@article {pmid40179700,
year = {2025},
author = {Shen, D and Guo, H and Zhang, F and Chen, X and Tong, X and Li, H and Wu, W and Mei, S},
title = {Highly-sensitive and logic platform based on spatially-constrained T7 transcription enhanced Cas13a for DNA repair enzyme detection and intracellular imaging.},
journal = {Biosensors & bioelectronics},
volume = {280},
number = {},
pages = {117406},
doi = {10.1016/j.bios.2025.117406},
pmid = {40179700},
issn = {1873-4235},
abstract = {The activity of DNA repair enzymes, particularly Flap endonuclease 1 (FEN1) and apurinic/apyrimidinic endonuclease 1 (APE1), plays a critical role in disease prevention, diagnosis, and prognosis. Accurate detection of these enzymes is therefore essential. Recent advancements in CRISPR-Cas technology, particularly its programmable and trans-cleavage activity, have paved the way for the development of innovative detection methods. However, there is a need for a simple, low-background, highly sensitive detection platform with logical capabilities for FEN1 and APE1. In this study, we present a novel detection platform that integrates spatially constrained T7 transcription with the CRISPR-Cas13a system. This biosensor minimizes background interference and achieves high sensitivity, with limits of detection as low as 5 × 10[-7] U/μL for FEN1 and 2 × 10[-8] U/μL for APE1, making it one of the most sensitive methods available for detecting these enzymes. The platform supports both OR and logic detection, offering enhanced versatility. It demonstrates robustness by detecting FEN1 activity at concentrations as low as 1 cell/μL and screening enzyme inhibitors. Additionally, the system was successfully used for intracellular imaging of FEN1 activity in cells and reliably measured APE1 activity in ovarian tissue samples, confirming its clinical applicability. This biosensor represents a promising tool for detecting FEN1 and APE1, further expanding the potential of CRISPR-Cas13a in diagnostic applications.},
}

@article {pmid40163950,
year = {2025},
author = {Fang, L and Yang, X and Li, Y and Xue, C and Li, Z and Jiang, H and Li, X and Lu, S and Wang, D and He, H and Huang, Z and Guo, X and Luo, G},
title = {SPECIAL: Phosphorothioate dNTP assisted RPA equipped with CRISPR/Cas12a amplifier enables high-specific nucleic acid testing.},
journal = {Biosensors & bioelectronics},
volume = {279},
number = {},
pages = {117421},
doi = {10.1016/j.bios.2025.117421},
pmid = {40163950},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Humans ; *Recombinases/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Recombinase polymerase amplification (RPA) is one of the most widely used isothermal amplification methods and considered to be a promising tool for point-of-care testing (POCT) molecular diagnosis. However, RPA is prone to have nonspecific amplification occur, due to the poor recognition accuracy of polymerase and recombinase, which severely hindered its clinical application. It is important to improve the specificity of RPA further. Herein, we developed a novel nucleic acid testing method termed phosphorothioate dNTP (dNTPαS) assisted RPA (S-RPA) that employs dNTPαS as substrates to suppress nonspecific amplification effectively. We found that dNTPαS could improve the recognition accuracy of Bsu polymerase and recombinase, thereby enhancing their amplification specificity. Our S-RPA provided much higher specificity (approximately 40 % improvement compared to classical RPA), realizing detection target with single nucleotide mutation. Based on its outstanding performance, we further combined the S-RPA with CRISPR/Cas12a to achieve highly specific and sensitive fluorescence detection, namely S-RPA equipped with CRISPR/Cas12a amplifier (SPECIAL). Our SPECIAL was more sensitive (10-fold higher) than the classical RPA-CRISPR/Cas12a assay, offering 100 % agreement with the qPCR during clinical validation. In summary, a strategy based on dNTPαS was established to enhance the specificity of RPA, thereby improving its practicability and providing a potential POCT tool for molecular diagnosis.},
}

*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
*Biosensing Techniques/methods
Humans
*Recombinases/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40179192,
year = {2025},
author = {Martin-Rufino, JD and Caulier, A and Lee, S and Castano, N and King, E and Joubran, S and Jones, M and Goldman, SR and Arora, UP and Wahlster, L and Lander, ES and Sankaran, VG},
title = {Transcription factor networks disproportionately enrich for heritability of blood cell phenotypes.},
journal = {Science (New York, N.Y.)},
volume = {388},
number = {6742},
pages = {52-59},
doi = {10.1126/science.ads7951},
pmid = {40179192},
issn = {1095-9203},
mesh = {Humans ; *Transcription Factors/genetics/metabolism ; *Gene Regulatory Networks ; *Chromatin/metabolism/genetics ; Phenotype ; Single-Cell Analysis ; CRISPR-Cas Systems ; Genome, Human ; Genetic Variation ; *Blood Cells ; Cell Differentiation/genetics ; *Erythropoiesis/genetics ; Erythroid Cells/cytology ; },
abstract = {Most phenotype-associated genetic variants map to noncoding regulatory regions of the human genome, but their mechanisms remain elusive in most cases. We developed a highly efficient strategy, Perturb-multiome, to simultaneously profile chromatin accessibility and gene expression in single cells with CRISPR-mediated perturbation of master transcription factors (TFs). We examined the connection between TFs, accessible regions, and gene expression across the genome throughout hematopoietic differentiation. We discovered that variants within TF-sensitive accessible chromatin regions in erythroid differentiation, although representing <0.3% of the genome, show a ~100-fold enrichment for blood cell phenotype heritability, which is substantially higher than that for other accessible chromatin regions. Our approach facilitates large-scale mechanistic understanding of phenotype-associated genetic variants by connecting key cis-regulatory elements and their target genes within gene regulatory networks.},
}

Humans
*Transcription Factors/genetics/metabolism
*Gene Regulatory Networks
*Chromatin/metabolism/genetics
Phenotype
Single-Cell Analysis
CRISPR-Cas Systems
Genome, Human
Genetic Variation
*Blood Cells
Cell Differentiation/genetics
*Erythropoiesis/genetics
Erythroid Cells/cytology

@article {pmid40178896,
year = {2025},
author = {Kim, DY and Lee, SY and Ha, HJ and Park, HH},
title = {AcrIE7 inhibits the CRISPR-Cas system by directly binding to the R-loop single-stranded DNA.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {14},
pages = {e2423205122},
doi = {10.1073/pnas.2423205122},
pmid = {40178896},
issn = {1091-6490},
support = {2021R1A2C3003331//National Research Foundation of Korea (NRF)/ ; },
mesh = {*DNA, Single-Stranded/metabolism/chemistry/genetics ; *CRISPR-Cas Systems/genetics ; Protein Binding ; *Bacterial Proteins/metabolism/chemistry/genetics ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; },
abstract = {The CRISPR-Cas system is a well-known adaptive immune system in bacteria, and a prominent mechanism for evading this immunity involves anti-CRISPR (Acr) proteins, which employ various methods to neutralize the CRISPR-Cas system. In this study, using structural and biochemical analyses, we revealed that AcrIE7 binds to the single-stranded DNA in the R-loop formed when Cascade encounters the target DNA, thereby preventing Cas3 from cleaving the DNA. This represents a different inhibition strategy distinct from previously reported Acr mechanisms and offers insights into CRISPR-Cas inhibition.},
}

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

@article {pmid40176181,
year = {2025},
author = {Yamamoto, A and Tanaka, Y and Ishibashi, S and Ikeda, M and Sugita, K and Ono, M and Nishi, H and Kurata, M},
title = {Modified screening of MYC promotor region elements using the CRISPR library in ovarian cancer.},
journal = {Journal of ovarian research},
volume = {18},
number = {1},
pages = {68},
pmid = {40176181},
issn = {1757-2215},
support = {21K16800//KAKENHI/ ; },
mesh = {Humans ; Female ; *Ovarian Neoplasms/genetics/pathology ; *Promoter Regions, Genetic ; Proto-Oncogene Mas ; Cell Line, Tumor ; *Proto-Oncogene Proteins c-myc/genetics ; Gene Expression Regulation, Neoplastic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Gene Library ; },
abstract = {Ovarian cancer remains one of the most lethal gynecological malignancies owing to its high recurrence rate and chemotherapeutic resistance. MYC is a well-known proto-oncogene that is frequently amplified in ovarian cancer and has been implicated in drug resistance. Previously, we established a new promoter-reporter system combined with a CRISPR activation library to identify unknown MYC regulators, and M1AP was identified as a novel MYC regulator. However, considering the insufficient explanation for the absence of guide RNA (gRNA) of MYC, this present study explored methods to prevent the gRNA of MYC itself from binding. This study first modified the promoter-reporter vector to improve its quality, then conducted CRISPR screening and analyzed candidate genes as MYC promoter regulators using next-generation sequencing in OVSAHO ovarian cancer cells. Eighty-six genes had ≥ 1000 reads, and Pearson's correlation coefficient analysis was performed on the cBioPortal of the Cancer Genomics database. Fourteen genes were identified as candidate MYC regulators with positive and significant correlations with MYC. Seven genes, including CYP4v2, ASPH, ANP32D, PCED1A, ABI1, FUZ, and HOOK2, demonstrated significantly higher luciferase activity than the control genes. Four genes, including ABI1, PCED1A, HOOK2, and CYP4v2, activated the MYC promoter, which showed over twofold higher activity than the control when overexpressed using a vector. In conclusion, four genes that activate MYC promoters were identified in an ovarian cancer cell line using the CRISPR library system with a modified promoter-reporter tool. These results will prove helpful in the development of novel treatment strategies for ovarian cancer.},
}

Humans
Female
*Ovarian Neoplasms/genetics/pathology
*Promoter Regions, Genetic
Proto-Oncogene Mas
Cell Line, Tumor
*Proto-Oncogene Proteins c-myc/genetics
Gene Expression Regulation, Neoplastic
*Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR-Cas Systems
Gene Library

@article {pmid40176174,
year = {2025},
author = {Okesanya, OJ and Ahmed, MM and Ogaya, JB and Amisu, BO and Ukoaka, BM and Adigun, OA and Manirambona, E and Adebusuyi, O and Othman, ZK and Oluwakemi, OG and Ayando, OD and Tan, MIRS and Idris, NB and Kayode, HH and Oso, TA and Ahmed, M and Kouwenhoven, MBN and Ibrahim, AM and Lucero-Prisno, DE},
title = {Reinvigorating AMR resilience: leveraging CRISPR-Cas technology potentials to combat the 2024 WHO bacterial priority pathogens for enhanced global health security-a systematic review.},
journal = {Tropical medicine and health},
volume = {53},
number = {1},
pages = {43},
pmid = {40176174},
issn = {1348-8945},
abstract = {BACKGROUND: Antimicrobial resistance (AMR) poses a global health threat, particularly in low- and middle-income countries (LMICs). Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system technology offers a promising tool to combat AMR by targeting and disabling resistance genes in WHO bacterial priority pathogens. Thus, we systematically reviewed the potential of CRISPR-Cas technology to address AMR.

METHODS: This systematic review adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A comprehensive literature search was conducted using the Scopus and PubMed databases, focusing on publications from 2014 to June 2024. Keywords included "CRISPR/Cas," "antimicrobial resistance," and "pathogen." The eligibility criteria required original studies involving CRISPR/Cas systems that targeted AMR. Data were extracted from eligible studies, qualitatively synthesized, and assessed for bias using the Joanna Briggs Institute (JBI)-standardized tool.

RESULTS: Data from 48 eligible studies revealed diverse CRISPR-Cas systems, including CRISPR-Cas9, CRISPR-Cas12a, and CRISPR-Cas3, targeting various AMR genes, such as blaOXA-232, blaNDM, blaCTX-M, ermB, vanA, mecA, fosA3, blaKPC, and mcr-1, which are responsible for carbapenem, cephalosporin, methicillin, macrolide, vancomycin, colistin, and fosfomycin resistance. Some studies have explored the role of CRISPR in virulence gene suppression, including enterotoxin genes, tsst1, and iutA in Staphylococcus aureus and Klebsiella pneumoniae. Delivery mechanisms include bacteriophages, nanoparticles, electro-transformation, and conjugative plasmids, which demonstrate high efficiency in vitro and in vivo. CRISPR-based diagnostic applications have demonstrated high sensitivity and specificity, with detection limits as low as 2.7 × 10[2] CFU/mL, significantly outperforming conventional methods. Experimental studies have reported significant reductions in resistant bacterial populations and complete suppression of the targeted strains. Engineered phagemid particles and plasmid-curing systems have been shown to eliminate IncF plasmids, cured plasmids carrying vanA, mcr-1, and blaNDM with 94% efficiency, and restore antibiotic susceptibility. Gene re-sensitization strategies have been used to restore fosfomycin susceptibility in E. coli and eliminate blaKPC-2-mediated carbapenem resistance in MDR bacteria. Whole-genome sequencing and bioinformatics tools have provided deeper insights into CRISPR-mediated defense mechanisms. Optimization strategies have significantly enhanced gene-editing efficiencies, offering a promising approach for tackling AMR in high-priority WHO pathogens.

