Sillanpää, A and Nätkin, R and Niskanen, MT and Saralahti, AK and Nykter, M and Rämet, M},
title = {The inflammasome adaptor pycard is essential for immunity against Mycobacterium marinum infection in adult zebrafish.},
journal = {Disease models & mechanisms},
volume = {18},
number = {9},
pages = {},
pmid = {39916610},
issn = {1754-8411},
support = {//Tampereen Tuberkuloosisaatio/ ; //Maud Kuistilan Muistosaatio/ ; //Tampere University Doctoral School/ ; //Suomen Kulttuurirahasto/ ; //Finnish Society for Study of Infectious Diseases/ ; 277495//The Academy of Finland/ ; //The Competitive State Research Financing of the Expert Responsibility Area of Oulu University Hospital/ ; //Tampereen Tuberkuloosisäätiö/ ; //Maud Kuistilan Muistosäätiö/ ; //Väinö ja Laina Kiven Säätiö/ ; //Orion Research Foundation/ ; //National Doctoral Education Pilot Based on the Immune System/ ; //Instrumentariumin Tiedesäätiö/ ; //Sigrid Juséliuksen Säätiö/ ; 277495//Research Council of Finland/ ; //Competitive State Research Financing of the Expert Responsibility Area of Oulun Yliopistollinen Sairaala/ ; //Tampereen Yliopisto/ ; },
mesh = {Animals ; *Zebrafish/microbiology/immunology/genetics ; *Mycobacterium marinum/physiology/immunology ; *Mycobacterium Infections, Nontuberculous/immunology/microbiology/genetics ; *Zebrafish Proteins/metabolism/genetics ; *Inflammasomes/metabolism ; Neutrophils/metabolism/immunology ; *CARD Signaling Adaptor Proteins/metabolism ; Granuloma/pathology ; *Immunity ; CRISPR-Cas Systems/genetics ; Kidney/pathology ; Gene Knockout Techniques ; Larva/microbiology ; Gene Expression Profiling ; Transcriptome/genetics ; },
abstract = {Inflammasomes regulate the host response to intracellular pathogens including mycobacteria. We have previously shown that the course of Mycobacterium marinum infection in adult zebrafish (Danio rerio) mimics the course of tuberculosis in human. To investigate the role of the inflammasome adaptor pycard in zebrafish M. marinum infection, we produced two zebrafish knockout mutant lines for the pycard gene with CRISPR/Cas9 mutagenesis. Although the zebrafish larvae lacking pycard developed normally and had unaltered resistance against M. marinum, the loss of pycard led to impaired survival and increased bacterial burden in the adult zebrafish. Based on histology, immune cell aggregates, granulomas, were larger in pycard-deficient fish than in wild-type controls. Transcriptome analysis with RNA sequencing of a zebrafish haematopoietic tissue, kidney, suggested a role for pycard in neutrophil-mediated defence, haematopoiesis and myelopoiesis during infection. Transcriptome analysis of fluorescently labelled, pycard-deficient kidney neutrophils identified genes that are associated with compromised resistance, supporting the importance of pycard for neutrophil-mediated immunity against M. marinum. Our results indicate that pycard is essential for resistance against mycobacteria in adult zebrafish.},
}

Animals
*Zebrafish/microbiology/immunology/genetics
*Mycobacterium marinum/physiology/immunology
*Mycobacterium Infections, Nontuberculous/immunology/microbiology/genetics
*Zebrafish Proteins/metabolism/genetics
*Inflammasomes/metabolism
Neutrophils/metabolism/immunology
*CARD Signaling Adaptor Proteins/metabolism
Granuloma/pathology
*Immunity
CRISPR-Cas Systems/genetics
Kidney/pathology
Gene Knockout Techniques
Larva/microbiology
Gene Expression Profiling
Transcriptome/genetics