CONCLUSIONS: CRISPR-Cas technology has the potential to address AMR across priority WHO pathogens. While promising, challenges in optimizing in vivo delivery, mitigating potential resistance, and navigating ethical-regulatory barriers must be addressed to facilitate clinical translation.},
}

@article {pmid40175930,
year = {2025},
author = {Xiao, G and Liu, H and Xu, H and Shi, H and Liu, D and Ou, M and Liu, P and Zhang, G},
title = {Direct detection from sputum for drug-resistant Mycobacterium tuberculosis using a CRISPR-Cas14a-based approach.},
journal = {BMC microbiology},
volume = {25},
number = {1},
pages = {188},
pmid = {40175930},
issn = {1471-2180},
support = {82300128//National Natural Science Foundation of China/ ; 82170009//National Natural Science Foundation of China/ ; 2020YFA0907200//National Key Research and Development Program of China/ ; KCXFZ20211020163545004//Shenzhen Scientific and Technological Foundation/ ; JCYJ20210324130009024//Shenzhen Scientific and Technological Foundation/ ; JCYJ20220530163216036//Shenzhen Scientific and Technological Foundation/ ; RCJC20221008092726022//Shenzhen Scientific and Technological Foundation/ ; 0620220214//Science Fund for Distinguished Young Scholars of Guangdong Province/ ; 2020B1111170014//Science and Technology Planning Project of Guangdong Province/ ; SZZYSM202311009//Sanming Project of Medicine in Shenzen Municipality/ ; },
mesh = {*Sputum/microbiology ; *Mycobacterium tuberculosis/genetics/drug effects/isolation & purification ; Humans ; *CRISPR-Cas Systems ; *Tuberculosis, Multidrug-Resistant/microbiology/diagnosis ; Rifampin/pharmacology ; Microbial Sensitivity Tests ; Sensitivity and Specificity ; Antitubercular Agents/pharmacology ; Isoniazid/pharmacology ; Drug Resistance, Multiple, Bacterial/genetics ; Mutation ; },
abstract = {The increasing prevalence of multidrug-resistant tuberculosis (MDR-TB) highlights the urgent need for an efficient approach to identify Mycobacterium tuberculosis complex (MTBC) strains resistant to rifampicin (RIF) and isoniazid (INH). In response, we developed a CRISPR-Cas14a MTB RIF/INH platform that can detect the most common mutations associated with RIF and INH resistance. To evaluate the sensitivity and specificity of our CRISPR-Cas14a MTB RIF/INH platform, we carried out a comprehensive assessment using clinical isolates of M. tuberculosis and sputum samples from TB patients, making direct comparisons with phenotypic drug susceptibility testing (pDST). A total of 60 clinical isolates from TB patients were utilized, consisting of 18 RIF mono-resistant, 15 INH mono-resistant, 24 MDR isolates, and 3 fully susceptible isolates. Among the 42 RIF-resistant isolates, our platform accurately identified 39, achieving a sensitivity of 93.3% (95% CI, 80.0-98.5) and a specificity of 100% (95% CI, 81.6-100). Similarly, out of the 39 INH-resistant isolates, the platform successfully identified 38, demonstrating a sensitivity of 97.5% (95% CI, 86.5-99.9) and a specificity of 100% (95% CI, 83.8-100) when compared with pDST. Moreover, in the analysis of 55 sputum samples, our platform accurately identified RIF resistance in 10 out of 12 samples (85.7%) and INH resistance in all 11 samples (100%). Notably, excluding the nucleic acid extraction step, the entire testing procedure can be completed in approximately 1.5 h. These results suggest that the CRISPR-Cas14a MTB RIF/INH platform is a reliable and promising novel tool for detecting RIF and INH resistance in isolates or directly from sputum samples.},
}

*Sputum/microbiology
*Mycobacterium tuberculosis/genetics/drug effects/isolation & purification
Humans
*CRISPR-Cas Systems
*Tuberculosis, Multidrug-Resistant/microbiology/diagnosis
Rifampin/pharmacology
Microbial Sensitivity Tests
Sensitivity and Specificity
Antitubercular Agents/pharmacology
Isoniazid/pharmacology
Drug Resistance, Multiple, Bacterial/genetics
Mutation

@article {pmid40175837,
year = {2025},
author = {Moreno, DS and Cunha, J and de Melo, LDR and Tanaka, K and Bamba, T and Hasunuma, T and Azeredo, J and Domingues, L},
title = {CRISPR-Cas9 engineered Saccharomyces cerevisiae for endolysin delivery to combat Listeria monocytogenes.},
journal = {Applied microbiology and biotechnology},
volume = {109},
number = {1},
pages = {81},
pmid = {40175837},
issn = {1432-0614},
support = {UIDB/04469/2020//Fundação para a Ciência e a Tecnologia/ ; UI/BD/151411/2021//Fundação para a Ciência e a Tecnologia/ ; },
mesh = {*Listeria monocytogenes/drug effects ; *Saccharomyces cerevisiae/genetics/metabolism ; *Endopeptidases/genetics/metabolism/pharmacology ; *CRISPR-Cas Systems ; *Anti-Bacterial Agents/pharmacology/metabolism ; Bacteriophages/enzymology/genetics ; Listeriosis/prevention & control/microbiology ; },
abstract = {Listeriosis is an infection caused by the consumption of food contaminated with Listeria monocytogenes. It leads to febrile gastroenteritis, central nervous system infections, and even death in risk populations. Bacteriophage endolysins selectively kill bacteria hydrolyzing their cell walls and have emerged as a potential tool for listeriosis control. Ply511 is an anti-Listeria endolysin that has activity against all serovars of L. monocytogenes. The yeast Saccharomyces cerevisiae has been used to produce endolysins for biocontrol, but prior efforts relied on plasmids, which can lead to gene loss and include selection markers unsuitable for therapeutic use. Integration of endolysins in its genome has also been previously demonstrated, relying however, on selection markers for selection and maintenance of the modifications. This study explores S. cerevisiae as a generally regarded as safe (GRAS) platform for producing and displaying Ply511 through CRISPR-Cas9 integration, offering a marker-free and stable solution for Listeria biocontrol. Our results demonstrate that the surface display of Ply511 does not lead to bacterial reduction. In contrast, we show that yeast secreting endolysin significantly reduces L. monocytogenes in cells, supernatants, and cell extracts. The strongest effect was observed with concentrated spent supernatant and cell extract, which reduced L. monocytogenes below the lower limit of quantification. Additionally, the spent supernatant exhibited active anti-Listeria activity in milk. This study highlights yeast-secreted endolysins as a promising platform for listeriosis control and demonstrates the yeast secretion of endolysins can be used for the biocontrol of pathogenic bacteria. KEY POINTS: • S. cerevisiae was edited using CRISPR-Cas9 to display or secrete endolysin Ply511. • Cells, supernatants, and extracts of yeast secreting Ply511 act against L. monocytogenes. • Demonstrates the yeast-based delivery of endolysins to control L. monocytogenes.},
}

*Listeria monocytogenes/drug effects
*Saccharomyces cerevisiae/genetics/metabolism
*Endopeptidases/genetics/metabolism/pharmacology
*CRISPR-Cas Systems
*Anti-Bacterial Agents/pharmacology/metabolism
Bacteriophages/enzymology/genetics
Listeriosis/prevention & control/microbiology

@article {pmid40175136,
year = {2025},
author = {Adachi, Y},
title = {[Identification of genes regulating human CAR-T cell proliferation by genome-wide CRISPR screening].},
journal = {[Rinsho ketsueki] The Japanese journal of clinical hematology},
volume = {66},
number = {3},
pages = {145-152},
doi = {10.11406/rinketsu.66.145},
pmid = {40175136},
issn = {0485-1439},
mesh = {Humans ; Cell Proliferation ; *T-Lymphocytes/immunology/cytology ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Receptors, Chimeric Antigen/immunology ; Immunotherapy, Adoptive ; },
abstract = {In vivo expansion and long-term maintenance of CAR-T cells are considered to be the hallmark of treatment success after CD19 CAR-T cell therapy. Genome-wide CRISPR screening has emerged as a powerful tool for large-scale gene screens. Genome-wide CRISPR screening revealed that CUL5 gene knockout (KO) improved the proliferation of CD19 CAR-T cells. CUL5KO improved not only the proliferation but also the effector function of CAR-T cells. The JAK-STAT pathway was upregulated in CUL5KO CAR-T cells, and CUL5 was associated with the degradation of JAK3 upon activation through IL-2 signaling. CUL5KO CD19 CAR-T cells efficiently suppressed in vivo tumor progression as compared to control CD19 CAR-T cells.},
}

Humans
Cell Proliferation
*T-Lymphocytes/immunology/cytology
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
*Receptors, Chimeric Antigen/immunology
Immunotherapy, Adoptive

@article {pmid40172964,
year = {2025},
author = {Patel, AC and Sinha, S and Arantes, PR and Palermo, G},
title = {Unveiling Cas8 dynamics and regulation within a transposon-encoded Cascade-TniQ complex.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {14},
pages = {e2422895122},
doi = {10.1073/pnas.2422895122},
pmid = {40172964},
issn = {1091-6490},
support = {R01GM141329//HHS | National Institutes of Health (NIH)/ ; CHE-2144823//National Science Foundation (NSF)/ ; FG-2023-20431//Alfred P. Sloan Foundation (APSF)/ ; TC-24-063//Camille and Henry Dreyfus Foundation (Dreyfus Foundation)/ ; },
mesh = {*Vibrio cholerae/genetics/metabolism ; *DNA Transposable Elements/genetics ; *Bacterial Proteins/metabolism/genetics/chemistry ; CRISPR-Associated Proteins/metabolism/chemistry/genetics ; CRISPR-Cas Systems ; Protein Binding ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Protein Conformation ; Models, Molecular ; },
abstract = {The Vibrio cholerae Cascade-TniQ complex unveiled a new paradigm in biology, demonstrating that CRISPR-associated proteins can direct DNA transposition. Despite the tremendous potential of "knocking-in" genes at desired sites, the mechanisms underlying DNA binding and transposition remain elusive. In this system, a conformational change of the Cas8 protein is essential for DNA binding, yet how it occurs is unclear. Here, structural modeling and free energy simulations reconstruct the Cas8 helical bundle and reveal an open-closed conformational change that is key for the complex's function. We show that when Cascade-TniQ binds RNA, the Cas8 bundle changes conformation mediated by the interaction with the Cas7.1 protein. This interaction promotes the bundle's transition toward the open state, priming the complex for DNA binding. As the target DNA binds the guide RNA, the opening of the Cas8 bundle becomes more favorable, exposing positively charged residues and facilitating their interaction with DNA, which ultimately leads the DNA-binding process to completion. These outcomes provide a dynamic representation of a critical conformational change in one of the largest CRISPR systems and illustrate its role at critical steps of the Cascade-TniQ biophysical function, advancing our understanding of nucleic acid binding and transposition mechanisms.},
}

*Vibrio cholerae/genetics/metabolism
*DNA Transposable Elements/genetics
*Bacterial Proteins/metabolism/genetics/chemistry
CRISPR-Associated Proteins/metabolism/chemistry/genetics
CRISPR-Cas Systems
Protein Binding
RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
Protein Conformation
Models, Molecular

@article {pmid39970912,
year = {2025},
author = {Zilberzwige-Tal, S and Altae-Tran, H and Kannan, S and Wilkinson, ME and Vo, SC and Strebinger, D and Edmonds, KK and Yao, CJ and Mears, KS and Shmakov, SA and Makarova, KS and Macrae, RK and Koonin, EV and Zhang, F},
title = {Reprogrammable RNA-targeting CRISPR systems evolved from RNA toxin-antitoxins.},
journal = {Cell},
volume = {188},
number = {7},
pages = {1925-1940.e20},
doi = {10.1016/j.cell.2025.01.034},
pmid = {39970912},
issn = {1097-4172},
mesh = {*CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; *Toxin-Antitoxin Systems ; Cryoelectron Microscopy ; CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Evolution, Molecular ; Bacterial Proteins/metabolism/genetics/chemistry ; *Antitoxins/metabolism/chemistry/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Untranslated/metabolism/genetics ; },
abstract = {Despite ongoing efforts to study CRISPR systems, the evolutionary origins giving rise to reprogrammable RNA-guided mechanisms remain poorly understood. Here, we describe an integrated sequence/structure evolutionary tracing approach to identify the ancestors of the RNA-targeting CRISPR-Cas13 system. We find that Cas13 likely evolved from AbiF, which is encoded by an abortive infection-linked gene that is stably associated with a conserved non-coding RNA (ncRNA). We further characterize a miniature Cas13, classified here as Cas13e, which serves as an evolutionary intermediate between AbiF and other known Cas13s. Despite this relationship, we show that their functions substantially differ. Whereas Cas13e is an RNA-guided RNA-targeting system, AbiF is a toxin-antitoxin (TA) system with an RNA antitoxin. We solve the structure of AbiF using cryoelectron microscopy (cryo-EM), revealing basic structural alterations that set Cas13s apart from AbiF. Finally, we map the key structural changes that enabled a non-guided TA system to evolve into an RNA-guided CRISPR system.},
}