@article {pmid39909615,
year = {2025},
author = {Chignon, A and Lettre, G},
title = {Using omics data and genome editing methods to decipher GWAS loci associated with coronary artery disease.},
journal = {Atherosclerosis},
volume = {401},
number = {},
pages = {118621},
doi = {10.1016/j.atherosclerosis.2024.118621},
pmid = {39909615},
issn = {1879-1484},
mesh = {Humans ; *Genome-Wide Association Study ; *Coronary Artery Disease/genetics ; Genetic Predisposition to Disease ; *Gene Editing/methods ; Mendelian Randomization Analysis ; Quantitative Trait Loci ; Phenotype ; *Genomics/methods ; Animals ; CRISPR-Cas Systems ; Polymorphism, Single Nucleotide ; },
abstract = {Coronary artery disease (CAD) is due to atherosclerosis, a pathophysiological process that involves several cell-types and results in the accumulation of lipid-rich plaque that disrupt the normal blood flow through the coronary arteries to the heart. Genome-wide association studies have identified 1000s of genetic variants robustly associated with CAD or its traditional risk factors (e.g. blood pressure, blood lipids, type 2 diabetes, smoking). However, gaining biological insights from these genetic discoveries remain challenging because of linkage disequilibrium and the difficulty to interpret the functions of non-coding regulatory elements in the human genome. In this review, we present different statistical methods (e.g. Mendelian randomization) and molecular datasets (e.g. expression or protein quantitative trait loci) that have helped connect CAD-associated variants with genes, biological pathways, and cell-types or tissues. We emphasize that these various strategies make predictions, which need to be validated in orthologous systems. We discuss specific examples where the integration of omics data with GWAS results has prioritized causal CAD variants and genes. Finally, we review how targeted and genome-wide genome editing experiments using the CRISPR/Cas9 toolbox have been used to characterize new CAD genes in human cells. Researchers now have the statistical and bioinformatic methods, the molecular datasets, and the experimental tools to dissect comprehensively the loci that contribute to CAD risk in humans.},
}

Humans
*Genome-Wide Association Study
*Coronary Artery Disease/genetics
Genetic Predisposition to Disease
*Gene Editing/methods
Mendelian Randomization Analysis
Quantitative Trait Loci
Phenotype
*Genomics/methods
Animals
CRISPR-Cas Systems
Polymorphism, Single Nucleotide

@article {pmid38772706,
year = {2025},
author = {Abate-Shen, C and Politi, K},
title = {The Evolution of Mouse Models of Cancer: Past, Present, and Future.},
journal = {Cold Spring Harbor perspectives in medicine},
volume = {15},
number = {5},
pages = {},
pmid = {38772706},
issn = {2157-1422},
mesh = {Animals ; Mice ; *Disease Models, Animal ; *Neoplasms/genetics ; Humans ; Gene Editing ; CRISPR-Cas Systems ; History, 21st Century ; History, 20th Century ; },
abstract = {In the nearly 50 years since the original models of cancer first hit the stage, mouse models have become a major contributor to virtually all aspects of cancer research, and these have evolved well beyond simple transgenic or xenograft models to encompass a wide range of more complex models. As the sophistication of mouse models has increased, an explosion of new technologies has expanded the potential to both further develop and apply these models to address major challenges in cancer research. In the current era, cancer modeling has expanded to include nongermline genetically engineered mouse models (GEMMs), patient-derived models, organoids, and adaptations of the models better suited for cancer immunology research. New technologies that have transformed the field include the application of CRISPR-Cas9-mediated genome editing, in vivo imaging, and single-cell analysis to cancer modeling. Here, we provide a historical perspective on the evolution of mouse models of cancer, focusing on how far we have come in a relatively short time and how new technologies will shape the future development of mouse models of cancer.},
}

Animals
Mice
*Disease Models, Animal
*Neoplasms/genetics
Humans
Gene Editing
CRISPR-Cas Systems
History, 21st Century
History, 20th Century

@article {pmid40324726,
year = {2025},
author = {Kumbhakar, R and Mondal, M and Thakro, V and Tripathi, S and Parida, SK},
title = {Shaping the future: Unravelling regulators modulating plant architecture for next-generation crops.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {},
number = {},
pages = {112534},
doi = {10.1016/j.plantsci.2025.112534},
pmid = {40324726},
issn = {1873-2259},
abstract = {Plant architecture traits in crops are modulated through intricate interactions of various genetic pathways, which helps them to adapt to diverse environmental conditions. Key developmental pathways involved in forming plant architecture include the LAZY-TAC (Tiller Angle Control) module regulating branch and tiller angle, the CLAVATA-WUSCHEL pathway controlling shoot apical meristem fate and the GID1-DELLA pathway governing plant height and tillering in major food crops. These pathways function in concert to shape the overall architecture of plants, which is essential for optimizing light capture, resource allocation, reproductive success and eventual crop yield enhancement. Presently, plant architecture of modern crops has been shaped especially by artificial selection of natural alleles that target yield traits. Recent advances in CRISPR-Cas-based genome editing and genomics-assisted breeding strategies have enabled precise genetic manipulation of natural alleles in the functionally relevant genes regulating plant architecture traits in crops. This will assist researchers to select and introgress superior natural alleles in popular cultivars strategically for restructuring their desirable plant-types suitable for mechanical harvesting as well as enhancing the crop yield potential.},
}

@article {pmid40324585,
year = {2025},
author = {Jiang, Y and Zheng, H and Bai, L and Wang, Y and Zhong, J and Zhang, W and Xu, T and Pan, Y and Tang, J and Lu, J and Zhang, B and Wu, Y},
title = {Genomic analysis and mobile genetic elements carriage of Clostridium perfringens type A.},
journal = {Anaerobe},
volume = {},
number = {},
pages = {102970},
doi = {10.1016/j.anaerobe.2025.102970},
pmid = {40324585},
issn = {1095-8274},
abstract = {OBJECTIVES: To explore a comprehensive genomic analysis of Clostridium perfringens type A strains from diverse regions in China, investigating their virulence genes, antibiotic resistance genes, and mobile genetic elements (MGEs) to inform strategies for infection control and resistance gene surveillance.

METHODS: We conducted whole-genome sequencing on 168 C. perfringens type A strains from nine provinces in China (2016-2021). Previously described alpha-toxin (PLC) sequence typing for C. perfringens was used for comparisons with core genome multilocus sequence typing. Virulence genes, antibiotic resistance genes, and MGEs, including CRISPR/Cas, prophages, and plasmids of C. perfringens type A were investigated by molecular and bioinformatic methods.

RESULTS: PLC type II contained the largest number of isolates (n=44). The same type strains were largely clustered in the same branches. Tetracycline resistance genes tetA(P) and tetB(P) had high prevalence in type A isolates. 395 prophages were predicted including 265 "incomplete," 55 "questionable," and 75 "intact" prophages. CRISPR/Cas systems were more common in isolates from humans (63%) than in those from animals and food (52% and 46%, respectively). Fifty-seven percent of strains likely had the tcp conjugation locus (tcpC to tcpH), and 12 isolates likely carried the conjugative pCW3 plasmid. Type A strains exhibited fewer plasmid-encoded toxins.

CONCLUSIONS: cgMLST analysis demonstrated some micro-evolution and regional transmission trends within type A, which exhibited partial correlated with PLC typing. This study highlights the need for enhanced surveillance of antimicrobial resistance and pathogenicity-associated MGEs in C. perfringens type A.},
}