*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/metabolism/genetics
*Toxin-Antitoxin Systems
Cryoelectron Microscopy
CRISPR-Associated Proteins/metabolism/genetics/chemistry
Evolution, Molecular
Bacterial Proteins/metabolism/genetics/chemistry
*Antitoxins/metabolism/chemistry/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
RNA, Untranslated/metabolism/genetics

@article {pmid40171918,
year = {2025},
author = {Wu, W and Miao, R and Li, Z and Fang, Z},
title = {CRISPR/Cas9-mediated editing of BADH2 and Wx genes for the development of novel aromatic and soft-textured black and red rice.},
journal = {Physiologia plantarum},
volume = {177},
number = {2},
pages = {e70194},
doi = {10.1111/ppl.70194},
pmid = {40171918},
issn = {1399-3054},
support = {Qiankehezhongyindi (2023) 008//the Key Laboratory of Molecular Breeding for Grain and Oil Crops in Guizhou Province/ ; qiankehechengguo (2024) General 116//the Guizhou Provincial Science and Technology Projects/ ; qiankehejichu-ZK (2021) General 128//the Guizhou Provincial Science and Technology Projects/ ; qiankehejichu-ZK (2022) Key 008//the Guizhou Provincial Science and Technology Projects/ ; qiankehepingtai-YWZ (2024) 004//the Guizhou Provincial Science and Technology Projects/ ; qiankehepingtairencai-BQW (2024) 001//the Guizhou Provincial Science and Technology Projects/ ; Qiandongnan Kehe Support (2023)06//the Qiandongnan Science and Technology Support Project/ ; Qianjiaoji (2023)007//the Key Laboratory of Functional Agriculture of Guizhou Provincial Department of Education/ ; qiankehepingtairencai-YQK (2023) 002//the Guizhou Provincial Excellent Young Talents Project of Science and Technology/ ; 32260498/32060064//the National Natural Science Foundation of China/ ; },
mesh = {*Oryza/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Amylose/metabolism/genetics ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified/genetics ; Odorants/analysis ; Gas Chromatography-Mass Spectrometry ; Pyrroles ; },
abstract = {Black and red rice are known for their rich nutritional content, yet most varieties suffer from a firm texture and insufficient fragrance. In this study, we aimed to develop a fragrant and soft-textured black and red rice variety using the CRISPR/Cas9 technology to knock out the OsWx gene, which is associated with amylose content (AC), and the OsBADH2 gene, responsible for rice aroma. Our results showed that, compared to wild-type, CRISPR lines of XHZ, HM, NWZ, and PGZ targeting OsWx and OsBADH2 exhibited a reduction in AC content, altered gel consistency, and a more than 50% increase in gel consistency. Headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) analysis revealed that the 2-acetyl-1-pyrroline (2-AP) content in the grains of xhz-c[BADH2 Wx] and hm-c[BADH2 Wx] reached 189.04 μg kg[-1] and 309.03 μg kg[-1], respectively. Furthermore, we observed a slight increase in anthocyanins and proanthocyanidins in these co-edited lines, without significant effects on their agronomic traits. Furthermore, to investigate the genes involved in the quality formation of black and red rice for the knockout of OsBADH2 and OsWx, we conducted RNA-seq analysis. The results indicated that knockout of OsBADH2 and OsWx affected the expression of genes involved in carotenoid biosynthesis, multiple amino acid metabolism genes, and endosperm starch and sucrose metabolic pathways. These findings suggest that the CRISPR/Cas9 technology can effectively target OsBADH2 and OsWx to develop high-quality black and red rice varieties with enhanced aroma and softer texture, providing a new strategy for the improvement of colored rice.},
}

*Oryza/genetics/metabolism
*CRISPR-Cas Systems
*Gene Editing/methods
*Amylose/metabolism/genetics
*Plant Proteins/genetics/metabolism
Plants, Genetically Modified/genetics
Odorants/analysis
Gas Chromatography-Mass Spectrometry
Pyrroles

@article {pmid40127118,
year = {2025},
author = {Dhinoja, S and Qaryoute, AA and Deebani, A and De Maria, A and Jagadeeswaran, P},
title = {CRISPR/Cas9 mediated generation of zebrafish f9a mutant as a model for hemophilia B.},
journal = {Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis},
volume = {36},
number = {3},
pages = {90-98},
doi = {10.1097/MBC.0000000000001355},
pmid = {40127118},
issn = {1473-5733},
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; *Hemophilia B/genetics ; *Factor IX/genetics/metabolism ; Disease Models, Animal ; Mutation ; Gene Knockout Techniques ; Humans ; },
abstract = {AIM: This study aimed to develop a zebrafish model for hemophilia B by creating a f9a knockout, as f9a has previously demonstrated functional similarity to human Factor IX.

METHODS: Using CRISPR/Cas9 technology, two gRNAs targeting exon 8 of the f9a gene, were injected along with Cas9 protein into single-cell zebrafish wild-type embryos. DNA was harvested from the tail tips of the resulting adult zebrafish and screened for mutations using PCR. The founder mutant was crossed with wild-type fish to confirm heritability and subsequently reared to homozygosity. Homozygous mutants were analyzed through quantitative RT-PCR and Western blot to assess f9a RNA and F9a protein levels, respectively. Functional assays like kinetic partial thromboplastin time (kPTT), bleeding assay in adult mutants, and venous laser injury on mutant larvae were performed to assess the hemostatic role.

RESULTS: Around 61 adults from the CRISPR/Cas9 knockouts were screened, which resulted in a mutant line with a 72 bp deletion in the exon 8 encoding catalytic domain. Quantitative RT-PCR and Western Blot analysis showed reduced levels of f9a RNA and F9a protein in the homozygous mutants compared to wild-type siblings. At five dpf, f9a homozygous mutant larvae demonstrated prolonged venous occlusion times in a laser injury assay. Additionally, plasma from the mutants displayed delayed fibrin formation in kPTT assays and exhibited increased bleeding after mechanical injury.

CONCLUSION: This study created a zebrafish f9a knockout model that mimics the bleeding phenotype observed in hemophilia B patients, which will be valuable for evaluating novel therapeutic approaches for hemophilia B.},
}

Animals
*Zebrafish/genetics
*CRISPR-Cas Systems
*Hemophilia B/genetics
*Factor IX/genetics/metabolism
Disease Models, Animal
Mutation
Gene Knockout Techniques
Humans

@article {pmid40110772,
year = {2025},
author = {Yang, W and Peng, M and Wang, Y and Zhang, X and Li, W and Zhai, X and Wu, Z and Hu, P and Chen, L},
title = {Deletion of hepcidin disrupts iron homeostasis and hematopoiesis in zebrafish embryogenesis.},
journal = {Development (Cambridge, England)},
volume = {152},
number = {7},
pages = {},
doi = {10.1242/dev.204307},
pmid = {40110772},
issn = {1477-9129},
support = {32200414//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Zebrafish/embryology/genetics/metabolism ; *Hepcidins/genetics/metabolism ; *Hematopoiesis/genetics ; *Iron/metabolism ; *Homeostasis/genetics ; *Embryonic Development/genetics ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Developmental ; Ferroptosis/genetics ; Gene Deletion ; Embryo, Nonmammalian/metabolism ; Gene Knockout Techniques ; GATA1 Transcription Factor/metabolism/genetics ; },
abstract = {Iron is essential for cell growth and hematopoiesis, which is regulated by hepcidin (hamp). However, the role of hamp in zebrafish hematopoiesis remains unclear. Here, we have created a stable hamp knockout zebrafish model using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 system (CRISPR/Cas9 system). Our study revealed that hamp deletion led to maternal iron overload in embryos, significantly downregulating hemoglobin genes and reducing hemoglobin content. Single-cell RNA sequencing identified abnormal expression patterns in blood progenitor cells, with a specific progenitor subtype showing increased ferroptosis and delayed development. By crossing hamp knockout zebrafish with a gata1+ line (blood cells labeled fish line), we confirmed ferroptosis in blood progenitor cells. These findings underscore the crucial role of hamp in iron regulation and hematopoiesis, offering novel insights into developmental biology and potential therapeutic targets for blood disorders.},
}

Animals
*Zebrafish/embryology/genetics/metabolism
*Hepcidins/genetics/metabolism
*Hematopoiesis/genetics
*Iron/metabolism
*Homeostasis/genetics
*Embryonic Development/genetics
*Zebrafish Proteins/genetics/metabolism
CRISPR-Cas Systems/genetics
Gene Expression Regulation, Developmental
Ferroptosis/genetics
Gene Deletion
Embryo, Nonmammalian/metabolism
Gene Knockout Techniques
GATA1 Transcription Factor/metabolism/genetics

@article {pmid40054857,
year = {2025},
author = {Kougnassoukou Tchara, PE and Loehr, J and Lambert, JP},
title = {Coupling Proximity Biotinylation with Genomic Targeting to Characterize Locus-Specific Changes in Chromatin Environments.},
journal = {Journal of proteome research},
volume = {24},
number = {4},
pages = {1845-1860},
doi = {10.1021/acs.jproteome.4c00931},
pmid = {40054857},
issn = {1535-3907},
mesh = {Humans ; *Chromatin/genetics/metabolism/chemistry ; Biotinylation/methods ; Cell Line, Tumor ; Azepines/pharmacology ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Triazoles/pharmacology ; *Proteomics/methods ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; Melanoma/genetics ; Genetic Loci ; Bromodomain Containing Proteins ; Proteins ; },
abstract = {Regulating gene expression involves significant changes in the chromatin environment at the locus level, especially at regulatory sequences. However, their modulation following pharmacological treatments or pathological conditions remain mostly undetermined. Here, we report versatile locus-specific proteomics tools to address this knowledge gap, which combine the targeting ability of the CRISPR/Cas9 system and the protein-labeling capability of the highly reactive biotin ligases TurboID (in CasTurbo) and UltraID (in CasUltra). CasTurbo and CasUltra enabled rapid chromatin protein labeling at repetitive sequences like centromeres and telomeres, as well as nonamplified genes. We applied CasUltra to A375 melanoma cell lines to decipher the protein environment of the MYC promoter and characterize the molecular effects of the bromodomain inhibitor JQ1, which targets bromodomain and extra-terminal (BET) proteins that regulate MYC expression. We quantified the consequences of BET protein displacement from the MYC promoter and found that it was associated with a considerable reorganization of the chromatin composition. Additionally, BET protein retention at the MYC promoter was consistent with a model of increased JQ1 resistance. Thus, through the combination of proximity biotinylation and CRISPR/Cas9 genomic targeting, CasTurbo and CasUltra have successfully demonstrated their utility in profiling the proteome associated with a genomic locus in living cells.},
}

Humans
*Chromatin/genetics/metabolism/chemistry
Biotinylation/methods
Cell Line, Tumor
Azepines/pharmacology
Promoter Regions, Genetic
CRISPR-Cas Systems
Triazoles/pharmacology
*Proteomics/methods
Proto-Oncogene Proteins c-myc/genetics/metabolism
Melanoma/genetics
Genetic Loci
Bromodomain Containing Proteins
Proteins

@article {pmid40014809,
year = {2025},
author = {Li, M and Lin, Y and Cheng, Q and Wei, T},
title = {Prime Editing: A Revolutionary Technology for Precise Treatment of Genetic Disorders.},
journal = {Cell proliferation},
volume = {58},
number = {4},
pages = {e13808},
doi = {10.1111/cpr.13808},
pmid = {40014809},
issn = {1365-2184},
support = {2023ZD0500601//Noncommunicable Chronic Diseases-National Science and Technology Major Project/ ; 2023IOZ0204//Initiative Scientific Research Program, Institute of Zoology, Chinese Academy of Sciences/ ; 22JCZXJC00030//Beijing-Tianjin-Hebei Basic Research Cooperation Project/ ; NCTIB2023XB02001//the "Open Competition to Select the Best Candidates" Key Technology Program for Nucleic Acid Drugs of NCTIB/ ; //the Fundamental Research Funds for the Central Universities, Peking University/ ; 2023000CC0040//the Beijing Life Science Academy/ ; 2023M740101//China Postdoctoral Science Foundation/ ; },
mesh = {Humans ; *Gene Editing/methods ; *Genetic Diseases, Inborn/therapy/genetics ; *Genetic Therapy/methods ; Animals ; CRISPR-Cas Systems/genetics ; },
abstract = {Genetic diseases have long posed significant challenges, with limited breakthroughs in treatment. Recent advances in gene editing technologies offer new possibilities in gene therapy for the treatment of inherited disorders. However, traditional gene editing methods have limitations that hinder their potential for clinical use, such as limited editing capabilities and the production of unintended byproducts. To overcome these limitations, prime editing (PE) has been developed as a powerful tool for precise and efficient genome modification. In this review, we provide an overview of the latest advancements in PE and its potential applications in the treatment of inherited disorders. Furthermore, we examine the current delivery vehicles employed for delivering PE systems in vitro and in vivo, and analyze their respective benefits and limitations. Ultimately, we discuss the challenges that need to be addressed to fully unlock the potential of PE for the remission or cure of genetic diseases.},
}

Humans
*Gene Editing/methods
*Genetic Diseases, Inborn/therapy/genetics
*Genetic Therapy/methods
Animals
CRISPR-Cas Systems/genetics

@article {pmid39731215,
year = {2025},
author = {Zhou, Z and Chen, Y and Ba, Y and Xu, H and Zuo, A and Liu, S and Zhang, Y and Weng, S and Ren, Y and Luo, P and Cheng, Q and Zuo, L and Zhu, S and Zhou, X and Zhang, C and Chen, Y and Han, X and Pan, T and Liu, Z},
title = {Revolutionising Cancer Immunotherapy: Advancements and Prospects in Non-Viral CAR-NK Cell Engineering.},
journal = {Cell proliferation},
volume = {58},
number = {4},
pages = {e13791},
doi = {10.1111/cpr.13791},
pmid = {39731215},
issn = {1365-2184},
support = {221100310100//Henan Provincial Science and Technology Research Project/ ; },
mesh = {Humans ; *Killer Cells, Natural/immunology/transplantation ; *Neoplasms/therapy/immunology ; *Receptors, Chimeric Antigen/immunology/genetics/metabolism ; *Cell Engineering/methods ; *Immunotherapy, Adoptive/methods ; *Immunotherapy/methods ; Gene Editing ; Animals ; CRISPR-Cas Systems ; },
abstract = {The recent advancements in cancer immunotherapy have spotlighted the potential of natural killer (NK) cells, particularly chimeric antigen receptor (CAR)-transduced NK cells. These cells, pivotal in innate immunity, offer a rapid and potent response against cancer cells and pathogens without the need for prior sensitization or recognition of peptide antigens. Although NK cell genetic modification is evolving, the viral transduction method continues to be inefficient and fraught with risks, often resulting in cytotoxic outcomes and the possibility of insertional mutagenesis. Consequently, there has been a surge in the development of non-viral transfection technologies to overcome these challenges in NK cell engineering. Non-viral approaches for CAR-NK cell generation are becoming increasingly essential. Cutting-edge techniques such as trogocytosis, electroporation, lipid nanoparticle (LNP) delivery, clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) gene editing and transposons not only enhance the efficiency and safety of CAR-NK cell engineering but also open new avenues for novel therapeutic possibilities. Additionally, the infusion of technologies already successful in CAR T-cell therapy into the CAR-NK paradigm holds immense potential for further advancements. In this review, we present an overview of the potential of NK cells in cancer immunotherapies, as well as non-viral transfection technologies for engineering NK cells.},
}

Humans
*Killer Cells, Natural/immunology/transplantation
*Neoplasms/therapy/immunology
*Receptors, Chimeric Antigen/immunology/genetics/metabolism
*Cell Engineering/methods
*Immunotherapy, Adoptive/methods
*Immunotherapy/methods
Gene Editing
Animals
CRISPR-Cas Systems

@article {pmid40171792,
year = {2025},
author = {Diril, MK and Esmen, K and Sehitogullari, T and Őztürk, G},
title = {Generation of albino C57BL/6J mice by CRISPR embryo editing of the mouse tyrosinase locus.},
journal = {Journal of genetics},
volume = {104},
number = {},
pages = {},
pmid = {40171792},
issn = {0973-7731},
mesh = {Animals ; *Gene Editing/methods ; *Monophenol Monooxygenase/genetics ; *CRISPR-Cas Systems ; Mice ; *Mice, Inbred C57BL ; Embryo, Mammalian/metabolism ; Zygote/metabolism ; Electroporation/methods ; Female ; Gene Knock-In Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genetic Loci ; INDEL Mutation ; },
abstract = {After the arrival of the CRISPR/Cas9 genome editing technology, genetic engineering of model organisms has become much faster and more efficient. The development of genetically modified mouse models is also facilitated by the application of various CRISPR methodologies. Although the very first studies utilized pronuclear injection (PNI) of Cas9 mRNA and sgRNAs into the zygote stage embryos to create knockout and knockin mutations, the repertoire of techniques and collection of reagents for CRISPR editing has rapidly expanded. This presents researchers in the field with a versatility of choices for genetic engineering. However, there are not many comparative studies that analysed the efficacy of gene editing when Cas9 and sgRNA/ssDNA oligos were transferred to the embryos by different methodologies. Here, we aimed to compare two different methods, electroporation and PNI. One of the recent developments gaining wide use in mouse model research is the application of electroporation for the introduction of Cas9/sgRNA ribonucleoprotein complexes into zygote stage embryos. Here, we have used this technique to generate albino coat-coloured C57BL/6J mice by targeted inactivation of the mouse tyrosinase gene through indel or knockin mutations. We have also applied the PNI protocol with the same set of reagents, to compare the efficiency of the two techniques in generation of indel and knockin mutations. Although PNI results in signifi- cantly higher efficiency for knockin mutations, it requires specialized equipment setup and advanced training in embryo micromanipulation and microinjection. Therefore, for the generation of simple gene knockouts by indel mutations, electroporation can be used.},
}

Animals
*Gene Editing/methods
*Monophenol Monooxygenase/genetics
*CRISPR-Cas Systems
Mice
*Mice, Inbred C57BL
Embryo, Mammalian/metabolism
Zygote/metabolism
Electroporation/methods
Female
Gene Knock-In Techniques/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Genetic Loci
INDEL Mutation

@article {pmid40171546,
year = {2025},
author = {Kaur, N and Qadir, M and Francis, DV and Alok, A and Tiwari, S and Ahmed, ZFR},
title = {CRISPR/Cas9: a sustainable technology to enhance climate resilience in major Staple Crops.},
journal = {Frontiers in genome editing},
volume = {7},
number = {},
pages = {1533197},
pmid = {40171546},
issn = {2673-3439},
abstract = {Climate change is a global concern for agriculture, food security, and human health. It affects several crops and causes drastic losses in yield, leading to severe disturbances in the global economy, environment, and community. The consequences on important staple crops, such as rice, maize, and wheat, will worsen and create food insecurity across the globe. Although various methods of trait improvements in crops are available and are being used, clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) mediated genome manipulation have opened a new avenue for functional genomics and crop improvement. This review will discuss the progression in crop improvement from conventional breeding methods to advanced genome editing techniques and how the CRISPR/Cas9 technology can be applied to enhance the tolerance of the main cereal crops (wheat, rice, and maize) against any harsh climates. CRISPR/Cas endonucleases and their derived genetic engineering tools possess high accuracy, versatile, more specific, and easy to design, leading to climate-smart or resilient crops to combat food insecurity and survive harsh environments. The CRISPR/Cas9-mediated genome editing approach has been applied to various crops to make them climate resilient. This review, supported by a bibliometric analysis of recent literature, highlights the potential target genes/traits and addresses the significance of gene editing technologies in tackling the vulnerable effects of climate change on major staple crops staple such as wheat, rice, and maize.},
}

@article {pmid40170307,
year = {2025},
author = {Mao, Y and Chu, G and Liang, Q and Liu, Y and Yang, Y and Liao, X and Wang, M},
title = {[Artificial intelligence-assisted design, mining, and modification of CRISPR-Cas systems].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {3},
pages = {949-967},
doi = {10.13345/j.cjb.240865},
pmid = {40170307},
issn = {1872-2075},
mesh = {*CRISPR-Cas Systems ; *Artificial Intelligence ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genetic Engineering/methods ; Data Mining ; Synthetic Biology/methods ; },
abstract = {With the rapid advancement of synthetic biology, CRISPR-Cas systems have emerged as a powerful tool for gene editing, demonstrating significant potential in various fields, including medicine, agriculture, and industrial biotechnology. This review comprehensively summarizes the significant progress in applying artificial intelligence (AI) technologies to the design, mining, and modification of CRISPR-Cas systems. AI technologies, especially machine learning, have revolutionized sgRNA design by analyzing high-throughput sequencing data, thereby improving the editing efficiency and predicting off-target effects with high accuracy. Furthermore, this paper explores the role of AI in sgRNA design and evaluation, highlighting its contributions to the annotation and mining of CRISPR arrays and Cas proteins, as well as its potential for modifying key proteins involved in gene editing. These advancements have not only improved the efficiency and precision of gene editing but also expanded the horizons of genome engineering, paving the way for intelligent and precise genome editing.},
}

*CRISPR-Cas Systems
*Artificial Intelligence
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Genetic Engineering/methods
Data Mining
Synthetic Biology/methods

@article {pmid40170086,
year = {2025},
author = {Wang, H and Qi, Z and Wang, J and He, Z and Lu, L and Chen, Z and Shao, Y and Wang, G and Wang, Z and Tu, J and Song, X},
title = {Rapid and visual detection of transmissible gastroenteritis virus using a CRISPR/Cas12a system combined with loop-mediated isothermal amplification.},
journal = {BMC veterinary research},
volume = {21},
number = {1},
pages = {234},
pmid = {40170086},
issn = {1746-6148},
support = {GXXT-2022-057//University Synergy Innovation Program of Anhui Province/ ; YQYB2023001//Anhui Provincial University Excellent Young Talents Support Program/ ; 2023SJY01//Research Funds of Joint Research Center for Food Nutrition and Health of IHM/ ; },
mesh = {*Transmissible gastroenteritis virus/genetics ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods/veterinary ; Animals ; *Gastroenteritis, Transmissible, of Swine/diagnosis/virology ; Swine ; Sensitivity and Specificity ; Molecular Diagnostic Techniques/methods/veterinary ; Endodeoxyribonucleases/genetics ; },
abstract = {BACKGROUND: Transmissible gastroenteritis (TGE) is a highly contagious intestinal disease caused by transmissible gastroenteritis virus (TGEV). The primary techniques for identifying TGEV involve enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and fluorescent quantitative PCR (qPCR). However, these approaches are complex, demanding specialized tools and significant time. Therefore, a precise, swift, and effective differential diagnosis method is crucial for TGEV prevention. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR) and Cas-associated proteins have become popular for their high specificity, unique cleavage activity, and ease of detection. CRISPR-Cas12a, a novel RNA-guided nucleic acid endonuclease, is emerging as a powerful molecular scissor.

RESULTS: In this study, we designed three pairs of crRNA targeting the N gene of TGEV. Following the selection of the most appropriate crRNA, we established the loop-mediated isothermal (LAMP) amplification method with a sensitivity of 10[2] copies/µL. And based on this, we established the CRISPR-Cas12a fluorescence assay with a sensitivity of 10[0] copies/µL. Furthermore, we established a CRISPR/Cas12a lateral-flow dipstick assay with a sensitivity of 10[2] copies/µL. Importantly, none of these methods exhibited cross-reactivity with other related viruses, enabling quicker and more straightforward observation of experimental results.