@article {pmid40319267,
year = {2025},
author = {García-Calvo, L and Kummen, C and Rustad, S and Rønning, SB and Fagerlund, A},
title = {A toolkit for facilitating markerless integration of expression cassettes in Komagataella phaffii via CRISPR/Cas9.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {97},
pmid = {40319267},
issn = {1475-2859},
support = {314743//Norwegian Fund for Research Fees for Agricultural Products (FFL)/ ; 314743//Norwegian Fund for Research Fees for Agricultural Products (FFL)/ ; 314743//Norwegian Fund for Research Fees for Agricultural Products (FFL)/ ; 314743//Norwegian Fund for Research Fees for Agricultural Products (FFL)/ ; 336136//The Research Council of Norway/ ; 336136//The Research Council of Norway/ ; 336136//The Research Council of Norway/ ; 336136//The Research Council of Norway/ ; 336136//The Research Council of Norway/ ; },
mesh = {*CRISPR-Cas Systems ; *Saccharomycetales/genetics/metabolism ; *Gene Editing/methods ; Plasmids/genetics ; Recombinant Proteins/genetics/biosynthesis ; Green Fluorescent Proteins/genetics/metabolism ; },
abstract = {BACKGROUND: The yeast Komagataella phaffii (formerly known as Pichia pastoris) has been widely used for functional expression of recombinant proteins, including plant and animal food proteins. CRISPR/Cas9 genome editing systems can be used for insertion of heterologous genes without the use of selection markers. The study aimed to create a convenient markerless knock-in method for integrating expression cassettes into the chromosome of K. phaffii using CRISPR/Cas9 technology. The approach was based on the hierarchical, modular, Golden Gate assembly employing the GoldenPiCS toolkit. Furthermore, the aim was to evaluate the system's efficiency and suitability for producing secreted recombinant food proteins.

RESULTS: Three Cas9/sgRNA plasmids were constructed, along with corresponding donor helper plasmids containing homology regions for chromosomal integration via homology-directed repair. The integration efficiency of an enhanced green fluorescent protein (eGFP) expression cassette was assessed at three genomic loci (04576, PFK1, and ROX1). The 04576 locus showed the highest integration efficiency, while ROX1 had the highest transformation efficiency. Whole genome sequencing revealed variable copy numbers of eGFP expression cassettes among clones, corresponding with increasing levels of fluorescence. Furthermore, the system's applicability for producing recombinant food proteins was validated by successfully expressing and secreting chicken ovalbumin. This constitutes the first report of CRISPR/Cas9 applied to produce recombinant chicken ovalbumin.

CONCLUSIONS: The adapted GoldenPiCS toolkit combined with CRISPR/Cas9 technology enabled efficient and precise genome integration in K. phaffii. This approach holds promise for expanding the production of high-value recombinant proteins. Future research should focus on optimizing integration sites and improving cloning procedures to enhance the system's efficiency and versatility.},
}

*CRISPR-Cas Systems
*Saccharomycetales/genetics/metabolism
*Gene Editing/methods
Plasmids/genetics
Recombinant Proteins/genetics/biosynthesis
Green Fluorescent Proteins/genetics/metabolism

@article {pmid40319033,
year = {2025},
author = {Cheng, B and Peng, SI and Jia, YY and Tong, E and Atwood, SX and Sun, BK},
title = {Comprehensive secretome profiling and CRISPR screen identifies SFRP1 as a key inhibitor of epidermal progenitor proliferation.},
journal = {Cell death & disease},
volume = {16},
number = {1},
pages = {360},
pmid = {40319033},
issn = {2041-4889},
support = {R03AR075844//U.S. Department of Health & Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/ ; K08AR067853//U.S. Department of Health & Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/ ; },
mesh = {Humans ; Cell Proliferation ; *Keratinocytes/metabolism/cytology ; *Membrane Proteins/metabolism/genetics ; *Stem Cells/metabolism/cytology ; *Intercellular Signaling Peptides and Proteins/metabolism/genetics ; *Secretome/metabolism ; *Epidermis/metabolism ; Wnt Signaling Pathway ; CRISPR-Cas Systems/genetics ; *Epidermal Cells/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Secreted proteins are crucial for the structure and functions of the human epidermis, but the full repertoire of the keratinocyte secretome has not been experimentally defined. In this study, we performed mass spectrometry on conditioned media from primary human keratinocytes, identifying 406 proteins with diverse roles in adhesion, migration, proliferation, proteolysis, signal transduction, and innate immunity. To leverage this new dataset, we developed a novel colony formation assay-based CRISPR screen to investigate the functions of uncharacterized secreted proteins on epidermal stem cells. The screen identified six candidate proteins that promoted proliferation of epidermal progenitors and two proteins that inhibited it. Secreted frizzled-related protein-1 (SFRP1) was the most potent inhibitor. We discovered that SFRP1 restrained clonogenic keratinocyte proliferation by inhibiting Wnt signaling as well as blocking ectopic expression of leukemia inhibitory factor (LIF). Collectively, our study expands our knowledge of the keratinocyte secretome, establishes a novel CRISPR screen to assess the function of non-cell autonomous factors, and highlights SFRP1's role in regulating epidermal balance.},
}

Humans
Cell Proliferation
*Keratinocytes/metabolism/cytology
*Membrane Proteins/metabolism/genetics
*Stem Cells/metabolism/cytology
*Intercellular Signaling Peptides and Proteins/metabolism/genetics
*Secretome/metabolism
*Epidermis/metabolism
Wnt Signaling Pathway
CRISPR-Cas Systems/genetics
*Epidermal Cells/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid40318042,
year = {2025},
author = {Kang, J and Park, C and Lee, G and Koo, J and Oh, H and Kim, EH and Bae, E and Suh, JY},
title = {Structural Investigation of the Anti-CRISPR Protein AcrIE7.},
journal = {Proteins},
volume = {},
number = {},
pages = {},
doi = {10.1002/prot.26832},
pmid = {40318042},
issn = {1097-0134},
support = {NRF-2022R1A2C1009804//National Research Foundation of Korea/ ; NRF-2021R1A2C1003917//National Research Foundation of Korea/ ; RS-2023-00207820//National Research Foundation of Korea/ ; A412550//Korea Basic Science Institute/ ; },
abstract = {The CRISPR-Cas system is an adaptive immune system in prokaryotes that provides protection against bacteriophages. As a countermeasure, bacteriophages have evolved various anti-CRISPR proteins that neutralize CRISPR-Cas immunity. Here, we report the structural and functional investigation of AcrIE7, which inhibits the type I-E CRISPR-Cas system in Pseudomonas aeruginosa. We determined both crystal and solution structures of AcrIE7, which revealed a novel helical fold. In binding assays using various biochemical methods, AcrIE7 did not tightly interact with a single Cas component in the type I-E Cascade complex or the CRISPR adaptation machinery. In contrast, AlphaFold modeling with our experimentally determined AcrIE7 structure predicted that AcrIE7 interacts with Cas3 in the type I-E CRISPR-Cas system in P. aeruginosa. Our findings are consistent with a model where AcrIE7 inhibits Cas3 and also highlight the effectiveness and limitations of AlphaFold modeling.},
}

@article {pmid40317571,
year = {2025},
author = {da Silva, GC and Rossi, CC},
title = {The Arms Race Between Actinobacillus pleuropneumoniae and Its Genetic Environment: A Comprehensive Analysis of Its Defensome and Mobile Genetic Elements.},
journal = {Molecular microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1111/mmi.15374},
pmid = {40317571},
issn = {1365-2958},
support = {APQ-03498-22//Fundação de Amparo à Pesquisa do Estado de Minas Gerais/ ; APQ-01339-25//Fundação de Amparo à Pesquisa do Estado de Minas Gerais/ ; 408564/2023-7//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; },
abstract = {Actinobacillus pleuropneumoniae is the causative agent of pleuropneumonia in swine, a highly contagious and economically significant disease. The genetic variability of A. pleuropneumoniae complicates disease control efforts, as it enables rapid adaptation to various stressors, including antimicrobial treatments. To better understand the molecular mechanisms underlying this adaptability, we investigated the role of the bacterial defensome and its relationship with mobile genetic elements (MGEs), such as prophages, plasmids, and integrative conjugative elements (ICEs). Using bioinformatic tools, we identified a diverse and rich defensome in A. pleuropneumoniae, with an average of 16 different defense systems per strain. We found that CRISPR-Cas systems, along with other defense mechanisms, are actively involved in restricting the entry of foreign genetic material, playing a crucial role in bacterial adaptation. Additionally, we characterized several novel prophages and examined their distribution across different strains, revealing their potential contribution to the bacterium's evolutionary success. Our findings underscore the complex interplay between the bacterium's defense systems and MGEs, shedding light on how A. pleuropneumoniae maintains genetic diversity while also safeguarding itself against external threats. These insights provide a better understanding of the genetic factors that influence the pathogen's adaptability and highlight potential avenues for more effective disease control strategies.},
}