CONCLUSIONS: We have successfully developed a CRISPR-Cas12a fluorescence assay and a CRISPR/Cas12a lateral-flow dipstick assay for clinical TGEV detection. Overall, we created a portable, quick, and sensitive TGEV assay with strong specificity utilizing the CRISPR-Cas12a system.},
}

*Transmissible gastroenteritis virus/genetics
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods/veterinary
Animals
*Gastroenteritis, Transmissible, of Swine/diagnosis/virology
Swine
Sensitivity and Specificity
Molecular Diagnostic Techniques/methods/veterinary
Endodeoxyribonucleases/genetics

@article {pmid40169710,
year = {2025},
author = {Palomino, SM and Gabriel, KA and Mwirigi, JM and Cervantes, A and Horton, P and Funk, G and Moutal, A and Martin, LF and Khanna, R and Price, TJ and Patwardhan, A},
title = {Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {11116},
pmid = {40169710},
issn = {2045-2322},
support = {NS116694/NS/NINDS NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Ganglia, Spinal/metabolism/cytology ; *Neurons/metabolism ; *TRPV Cation Channels/genetics/metabolism ; Cells, Cultured ; Transfection/methods ; },
abstract = {CRISPR-Cas9 is now the leading method for genome editing and is advancing for the treatment of human disease. CRIPSR has promise in treating neurological diseases, but traditional viral-vector-delivery approaches have neurotoxicity limiting their use. Here we describe a simple method for non-viral transfection of primary human DRG (hDRG) neurons for CRISPR-Cas9 editing. We edited TRPV1, NTSR2, and CACNA1E using a lipofection method with CRISPR-Cas9 plasmids containing reporter tags (GFP or mCherry). Transfection was successfully demonstrated by the expression of the reporters two days post-administration. CRISPR-Cas9 editing was confirmed at the genome level with a T7-endonuclease-I assay; protein level with immunocytochemistry and Western blot; and functional level through capsaicin-induced Ca[2+] accumulation in a high-throughput compatible fluorescent imaging plate reader (FLIPR) system. This work establishes a reliable, target specific, non-viral CRISPR-Cas9-mediated genetic editing in primary human neurons with potential for future clinical application for sensory diseases.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*Ganglia, Spinal/metabolism/cytology
*Neurons/metabolism
*TRPV Cation Channels/genetics/metabolism
Cells, Cultured
Transfection/methods

@article {pmid40166066,
year = {2025},
author = {Saeed, U and Piracha, ZZ and Khan, M and Tariq, MN and Gilani, SS and Raza, M and Munusamy, R and Bose, N and Ozsahin, DU and Özşahin, İ and Nauli, SM},
title = {Cracking the code of HBV persistence: cutting-edge approaches to targeting cccDNA in chronic hepatitis B with or without pyogenic liver Abscesses.},
journal = {Frontiers in medicine},
volume = {12},
number = {},
pages = {1504736},
pmid = {40166066},
issn = {2296-858X},
abstract = {Chronic Hepatitis B Virus (HBV) infection remains a formidable global health challenge, driving severe liver complications such as hepatocellular carcinoma (HCC) and pyogenic liver abscesses (PLA). At the core of HBV persistence lies covalently closed circular DNA (cccDNA), a viral reservoir that fuels ongoing infection despite antiviral treatments. This review highlights molecular mechanisms governing cccDNA formation, maintenance, and clearance, spotlighting innovative therapeutic strategies to disrupt this key viral element. We explore cutting-edge approaches, including epigenetic modulation to silence cccDNA, RNA interference (RNAi) for viral RNA degradation, and CRISPR/Cas genome editing to excise cccDNA directly. Additionally, emerging antiviral therapies and immunotherapies, such as therapeutic vaccines and immune checkpoint inhibitors, offer new avenues for enhanced treatment efficacy. Special attention is given to the clinical complexities of managing HBV in patients with co-morbid conditions like HCC and PLA, emphasizing the necessity of a multidisciplinary approach. The interplay between antibacterial and antiviral therapies in PLA-associated HBV cases is critically examined to prevent treatment antagonism, ensuring optimal patient outcomes. Advanced therapeutic strategies, including nucleos(t)ide analogs, interferon therapy, and novel genomic interventions, are explored in both isolated HBV infection and PLA co-infections. Personalized regimens remain pivotal in enhancing therapeutic efficacy and long-term disease control. Current review advocates for a shift toward precision medicine, highlighting the critical need for interdisciplinary collaboration to bridge molecular discoveries with clinical innovations. Ultimately, these advancements promise to revolutionize the management of chronic HBV, paving the way for potential cures and improved patient outcomes.},
}

@article {pmid40165637,
year = {2025},
author = {Guo, Q and Shen, Q and Hao, Q and Jiang, XL and Zou, LP and Xue, YP and Zheng, YG},
title = {Resolving the Trade-Off Between Toxicity and Efficiency of CRISPR-Cas9 System for Genome Editing Within Escherichia coli.},
journal = {Biotechnology journal},
volume = {20},
number = {4},
pages = {e70010},
doi = {10.1002/biot.70010},
pmid = {40165637},
issn = {1860-7314},
support = {2021YFC2102100//National Key Research and Development Program/ ; 2024C03011//Zhejiang Provincial Science and Technology Plan Project/ ; },
mesh = {*Escherichia coli/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Plasmids/genetics ; Genome, Bacterial/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Efficient gene editing of Escherichia coli BL21 (DE3) holds significant practical value as a host for heterologous protein expression. Recently reported CRISPR-Cas9 editing systems for this strain exhibit a trade-off between efficiency and toxicity. In this study, we addressed this trade-off by employing the strategy to transiently induce Cas9 expression in the high-copy plasmid during the editing stage. Furthermore, we demonstrated that eliminating the sgRNA-expressing plasmid using a temperature-sensitive replicon, combined with SacB for removing the Cas9-expressing plasmid, exhibited higher efficiency compared to previously reported strategies for editing system removal. We assigned this optimized CRISPR-Cas9 genome editing system as the pEBcas9/pEBsgRNA system, which has successfully achieved efficient five rounds of genome editing and simultaneous editing of multiple loci in E. coli BL21 (DE3). Using this system, we identified several loci suitable for multi-copy integrated expression of exogenous genes. Overall, the pEBcas9/pEBsgRNA system may facilitate the application of E. coli in both industrial and academic fields.},
}

*Escherichia coli/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Plasmids/genetics
Genome, Bacterial/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40165508,
year = {2025},
author = {Zhang, T and Zhao, Y and Zhu, C and Zhu, X and Zhu, X and Qiu, Y and Nie, Z and Lei, C},
title = {CRISPR/Cas12a Protein Switch Powered Label-Free Electrochemical Biosensor for Sensitive Viral Protease Detection.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c00547},
pmid = {40165508},
issn = {1520-6882},
abstract = {Viral proteases are critical molecular targets in viral pathogenesis, representing pivotal biomarkers for understanding viral infection mechanisms and developing antiviral therapeutics. This study introduces a label-free electrochemical biosensor that enables sensitive viral protease detection by integrating protease-responsive CRISPR/Cas protein switches (CasPSs) with a hemin aptamer-functionalized electrochemical interface. The biosensor's mechanism relies on viral protease-mediated proteolysis, which leads to the release of active Cas12a proteins from CasPSs and generates amplified electrochemical responses through continuous cleavage of immobilized redox-active hemin/aptamer complexes. This biosensor achieved specific hepatitis C virus NS3/4A protease sensing with femtomolar sensitivity and could be readily expanded to other viral proteases by replacing the CasPS module. The feasibility of this biosensor was demonstrated by monitoring enterovirus 71 3C protease activities in virus-infected cell samples with different viral loads and postinfection times. This study provides a promising strategy for integrating CRISPR biosensing with electrochemical platforms, offering a helpful analytical tool for viral infection monitoring and antiviral drug screening.},
}

@article {pmid40126138,
year = {2025},
author = {Pirali, A and Jafarpour, F and Hajian, M and Hosseini Moghaddam, SH and Moradi, R and Tanhaie-Vash, N and Rahimi Andani, M and Izadi, T and Shiralian-Esfahani, H and Safaeinejad, Z and Kues, W and Nasr-Esfahani, MH and Eghbalsaied, S},
title = {Editing the CYP19 Gene in Goat Embryos Using CRISPR/Cas9 and Somatic Cell Nuclear Transfer Techniques.},
journal = {Cellular reprogramming},
volume = {27},
number = {2},
pages = {86-93},
doi = {10.1089/cell.2024.0109},
pmid = {40126138},
issn = {2152-4998},
mesh = {Animals ; *Nuclear Transfer Techniques/veterinary ; *CRISPR-Cas Systems ; *Aromatase/genetics ; *Gene Editing/methods ; *Goats/genetics/embryology ; Female ; Blastocyst/metabolism/cytology ; *Embryo, Mammalian/metabolism ; Embryonic Development/genetics ; Gene Knockout Techniques ; Male ; Cloning, Organism ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) system is revolutionizing genome engineering and is expected to bring significant advancements in livestock traits, including the treatment of genetic diseases. This study focuses on CRISPR/Cas9-mediated modifications of the CYP19 gene, which encodes aromatase, an enzyme crucial for converting testosterone to estrogen and essential for steroid metabolism. Guide RNAs (gRNAs) were designed to target the CYP19 gene and cloned into the pX459 vector. The recombinant plasmid was then electrotransfected into fibroblast cells from a Lori-Bakhtiari buck, and these transfected cells were used for embryo production via somatic cell nuclear transfer (SCNT). The cloned embryos were evaluated for their progression through embryonic stages, showing no significant difference in blastocyst development between knock-out and unedited groups. The knockout efficiency was 78.4% in cells and 68.9% in goat blastocysts, demonstrating the successful depletion of CYP19. We successfully achieved a high rate of CYP19 gene-edited embryos through the combined application of cell electrotransfection and SCNT technologies, while maintaining the normal developmental rate of the embryos. These embryos can be used for transfer to generate knock-out goats, providing a foundation for further studies on CYP19's role in male fertility and production traits.},
}

Animals
*Nuclear Transfer Techniques/veterinary
*CRISPR-Cas Systems
*Aromatase/genetics
*Gene Editing/methods
*Goats/genetics/embryology
Female
Blastocyst/metabolism/cytology
*Embryo, Mammalian/metabolism
Embryonic Development/genetics
Gene Knockout Techniques
Male
Cloning, Organism

@article {pmid40022449,
year = {2025},
author = {Wallace, KA and Gerstenberg, TL and Ennis, CL and Perez-Bermejo, JA and Partridge, JR and Bandoro, C and Matern, WM and Andreoletti, G and Krassovsky, K and Kabir, S and Lalisan, CD and Churi, AR and Chew, GM and Corbo, L and Vincelette, JE and Klasson, TD and Silva, BJ and Strukov, YG and Quejarro, BJ and Hill, KA and Treusch, S and Grogan, JL and Dever, DP and Porteus, MH and Wienert, B},
title = {A differentiated β-globin gene replacement strategy uses heterologous introns to restore physiological expression.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {4},
pages = {1407-1419},
doi = {10.1016/j.ymthe.2025.02.036},
pmid = {40022449},
issn = {1525-0024},
mesh = {*beta-Globins/genetics/metabolism ; *Introns ; Humans ; CRISPR-Cas Systems ; *beta-Thalassemia/genetics/therapy ; Animals ; *Anemia, Sickle Cell/genetics/therapy ; Mice ; Genetic Therapy/methods ; Gene Editing ; Recombinational DNA Repair ; },
abstract = {β-Hemoglobinopathies are common monogenic disorders. In sickle cell disease (SCD), a single mutation in the β-globin (HBB) gene results in dysfunctional hemoglobin protein, while in β-thalassemia, over 300 mutations distributed across the gene reduce β-globin levels and cause severe anemia. Genetic engineering replacing the whole HBB gene through homology-directed repair (HDR) is an ideal strategy to restore a benign genotype and rescue HBB expression for most genotypes. However, this is technically challenging because (1) the insert must not be homologous to the endogenous gene and (2) synonymous codon-optimized, intron-less sequences may not reconstitute adequate β-globin levels. Here, we developed an HBB gene replacement strategy using CRISPR-Cas9 that successfully addresses these challenges. We determined that a DNA donor containing a diverged HBB coding sequence and heterologous introns to avoid sequence homology provides proper physiological expression. We identified a DNA donor that uses truncated γ-globin introns, results in 34% HDR, and rescues β-globin expression in in vitro models of SCD and β-thalassemia in hematopoietic stem and progenitor cells (HSPCs). Furthermore, while HDR allele frequency dropped in vivo, it was maintained at ∼15%, demonstrating editing of long-term repopulating HSPCs. In summary, our HBB gene replacement strategy offers a differentiated approach by restoring naturally regulated adult hemoglobin expression.},
}

*beta-Globins/genetics/metabolism
*Introns
Humans
CRISPR-Cas Systems
*beta-Thalassemia/genetics/therapy
Animals
*Anemia, Sickle Cell/genetics/therapy
Mice
Genetic Therapy/methods
Gene Editing
Recombinational DNA Repair