@article {pmid40317300,
year = {2025},
author = {Rathore, RS and Jiang, W and Sedeek, K and Mahfouz, M},
title = {Harnessing neo-domestication of wild pigmented rice for enhanced nutrition and sustainable agriculture.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {138},
number = {5},
pages = {108},
pmid = {40317300},
issn = {1432-2242},
mesh = {*Oryza/genetics ; Gene Editing ; *Domestication ; *Plant Breeding ; *Agriculture ; Nutritive Value ; Genetic Variation ; CRISPR-Cas Systems ; Crops, Agricultural/genetics ; },
abstract = {Advances in precision gene editing have enabled the rapid domestication of wild crop relatives, a process known as neo-domestication. During domestication, breeding rice for maximum productivity under optimal growth conditions reduced genetic diversity, eliminating variants for stress tolerance and grain nutrients. Wild rice varieties have rich genetic diversity, including variants for disease resistance, stress tolerance, and grain nutritional quality. For example, the grain of pigmented wild rice has abundant antioxidants (anthocyanins, proanthocyanidins, and flavonoids), but low yield, poor plant architecture, and long life cycle limit its cultivation. In this review, we address the neo-domestication of wild pigmented rice, focusing on recent progress, CRISPR-Cas editing toolboxes, selection of key candidate genes for domestication, identifying species with superior potential via generating genomic and multi-omics resources, efficient crop transformation methods and highlight strategies for the promotion and application pigmented rice. We also address critical outstanding questions and potential solutions to enable efficient neo-domestication of wild pigmented rice and thus enhance food security and nutrition.},
}

*Oryza/genetics
Gene Editing
*Domestication
*Plant Breeding
*Agriculture
Nutritive Value
Genetic Variation
CRISPR-Cas Systems
Crops, Agricultural/genetics

@article {pmid40316660,
year = {2025},
author = {Chen, Y and Ren, J and Yang, X and Li, Z and Wu, W and Luo, G and Xu, Y and Yang, H and Wang, Q and Zhang, Q},
title = {The rapid detection of human HLA-B*27 gene based on BASIC isothermal detection.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {15427},
pmid = {40316660},
issn = {2045-2322},
support = {82472386//National Natural Science Foundation of China/ ; 23LCYJ038//Sichuan Health Commission/ ; 2023HT073//Industry-University-Research Innovation Fund for Chinese Universities/ ; S21077//Sichuan Medical Association/ ; 23JCYJPT0032, 20SXZRKX0010, 20SXQT0109 and 202001207//Nanchong Science and Technology Fund/ ; 2024CX001, 2023PTZK016 and ZX-51130001-2021-023//Research Fund of Affiliated Hospital of North Sichuan Medical College/ ; 22SXQT0307//Special Fund of the Strategic Science and Technology Cooperation between Nanchong Municipal Science and Technology Bureau and Universities/ ; 2024NSFSC1546//National Natural Science Foundation of Sichuan Province/ ; },
mesh = {Humans ; *HLA-B27 Antigen/genetics ; *Spondylitis, Ankylosing/genetics/diagnosis ; CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Genotype ; },
abstract = {Ankylosing spondylitis (AS) is a chronic, inflammatory arthritis of the spine and peripheral joints which is known to have a strong association with the human leukocyte antigen B27 (HLA-B27). Quantitative real-time PCR and flow cytometry are the predominant methods for HLA-B27 gene and antigen, respectively, which are too time-consuming and labor-intensive to realize rapid analysis. Therefore, a rapid diagnostic tool is highly required. In this study, we developed a rapid HLA-B*27 detection platform (namely BASIC) by combining our previously invented BASIS isothermal amplification method with the widely used CRISPR/Cas12a signal output tool. The BASIS can efficiently amplify all HLA-B*27 genotypes by using a set of universal primers, which target the conserved regions. The amplicons are subsequently applied to CRISPR/Cas12a analysis. The CRISPR/Cas12a recognizes the pathogenic HLA-B*27 amplicons specifically by using a well-designed gRNA, thereby achieving fluorescence signal output. Our results showed that the BASIC can be completed in 1 h with analytical sensitivity up to 100 aM. It could resist interference of homologous genes, hemoglobin, bilirubin, and triglyceride. For clinical sample detection, the BASIC offered completely consistent results with qPCR. Given the advantages of sensitivity, specificity, simplicity and rapidity, the BASIC was demonstrated a promising HLA-B*27 gene rapid detection tool for the early screening and diagnosis of AS.},
}

Humans
*HLA-B27 Antigen/genetics
*Spondylitis, Ankylosing/genetics/diagnosis
CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
*Molecular Diagnostic Techniques/methods
Sensitivity and Specificity
Genotype

@article {pmid40316524,
year = {2025},
author = {Berman, A and Su, N and Li, Z and Landau, U and Chakraborty, J and Gerbi, N and Liu, J and Qin, Y and Yuan, B and Wei, W and Yanai, O and Mayrose, I and Zhang, Y and Shani, E},
title = {Construction of multi-targeted CRISPR libraries in tomato to overcome functional redundancy at genome-scale level.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {4111},
pmid = {40316524},
issn = {2041-1723},
support = {757683//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 101118769//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Euratom (H2020 Euratom Research and Training Programme 2014-2018)/ ; 101113412//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Euratom (H2020 Euratom Research and Training Programme 2014-2018)/ ; 3419/20//Israel Science Foundation (ISF)/ ; 1462/24//Israel Science Foundation (ISF)/ ; },
mesh = {*Solanum lycopersicum/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Plant ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Library ; Plants, Genetically Modified ; Phenotype ; Plant Breeding ; Clustered Regularly Interspaced Short Palindromic Repeats ; Mutation ; },
abstract = {Genetic variance is vital for breeding programs and mutant screening, yet traditional mutagenesis methods wrestle with genetic redundancy and a lack of specificity in gene targeting. CRISPR-Cas9 offers precise, site-specific gene editing, but its application in crop improvement has been limited by scalability challenges. In this study, we develop genome-wide multi-targeted CRISPR libraries in tomato, enhancing the scalability of CRISPR gene editing in crops and addressing the challenges of redundancy while maintaining its precision. We design 15,804 unique single guide RNAs (sgRNAs), each targeting multiple genes within the same gene families. These sgRNAs are classified into 10 sub-libraries based on gene function. We generate approximately 1300 independent CRISPR lines and successfully identify mutants with distinct phenotypes related to fruit development, fruit flavor, nutrient uptake, and pathogen response. Additionally, we develop CRISPR-GuideMap, a double-barcode tagging system to enable large-scale sgRNA tracking in generated plants. Our results demonstrate that multi-targeted CRISPR libraries are scalable and effective for large-scale gene editing and offer an approach to overcome gene functional redundancy in basic plant research and crop breeding.},
}

*Solanum lycopersicum/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Genome, Plant
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Library
Plants, Genetically Modified
Phenotype
Plant Breeding
Clustered Regularly Interspaced Short Palindromic Repeats
Mutation

@article {pmid40316390,
year = {2025},
author = {Shixing, X and Shengjun, B and He, S and Xinyue, Z and Xingdong, Z and Xiaoying, Z and Leng, H and Enyong, D and Wan, J},
title = {A fluorescence biosensor for detecting LncRNA MALAT1 based on isothermal amplification by cyclic extension.},
journal = {Analytica chimica acta},
volume = {1357},
number = {},
pages = {344076},
doi = {10.1016/j.aca.2025.344076},
pmid = {40316390},
issn = {1873-4324},
mesh = {*RNA, Long Noncoding/analysis/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Fluorescence ; Limit of Detection ; DNA Probes/chemistry/genetics ; },
abstract = {BACKGROUND: Long non-coding RNA (lncRNA) Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1), a crucial regulator of gene expression, has emerged as a highly promising biomarker in the progression of various cancers. The clinical detection of lncRNA MALAT1 primarily relies on Reverse Transcription-Polymerase Chain Reaction (RT-PCR), which requires skilled operators and large, expensive thermal cycling equipment. These limitations have restricted the application of RT-PCR, particularly in resource-constrained settings.