@article {pmid39955618,
year = {2025},
author = {Baatz, F and Ghosh, A and Herbst, J and Polten, S and Meyer, J and Rhiel, M and Maetzig, T and Geffers, R and Rothe, M and Bastone, AL and John-Neek, P and Frühauf, J and Eiz-Vesper, B and Bonifacius, A and Falk, CS and Kaisenberg, CV and Cathomen, T and Schambach, A and van den Brink, MRM and Hust, M and Sauer, MG},
title = {Targeting BCL11B in CAR-engineered lymphoid progenitors drives NK-like cell development with prolonged anti-leukemic activity.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {4},
pages = {1584-1607},
doi = {10.1016/j.ymthe.2025.02.024},
pmid = {39955618},
issn = {1525-0024},
mesh = {Animals ; Humans ; Mice ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; CRISPR-Cas Systems ; *Killer Cells, Natural/immunology/metabolism/cytology ; *Lymphoid Progenitor Cells/metabolism/immunology/cytology ; *Repressor Proteins/genetics/metabolism ; *Tumor Suppressor Proteins/genetics/metabolism ; *Leukemia/therapy/immunology/genetics ; Immunotherapy, Adoptive ; Cell Differentiation ; },
abstract = {Chimeric antigen receptor (CAR)-induced suppression of the transcription factor B cell CLL/lymphoma 11B (BCL11B) propagates CAR-induced killer (CARiK) cell development from lymphoid progenitors. Here, we show that CRISPR-Cas9-mediated Bcl11b knockout in human and murine early lymphoid progenitors distinctively modulates this process either alone or in combination with a CAR. Upon adoptive transfer into hematopoietic stem cell recipients, Bcl11b-edited progenitors mediated innate-like antigen-independent anti-leukemic immune responses. With CAR expression allowing for additional antigen-specific responses, the progeny of double-edited lymphoid progenitors acquired prolonged anti-leukemic activity in vivo. These findings give important insights into how Bcl11b targeting can be used to tailor anti-leukemia functionality of CAR-engineered lymphoid progenitor cells.},
}

Animals
Humans
Mice
*Receptors, Chimeric Antigen/genetics/immunology/metabolism
CRISPR-Cas Systems
*Killer Cells, Natural/immunology/metabolism/cytology
*Lymphoid Progenitor Cells/metabolism/immunology/cytology
*Repressor Proteins/genetics/metabolism
*Tumor Suppressor Proteins/genetics/metabolism
*Leukemia/therapy/immunology/genetics
Immunotherapy, Adoptive
Cell Differentiation

@article {pmid39935177,
year = {2025},
author = {Cheng, Y and Zhang, J and Mu, W and Ye, S and Cheng, J and Zhu, L and Wang, G and Cao, Y and Li, D and Hu, G and Huang, L and Wang, J and Zhou, J},
title = {Dasatinib-resistant universal CAR-T cells proliferate in the presence of host immune cells and exhibit antitumor activity.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {4},
pages = {1535-1551},
doi = {10.1016/j.ymthe.2025.02.012},
pmid = {39935177},
issn = {1525-0024},
mesh = {*Dasatinib/pharmacology ; Animals ; Humans ; Mice ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; *Immunotherapy, Adoptive/methods ; Xenograft Model Antitumor Assays ; Cell Line, Tumor ; Cell Proliferation/drug effects ; *T-Lymphocytes/immunology/metabolism/drug effects ; *Drug Resistance, Neoplasm ; Protein Kinase Inhibitors/pharmacology ; Receptors, Antigen, T-Cell/genetics ; Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics ; CRISPR-Cas Systems ; Mutation ; },
abstract = {The universal chimeric antigen receptor T cell (UCAR-T) immunotherapy derived from healthy donors holds great promise in pan-cancer treatment. However, UCAR-T cell therapy faces a challenge in the rapid elimination of allogeneic cells by the host immune system. To address this, we introduced a T316I mutation in the leukocyte-specific protein tyrosine kinase (LCK) locus in CAR-T cells using the cytosine base editor (CBE) system. Concurrently, we disrupted endogenous T cell receptor alpha chain (TRAC) and beta-2 microglobulin (B2M) with the CRISPR-Cas9 system, along with dasatinib to overcome host immune rejection, an Src family kinase (SFK) inhibitor. The resulting LCK mutated UCAR-T (KM UCAR-T) cells exhibited normal phenotypes in activation, proliferation, differentiation, and tumor cytotoxicity in vitro. Moreover, KM UCAR-T cells demonstrated sustained expansion in mixed lymphocyte reactions (MLR) when incubated with T cells or peripheral blood mononuclear cells (PBMCs) from HLA-mismatched donors upon dasatinib treatment. Additionally, we illustrated that KM UCAR-T cells displayed antitumor activity in a xenograft murine model and verified the expansion and cytotoxicity of KM UCAR-T over traditional UCAR-T in the presence of allogeneic PBMCs when treated with dasatinib in vivo. These findings offer a novel strategy for UCAR-T cells to resist host immune rejection and achieve sustained expansion.},
}

*Dasatinib/pharmacology
Animals
Humans
Mice
*Receptors, Chimeric Antigen/genetics/immunology/metabolism
*Immunotherapy, Adoptive/methods
Xenograft Model Antitumor Assays
Cell Line, Tumor
Cell Proliferation/drug effects
*T-Lymphocytes/immunology/metabolism/drug effects
*Drug Resistance, Neoplasm
Protein Kinase Inhibitors/pharmacology
Receptors, Antigen, T-Cell/genetics
Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/genetics
CRISPR-Cas Systems
Mutation

@article {pmid39921871,
year = {2025},
author = {Lv, J and Fan, Q and Zhang, Y and Zhou, X and Yu, P and Yu, X and Xin, C and Hong, J and Cheng, Y},
title = {A Serum Resistant Polymer with Exceptional Endosomal Escape and mRNA Delivery Efficacy for CRISPR Gene Therapy.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {13},
pages = {e2413006},
doi = {10.1002/advs.202413006},
pmid = {39921871},
issn = {2198-3844},
support = {2019YFA0904500//National Key R&D Program of China/ ; 2023YFA0915000//National Key R&D Program of China/ ; 52373134//National Natural Science Foundation of China/ ; 82171102//National Natural Science Foundation of China/ ; 82271044//National Natural Science Foundation of China/ ; 2019YFA0904500//National Key Research and Development Program of China/ ; 2023YFA0915000//National Key Research and Development Program of China/ ; 22Y21900900//Shanghai Medical Innovation Research Program/ ; },
mesh = {Animals ; *RNA, Messenger/genetics/metabolism/administration & dosage ; *Genetic Therapy/methods ; Mice ; *CRISPR-Cas Systems/genetics ; Humans ; *Endosomes/metabolism ; *Polymers/chemistry ; Gene Editing/methods ; Nanoparticles/chemistry ; *Gene Transfer Techniques ; Choroidal Neovascularization/therapy/genetics ; },
abstract = {Nanoparticle-based mRNA delivery offers a versatile platform for innovative therapies. However, most of the current delivery systems are limited by poor serum tolerance, suboptimal endosomal escape and mRNA delivery efficacy. Herein, a highly efficient mRNA-delivering material is identified from a library of fluoropolymers. The lead material FD17 shows exceptional serum stability and endosomal escape, enabling efficient mRNA delivery into various cell types, surpassing commercial mRNA delivery reagents such as Lipofectamine 3000. The formed mRNA nanoparticles adsorb abundant serum albumin on the surface, which facilitates cellular uptake via scavenger receptor-mediated endocytosis. FD17 enables the delivery of mRNAs encoding CRE, Cas9, and base editor hyCBE for efficient genome editing. The material mediates CRISPR/Cas9 gene therapy via intraocular injection effectively down-regulates vascular endothelial growth factor A in retinal pigment epithelial cells of mice, yielding promising therapeutic responses against laser-induced choroidal neovascularization. The discovered material in this study shows great promise for the development of mRNA therapeutics to combat a wide range of diseases.},
}

Animals
*RNA, Messenger/genetics/metabolism/administration & dosage
*Genetic Therapy/methods
Mice
*CRISPR-Cas Systems/genetics
Humans
*Endosomes/metabolism
*Polymers/chemistry
Gene Editing/methods
Nanoparticles/chemistry
*Gene Transfer Techniques
Choroidal Neovascularization/therapy/genetics

@article {pmid39881543,
year = {2025},
author = {Tachida, Y and Manian, KV and Butcher, R and Levy, JM and Pendse, N and Hennessey, E and Liu, DR and Pierce, EA and Liu, Q and Comander, J},
title = {Systematic empirical evaluation of individual base editing targets: Validating therapeutic targets in USH2A and comparison of methods.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {4},
pages = {1466-1484},
doi = {10.1016/j.ymthe.2025.01.042},
pmid = {39881543},
issn = {1525-0024},
mesh = {Humans ; *Gene Editing/methods ; Animals ; *Extracellular Matrix Proteins/genetics ; Mice ; Mutation ; Genetic Therapy/methods ; *Usher Syndromes/genetics/therapy ; High-Throughput Nucleotide Sequencing ; Disease Models, Animal ; Dependovirus/genetics ; CRISPR-Cas Systems ; },
abstract = {Base editing shows promise for the correction of human mutations at a higher efficiency than other repair methods and is especially attractive for mutations in large genes that are not amenable to gene augmentation therapy. Here, we demonstrate a comprehensive workflow for in vitro screening of potential therapeutic base editing targets for the USH2A gene and empirically validate the efficiency of adenine and cytosine base editor/guide combinations for correcting 35 USH2A mutations. Editing efficiency and bystander edits are compared between different target templates (plasmids vs. transgenes) and assays (next-generation sequencing vs. Sanger), as well as comparisons between unbiased empirical results and computational predictions. Based on these observations, practical assay recommendations are discussed. Finally, a humanized knockin mouse model was created with the best-performing target, the nonsense mutation c.11864G>A p.(Trp3955∗). Split-intein AAV9 delivery of editing reagents resulted in the restoration of USH2A protein and a correction rate of 65% ± 3% at the mutant base pair and of 52% ± 3% excluding bystander amino acid changes. This efficiency is higher than that seen in a retinal gene editing program testing in a clinical trial. These results demonstrate the effectiveness of this overall strategy to identify and test base editing reagents with the potential for human therapeutic applications.},
}

Humans
*Gene Editing/methods
Animals
*Extracellular Matrix Proteins/genetics
Mice
Mutation
Genetic Therapy/methods
*Usher Syndromes/genetics/therapy
High-Throughput Nucleotide Sequencing
Disease Models, Animal
Dependovirus/genetics
CRISPR-Cas Systems

@article {pmid39825209,
year = {2025},
author = {Jin, Q and Feng, X and Hong, M and Wang, K and Chen, X and Cheng, J and Kuang, Y and Si, X and Xu, M and Huang, X and Guang, S and Zhu, C},
title = {Peri-centrosomal localization of small interfering RNAs in C. elegans.},
journal = {Science China. Life sciences},
volume = {68},
number = {4},
pages = {895-911},
pmid = {39825209},
issn = {1869-1889},
mesh = {Animals ; *Caenorhabditis elegans/genetics/metabolism ; Caenorhabditis elegans Proteins/metabolism/genetics ; *Centrosome/metabolism ; *RNA, Small Interfering/metabolism/genetics ; RNA Interference ; RNA-Dependent RNA Polymerase/metabolism/genetics ; Argonaute Proteins/metabolism/genetics ; CRISPR-Cas Systems ; RNA-Binding Proteins ; },
abstract = {The centrosome is the microtubule-organizing center and a crucial part of cell division. Centrosomal RNAs (cnRNAs) have been reported to enable precise spatiotemporal control of gene expression during cell division in many species. Whether and how cnRNAs exist in C. elegans are unclear. Here, using the nuclear RNAi Argonaute protein NRDE-3 as a reporter, we observed potential peri-centrosome localized small interfering (si)RNAs in C. elegans. NRDE-3 was previously shown to associate with pre-mRNAs and pre-rRNAs via a process involving the presence of complementary siRNAs. We generated a GFP-NRDE-3 knock-in transgene through CRISPR/Cas9 technology and observed that NRDE-3 formed peri-centrosomal foci neighboring the tubulin protein TBB-2, other centriole proteins and pericentriolar material (PCM) components in C. elegans embryos. The peri-centrosomal accumulation of NRDE-3 depends on RNA-dependent RNA polymerase (RdRP)-synthesized 22G siRNAs and the PAZ domain of NRDE-3, which is essential for siRNA binding. Mutation of eri-1, ergo-1, or drh-3 significantly increased the percentage of pericentrosome-enriched NRDE-3. At the metaphase of the cell cycle, NRDE-3 was enriched in both the peri-centrosomal region and the spindle. Moreover, the integrity of centriole proteins and pericentriolar material (PCM) components is also required for the peri-centrosomal accumulation of NRDE-3. Therefore, we concluded that siRNAs could accumulate in the pericentrosomal region in C. elegans and suggested that the peri-centrosomal region may also be a platform for RNAi-mediated gene regulation.},
}

Animals
*Caenorhabditis elegans/genetics/metabolism
Caenorhabditis elegans Proteins/metabolism/genetics
*Centrosome/metabolism
*RNA, Small Interfering/metabolism/genetics
RNA Interference
RNA-Dependent RNA Polymerase/metabolism/genetics
Argonaute Proteins/metabolism/genetics
CRISPR-Cas Systems
RNA-Binding Proteins

@article {pmid39462750,
year = {2025},
author = {Liang, L and Tan, H and Liu, R},
title = {Emerging gene editing in industrial microbiology beyond CRISPR-Cas9.},
journal = {Trends in biotechnology},
volume = {43},
number = {4},
pages = {742-744},
doi = {10.1016/j.tibtech.2024.09.012},
pmid = {39462750},
issn = {1879-3096},
mesh = {*Gene Editing/methods/trends ; *CRISPR-Cas Systems/genetics ; *Industrial Microbiology/methods/trends ; },
abstract = {The CRISPR-Cas9 system has been widely applied for industrial microbiology but is not effective in certain microorganisms. This forum explores the strategies aimed at overcoming these challenges, including the use of the Cas12a system, Cas9 variants, and non-CRISPR techniques, to provide more effective strategies for expanding applications in microbial engineering.},
}

*Gene Editing/methods/trends
*CRISPR-Cas Systems/genetics
*Industrial Microbiology/methods/trends

@article {pmid39327106,
year = {2025},
author = {Yan, Y and Ahmed, HMM and Wimmer, EA and Schetelig, MF},
title = {Biotechnology-enhanced genetic controls of the global pest Drosophila suzukii.},
journal = {Trends in biotechnology},
volume = {43},
number = {4},
pages = {826-837},
doi = {10.1016/j.tibtech.2024.09.005},
pmid = {39327106},
issn = {1879-3096},
mesh = {Animals ; *Drosophila/genetics ; *Biotechnology/methods ; *Pest Control, Biological/methods ; Male ; CRISPR-Cas Systems ; Female ; Animals, Genetically Modified ; },
abstract = {Spotted wing Drosophila (Drosophila suzukii Matsumura, or SWD), an insect pest of soft-skinned fruits native to East Asia, has rapidly spread worldwide in the past 15 years. Genetic controls such as sterile insect technique (SIT) have been considered for the environmentally friendly and cost-effective management of this pest. In this review, we provide the latest developments for the genetic control strategies of SWD, including sperm-marking strains, CRISPR-based sex-ratio distortion, neoclassical genetic sexing strains, transgenic sexing strains, a sex-sorting incompatible male system, precision-guided SIT, and gene drives based on synthetic Maternal effect dominant embryonic arrest (Medea) or homing CRISPR systems. These strategies could either enhance the efficacy of traditional SIT or serve as standalone methods for the sustainable control of SWD.},
}

Animals
*Drosophila/genetics
*Biotechnology/methods
*Pest Control, Biological/methods
Male
CRISPR-Cas Systems
Female
Animals, Genetically Modified

@article {pmid40165374,
year = {2025},
author = {Butt, H and Sathish, S and London, E and Lee Johnson, T and Essawi, K and Leonard, A and Tisdale, JF and Demirci, S},
title = {Genome Editing Strategies for Targeted Correction of β-globin Mutation in Sickle Cell Disease: From Bench to Bedside.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2025.03.047},
pmid = {40165374},
issn = {1525-0024},
abstract = {Sickle cell disease (SCD) includes a range of genotypes that result in a clinical syndrome, where abnormal red blood cell (RBC) physiology leads to widespread complications affecting nearly every organ system. Treatment strategies for SCD can be broadly categorized into disease-modifying therapies and those aimed toward a cure. Although several disease-modifying drugs have been approved, they do not fully address the complexity and severity of SCD. Recent advances in allogeneic transplantation and autologous gene therapy show promising outcomes in terms of efficacy and safety. While these approaches have improved the lives of many patients, achieving a durable and comprehensive cure for all remains challenging. To address this, gene-editing technologies, including zinc finger nucleases, TALENs, CRISPR-Cas, base editing, and prime editing, have been explored both ex vivo and in vivo for targeted correction of the β-globin gene (HBB) in SCD. However, direct correction of HBB and its translation from the laboratory to the clinic presents ongoing limitations, with challenges involved in achieving robust mutation correction efficiency, off-target effects, and high costs of therapies. The optimal strategy for curing SCD remains uncertain, but several promising approaches are emerging. This review will touch on past, present and future developments in HBB correction.},
}

@article {pmid40165206,
year = {2025},
author = {Lipert, BA and Siemens, KN and Khan, A and Airey, R and Dam, GH and Lu, M and Flinterman, M and Yong, Q and Lee, TW and Hunter, FW and Jamieson, SMF},
title = {CRISPR screens with trastuzumab emtansine in HER2-positive breast cancer cell lines reveal new insights into drug resistance.},
journal = {Breast cancer research : BCR},
volume = {27},
number = {1},
pages = {48},
pmid = {40165206},
issn = {1465-542X},
support = {22/345//Health Research Council of New Zealand/ ; 22/345//Health Research Council of New Zealand/ ; 22/345//Health Research Council of New Zealand/ ; 22/345//Health Research Council of New Zealand/ ; 22/345//Health Research Council of New Zealand/ ; 22/345//Health Research Council of New Zealand/ ; 22/345//Health Research Council of New Zealand/ ; 1921 PG//Cancer Research Trust New Zealand/ ; 1921 PG//Cancer Research Trust New Zealand/ ; 1921 PG//Cancer Research Trust New Zealand/ ; 1921 PG//Cancer Research Trust New Zealand/ ; 1921 PG//Cancer Research Trust New Zealand/ ; 1921 PG//Cancer Research Trust New Zealand/ ; },
mesh = {Humans ; *Drug Resistance, Neoplasm/genetics ; *Breast Neoplasms/genetics/drug therapy/pathology/metabolism ; Female ; *Receptor, ErbB-2/metabolism/genetics ; *CRISPR-Cas Systems ; *Ado-Trastuzumab Emtansine/pharmacology/therapeutic use ; Cell Line, Tumor ; Trastuzumab/pharmacology/therapeutic use ; Gene Knockout Techniques ; Antineoplastic Agents, Immunological/pharmacology/therapeutic use ; },
abstract = {BACKGROUND: Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate that is an effective therapy for HER2-positive breast cancer; however, its efficacy is limited by drug resistance. While multiple mechanisms of resistance have been proposed, these are not yet well understood. Greater understanding of T-DM1 sensitivity and resistance could provide new combination strategies to overcome resistance or predictive biomarkers to guide therapy.

METHODS: We have conducted CRISPR/Cas9 functional genomics modifier screens in HER2-positive breast cancer cell lines to allow for unbiased discovery of T-DM1 sensitivity and resistance genes. Whole-genome knockout screens were carried out in MDA-MB-361 and MDA-MB-453 cells treated with T-DM1 and its payload cytotoxin DM1. Hits were validated in secondary T-DM1 screens using a focused single-guide RNA (sgRNA) library and subsequently by individual gene knockout.

RESULTS: The whole-genome CRISPR screens with T-DM1 and DM1 identified 599 genes as potential modifiers of T-DM1 sensitivity and resistance. Of these, 17 genes were significantly enriched and 3 genes depleted at P < 0.001 in either or both MDA-MB-361 and MDA-MB-453 libraries in the secondary screens. Among the top hits, were known T-DM1 sensitivity genes ERBB2 and SLC46A3, in addition to negative regulators of mTOR complex 1: TSC1 and TSC2. MDA-MB-453 clones with knockout of TSC1 or partial knockout of TSC2 were more resistant to T-DM1 than wild type cells in competition growth assays and to T-DM1 and other HER2 targeting therapies (T-DXd, lapatinib and neratinib) in growth inhibition assays, and had increased internalisation of T-DM1 at 6 h. T-DM1 and the mTOR inhibitor everolimus demonstrated synergistic activity at inhibiting cell proliferation at multiple T-DM1 concentrations across four HER2-positive breast cancer cell lines.

CONCLUSIONS: Our CRISPR screening approach with T-DM1 in HER2-positive breast cancer cell lines identified genes not previously implicated in T-DM1 sensitivity or resistance, including TSC1 and TSC2. These genes may inform new strategies to enhance T-DM1 therapy in the clinic.},
}

Humans
*Drug Resistance, Neoplasm/genetics
*Breast Neoplasms/genetics/drug therapy/pathology/metabolism
Female
*Receptor, ErbB-2/metabolism/genetics
*CRISPR-Cas Systems
*Ado-Trastuzumab Emtansine/pharmacology/therapeutic use
Cell Line, Tumor
Trastuzumab/pharmacology/therapeutic use
Gene Knockout Techniques
Antineoplastic Agents, Immunological/pharmacology/therapeutic use

@article {pmid40164595,
year = {2025},
author = {Feist, WN and Luna, SE and Ben-Efraim, K and Filsinger Interrante, MV and Amorin, A and Johnston, NM and Bruun, TUJ and Utz, A and Ghanim, HY and Lesch, BJ and McLaughlin, TM and Dudek, AM and Porteus, MH},
title = {Multilayered HIV-1 resistance in HSPCs through CCR5 Knockout and B cell secretion of HIV-inhibiting antibodies.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3103},
pmid = {40164595},
issn = {2041-1723},
mesh = {*Receptors, CCR5/immunology/genetics/metabolism ; Humans ; *HIV-1/immunology/genetics ; *HIV Infections/immunology/virology/therapy ; *B-Lymphocytes/immunology ; *HIV Antibodies/immunology ; *Hematopoietic Stem Cells/immunology/virology/metabolism ; Animals ; *CRISPR-Cas Systems ; Antibodies, Neutralizing/immunology ; Hematopoietic Stem Cell Transplantation ; Mice ; Gene Knockout Techniques ; Female ; },
abstract = {Allogeneic transplantation of CCR5 null hematopoietic stem and progenitor cells (HSPCs) is the only known cure for HIV-1 infection. However, this treatment is limited because of the rarity of CCR5-null matched donors, the morbidities associated with allogeneic transplantation, and the prevalence of HIV-1 strains resistant to CCR5 knockout (KO) alone. Here, we propose a one-time therapy through autologous transplantation of HSPCs genetically engineered ex vivo to produce both CCR5 KO cells and long-term secretion of potent HIV-1 inhibiting antibodies from B cell progeny. CRISPR-Cas9-engineered HSPCs engraft and reconstitute multiple hematopoietic lineages in vivo and can be engineered to express multiple antibodies simultaneously (in pre-clinical models). Human B cells engineered to express each antibody secrete neutralizing concentrations capable of inhibiting HIV-1 pseudovirus infection in vitro. This work lays the foundation for a potential one-time functional cure for HIV-1 through combining the long-term delivery of therapeutic antibodies against HIV-1 and the known efficacy of CCR5 KO HSPC transplantation.},
}

*Receptors, CCR5/immunology/genetics/metabolism
Humans
*HIV-1/immunology/genetics
*HIV Infections/immunology/virology/therapy
*B-Lymphocytes/immunology
*HIV Antibodies/immunology
*Hematopoietic Stem Cells/immunology/virology/metabolism
Animals
*CRISPR-Cas Systems
Antibodies, Neutralizing/immunology
Hematopoietic Stem Cell Transplantation
Mice
Gene Knockout Techniques
Female

@article {pmid40164474,
year = {2025},
author = {Guia, S and Fenis, A and Baudesson De Chanville, C and Galluso, J and Medjouel, H and Escaliere, B and Modelska, A and Vienne, M and Lopes, N and Pouchin, A and Rossi, B and Gauthier, L and Roulland, S and Vivier, E and Narni-Mancinelli, E},
title = {Genome-wide CRISPR/Cas9 screen reveals factors that influence the susceptibility of tumor cells to NK cell-mediated killing.},
journal = {Journal for immunotherapy of cancer},
volume = {13},
number = {3},
pages = {},
doi = {10.1136/jitc-2024-010699},
pmid = {40164474},
issn = {2051-1426},
mesh = {Humans ; *Killer Cells, Natural/immunology/metabolism ; *CRISPR-Cas Systems ; Animals ; Mice ; Cytotoxicity, Immunologic ; HCT116 Cells ; },
abstract = {BACKGROUND: Natural killer (NK) cells exhibit potent cytotoxic activity against various cancer cell types. Over the past five decades, numerous methodologies have been employed to elucidate the intricate molecular mechanisms underlying NK cell-mediated tumor control. While significant progress has been made in elucidating the interactions between NK cells and tumor cells, the regulatory factors governing NK cell-mediated tumor cell destruction are not yet fully understood. This includes the diverse array of tumor ligands recognized by NK cells and the mechanisms that NK cells employ to eliminate tumor cells.

METHODS: In this study, we employed a genome-wide CRISPR/Cas9 screening approach in conjunction with functional cytotoxicity assays to delineate the pathways modulating the susceptibility of colon adenocarcinoma HCT-116 cells to NK cell-mediated cytotoxicity.