RESULTS: In this study, we developed a novel signal amplification method, termed Isothermal Amplification by Cyclic Extension (IACE), based on the linear extension of a single-stranded DNA probe. IACE operates through the continuous extension of Probe 1 (a) into long single-stranded DNA with multiple repetitive sequences, facilitated by Probe 2 (a∗a∗) and Bst DNA polymerase. We found that the single-stranded DNA product of IACE could directly activate the CRISPR-Cas12a system without requiring a protospacer adjacent motif (PAM). By integrating IACE with a three-way junction structure and a nicking enzyme, we established a one-step signal amplification strategy for the detection of lncRNA MALAT1, achieving a detection limit as low as 37.5 fM using the CRISPR-Cas system.

SIGNIFICANCE: The biosensor developed in the present study simplifies workflows, minimizes contamination risks, and demonstrates exceptional detection performance in tumor patient samples, highlighting its potential to advance clinical tumor diagnostic approaches.},
}

*RNA, Long Noncoding/analysis/genetics
Humans
*Nucleic Acid Amplification Techniques/methods
*Biosensing Techniques/methods
Fluorescence
Limit of Detection
DNA Probes/chemistry/genetics

@article {pmid40316384,
year = {2025},
author = {Chang, Z and Zhou, J and Li, D and Wang, H and Chen, M and Liu, L and Ding, J and Wang, Y and Gao, Z and Sai, N},
title = {Selection and identification of the ssDNA aptamer against polymyxin B sulfate.},
journal = {Analytica chimica acta},
volume = {1357},
number = {},
pages = {344067},
doi = {10.1016/j.aca.2025.344067},
pmid = {40316384},
issn = {1873-4324},
mesh = {*Aptamers, Nucleotide/chemistry ; *DNA, Single-Stranded/chemistry ; SELEX Aptamer Technique ; *Polymyxin B/analysis ; Biosensing Techniques ; CRISPR-Cas Systems ; *Anti-Bacterial Agents/analysis ; Animals ; },
abstract = {BACKGROUND: Polymyxin B (PMB) is a potent antibiotic, and its sulfate form, Polymyxin B Sulfate (PMBS), is widely used. When PMBS is used as a veterinary drug, excessive use can lead to drug residues in animal bodies, which is a significant food safety issue and raises serious concerns. Therefore, there is an urgent need to strengthen research in this area. In this study, we obtained a ssDNA aptamer against PMBS using the Capture-SELEX method, which has notable advantages in small molecule selection. Then, we integrated the obtained aptamer with a biosensor based on the CRISPR/Cas14a system for a series of validations.

RESULTS: In this study, specific aptamers against PMBS were discovered via the Capture-SELEX process. The selection process consisted of ten rounds. The affinity of the candidate sequences was determined by Isothermal Titration Calorimetry (ITC), and the PMBS24 aptamer with the highest affinity was ultimately identified, with a dissociation constant (Kd) of 3.89 ± 0.46 μM. To further obtain high-affinity aptamers, we attempted to truncate the PMBS24 aptamer. However, the results demonstrated that the aptamer with the full-length sequence exhibited superior affinity. Subsequently, it was applied in the CRISPR/Cas14a-based sensor for the detection of PMBS. This method had a LOD of 0.99 ng mL[-1], with good specificity, and was successfully used for the detection in milk.

SIGNIFICANCE: An important recognition element of PMBS has been successfully obtained, filling a part of the gap in the detection of PMBS. Moreover, the aptamer has been applied to the CRISPR/Cas14a-based sensor for amplification-free PMBS detection, yielding favorable detection results. This also opens up the possibility of applying the aptamer to other similar sensors.},
}

*Aptamers, Nucleotide/chemistry
*DNA, Single-Stranded/chemistry
SELEX Aptamer Technique
*Polymyxin B/analysis
Biosensing Techniques
CRISPR-Cas Systems
*Anti-Bacterial Agents/analysis
Animals