RESULTS: Analysis of guide RNA distribution in HCT-116 cells that survived co-incubation with NK cells identified ICAM-1 as a pivotal player in the NKp44-mediated immune synapse, with NKp44 serving as an activating receptor crucial for the elimination of HCT-116 tumor cells by NK cells. Furthermore, disruption of genes involved in the apoptosis or interferon (IFN)-γ signaling pathways conferred resistance to NK cell attack. We further dissected that NK cell-derived IFN-γ promotes mitochondrial apoptosis in vitro and exerts control over B16-F10 lung metastases in vivo.

CONCLUSION: Monitoring ICAM-1 levels on the surface of tumor cells or modulating its expression should be considered in the context of NK cell-based therapy. Furthermore, promoting FasL expression on the NK cell surface is reaffirmed as an important strategy to enhance NK cell-mediated tumor killing, offering an additional avenue for therapeutic optimization. Additionally, considering the diffusion properties of IFN-γ, our findings highlight the potential of leveraging NK cell-derived IFN-γ to enhance direct tumor cell killing and facilitate bystander effects via cytokine diffusion, warranting further investigation.},
}

Humans
*Killer Cells, Natural/immunology/metabolism
*CRISPR-Cas Systems
Animals
Mice
Cytotoxicity, Immunologic
HCT116 Cells

@article {pmid40163287,
year = {2025},
author = {Oh, Y and Nagalakshmi, U and Dahlbeck, D and Koehler, N and Cho, MJ and Dinesh-Kumar, SP and Staskawicz, BJ},
title = {Heritable virus-induced germline editing in tomato.},
journal = {The Plant journal : for cell and molecular biology},
volume = {122},
number = {1},
pages = {e70115},
pmid = {40163287},
issn = {1365-313X},
support = {ios-2303522//National Science Foundation/ ; ios-2303523//National Science Foundation/ ; //Innovative Genomics Institute/ ; },
mesh = {*Solanum lycopersicum/genetics/virology ; *Gene Editing/methods ; *Plants, Genetically Modified/genetics ; Plant Viruses/genetics ; CRISPR-Cas Systems ; Caulimovirus/genetics ; Promoter Regions, Genetic/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Plant Proteins/genetics/metabolism ; Genome, Plant/genetics ; Oxidoreductases ; },
abstract = {Here, we report the successful implementation of heritable virus-induced genome editing (VIGE) in tomato (Solanum lycopersicum). We generated three transgenic tomato lines expressing Streptococcus pyogenes Cas9 (SpCas9) under the control of Cauliflower mosaic virus 35S (35S), S. lycopersicum ribosomal protein S5A (SlRPS5A), or S. lycopersicum YAO promoters (SlYAO). These three lines were tested for somatic and heritable editing using the tobacco rattle virus (TRV)-based system carrying guide RNAs (gRNAs) fused with mobile RNA sequences. TRV with gRNA targeted to Phytoene desaturase (SlPDS) and Downy mildew resistance 6 (SlDMR6) genes fused to mobile RNA sequences showed significant somatic editing efficiency in all three tomato lines expressing SpCas9. However, the progenies from the SlYAO promoter-driven SpCas9 tomato infected with TRV with gRNA targeted to SlDMR6 fused to the mobile RNA sequence resulted in monoallelic mutations with a frequency of 3%. Optimization of environmental conditions, such as reduced light intensity, significantly increased heritable editing frequencies, from 0% to 86% at the SlPDS and from 3% to 100% at the SlDMR6, including biallelic mutations. These findings underscore the use of appropriate promoters to express Cas nucleases and optimized environmental conditions to enhance heritable genome editing efficiency in tomato using VIGE. Furthermore, our method enables the generation of mutants without additional tissue culture or transformation once a SpCas9-expressing tomato line is established.},
}

*Solanum lycopersicum/genetics/virology
*Gene Editing/methods
*Plants, Genetically Modified/genetics
Plant Viruses/genetics
CRISPR-Cas Systems
Caulimovirus/genetics
Promoter Regions, Genetic/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Plant Proteins/genetics/metabolism
Genome, Plant/genetics
Oxidoreductases

@article {pmid40162959,
year = {2025},
author = {Shen, H and Li, Y and Tang, K and Liang, H and Xu, ZL and Liu, Y and Liu, W},
title = {Programmable AIESTA: All-in-One Isothermal Enzymatic Signal Transduction Amplifier for Portable Profiling.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c00934},
pmid = {40162959},
issn = {1520-6882},
abstract = {The Argonaute (Ago) protein exhibits high specificity in nucleic acid recognition and cleavage, making it highly promising for biosensing applications. Its potential is further enhanced by its independence from protospacer adjacent motif (PAM) requirements and the cost-effectiveness of using short DNA guides. Both Ago and CRISPR/Cas systems face challenges in signal amplification, which limit their ability to detect targets at ultralow concentrations. To overcome this limitation, a thermostable quadratic amplification system (T-QAS) was constructed by integrating a thermostable nicking-enzyme-mediated amplification (NEMA) strategy with TtAgo. The system leverages the high stability of T-QAS at elevated temperatures to enhance guide-target interactions and decrease false positives caused by nonspecific amplification. Additionally, nanozyme is integrated with T-QAS to construct the AIESTA platform (all-in-one isothermal enzymatic signal transduction amplifier), which is a single-tube visual sensing platform. Within the AIESTA system, T-QAS improves specificity through high operational temperatures and offers programmable functions, enabling the sensitive detection of miRNA and foodborne toxins. The combination of T-QAS and nanozyme makes AIESTA a candidate of point-of-care testing (POCT) field, showcasing the potential for biosensing in resource-limited and complex environments.},
}

@article {pmid40162949,
year = {2025},
author = {Jurgens, JA and Matos Ruiz, PM and King, J and Foster, EE and Berube, L and Chan, WM and Barry, BJ and Jeong, R and Rothman, E and Whitman, MC and MacKinnon, S and Rivera-Quiles, C and Pratt, BM and Easterbrooks, T and Mensching, FM and Di Gioia, SA and Pais, L and England, EM and de Berardinis, T and Magli, A and Koc, F and Asakawa, K and Kawakami, K and O'Donnell-Luria, A and Hunter, DG and Robson, CD and Bulyk, ML and Engle, EC},
title = {Gene Identification for Ocular Congenital Cranial Motor Neuron Disorders Using Human Sequencing, Zebrafish Screening, and Protein Binding Microarrays.},
journal = {Investigative ophthalmology & visual science},
volume = {66},
number = {3},
pages = {62},
pmid = {40162949},
issn = {1552-5783},
mesh = {*Zebrafish ; Animals ; Humans ; Zebrafish Proteins/genetics/metabolism ; Disease Models, Animal ; Transcription Factors/genetics/metabolism ; Homeodomain Proteins/genetics/metabolism ; Exome Sequencing ; CRISPR-Cas Systems ; },
abstract = {PURPOSE: To functionally evaluate novel human sequence-derived candidate genes and variants for unsolved ocular congenital cranial dysinnervation disorders (oCCDDs).

METHODS: Through exome and genome sequencing of a genetically unsolved human oCCDD cohort, we previously reported the identification of variants in many candidate genes. Here, we describe a parallel study that prioritized a subset of these genes (43 human genes, 57 zebrafish genes) using a G0 CRISPR/Cas9-based knockout assay in zebrafish and generated F2 germline mutants for 17. We tested the functionality of variants of uncertain significance in known and novel candidate transcription factor-encoding genes through protein binding microarrays.

RESULTS: We first demonstrated the feasibility of the G0 screen by targeting known oCCDD genes phox2a and mafba. Approximately 70% to 90% of gene-targeted G0 zebrafish embryos recapitulated germline homozygous null-equivalent phenotypes. Using this approach, we then identified three novel candidate oCCDD genes (SEMA3F, OLIG2, and FRMD4B) with putative contributions to human and zebrafish cranial motor development. In addition, protein binding microarrays demonstrated reduced or abolished DNA binding of human variants of uncertain significance in known and novel sequence-derived transcription factors PHOX2A (p.(Trp137Cys)), MAFB (p.(Glu223Lys)), and OLIG2 (p.(Arg156Leu)).

CONCLUSIONS: This study nominates three strong novel candidate oCCDD genes (SEMA3F, OLIG2, and FRMD4B) and supports the functionality and putative pathogenicity of transcription factor candidate variants PHOX2A p.(Trp137Cys), MAFB p.(Glu223Lys), and OLIG2 p.(Arg156Leu). Our findings support that G0 loss-of-function screening in zebrafish can be coupled with human sequence analysis and protein binding microarrays to aid in prioritizing oCCDD candidate genes/variants.},
}

*Zebrafish
Animals
Humans
Zebrafish Proteins/genetics/metabolism
Disease Models, Animal
Transcription Factors/genetics/metabolism
Homeodomain Proteins/genetics/metabolism
Exome Sequencing
CRISPR-Cas Systems

@article {pmid40014012,
year = {2025},
author = {Dort, EN and Feau, N and Hamelin, RC},
title = {Novel application of ribonucleoprotein-mediated CRISPR-Cas9 gene editing in plant pathogenic oomycete species.},
journal = {Microbiology spectrum},
volume = {13},
number = {4},
pages = {e0301224},
pmid = {40014012},
issn = {2165-0497},
support = {10106//Genome Canada (GC)/ ; CGS-D//Canadian Government | Natural Sciences and Engineering Research Council of Canada (NSERC)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Phytophthora/genetics/drug effects/pathogenicity ; *Plant Diseases/microbiology ; *Ribonucleoproteins/genetics/metabolism ; Fungicides, Industrial/pharmacology ; Oomycetes/genetics/pathogenicity/drug effects ; Virulence/genetics ; Hydrocarbons, Fluorinated ; Pyrazoles ; },
abstract = {CRISPR-Cas9 gene editing has become an important tool for the study of plant pathogens, allowing researchers to functionally characterize specific genes involved in phytopathogenicity, virulence, and fungicide resistance. Protocols for CRISPR-Cas9 gene editing have already been developed for Phytophthoras, an important group of oomycete plant pathogens; however, these efforts have exclusively focused on agricultural pathosystems, with research lacking for forest pathosystems. We sought to develop CRISPR-Cas9 gene editing in two forest pathogenic Phytophthoras, Phytophthora cactorum and P. ramorum, using a plasmid-ribonucleoprotein (RNP) co-transformation approach. Our gene target in both species was the ortholog of PcORP1, which encodes an oxysterol-binding protein that is the target of the fungicide oxathiapiprolin in the agricultural pathogen P. capsici. We delivered liposome complexes, each containing plasmid DNA and CRISPR-Cas9 RNPs, to Phytophthora protoplasts using a polyethylene glycol-mediated transformation protocol. We obtained two ORP1 mutants in P. cactorum but were unable to obtain any mutants in P. ramorum. The two P. cactorum mutants exhibited decreased resistance to oxathiapiprolin, as measured by their radial growth relative to wild-type cultures on oxathiapiprolin-supplemented medium. Our results demonstrate the potential for RNP-mediated CRISPR-Cas9 gene editing in P. cactorum and provide a foundation for future optimization of our protocol in other forest pathogenic Phytophthora species.IMPORTANCECRISPR-Cas9 gene editing has become a valuable tool for characterizing the genetics driving virulence and pathogenicity in plant pathogens. CRISPR-Cas9 protocols are now well-established in several Phytophthora species, an oomycete genus with significant economic and ecological impact globally. These protocols, however, have been developed for agricultural Phytophthora pathogens only; CRISPR-Cas9 systems have not yet been developed for any forest pathogenic Phytophthoras. In this study, we sought to establish CRISPR-Cas9 gene editing in two forest Phytophthora pathogens that cause widespread tree mortality: P. cactorum and P. ramorum. We successfully obtained gene mutations in P. cactorum and demonstrated a decrease in fungicide resistance, a trait that could impact the pathogen's ability to cause disease. However, the same protocol did not yield any mutants in P. ramorum. The results of our study will serve as a baseline for the development of CRISPR-Cas9 gene editing in forest Phytophthoras and other oomycetes.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Phytophthora/genetics/drug effects/pathogenicity
*Plant Diseases/microbiology
*Ribonucleoproteins/genetics/metabolism
Fungicides, Industrial/pharmacology
Oomycetes/genetics/pathogenicity/drug effects
Virulence/genetics
Hydrocarbons, Fluorinated
Pyrazoles

@article {pmid39998241,
year = {2025},
author = {Yang, F and Wu, Q and Zeng, X and Jiang, Q and Zhang, S and Wang, J and Zhang, Q and Li, F and Xu, D},
title = {The establishment and optimization of a Mycoplasma pneumoniae detection system based on ERA-CRISPR/Cas12a.},
journal = {Microbiology spectrum},
volume = {13},
number = {4},
pages = {e0323524},
pmid = {39998241},
issn = {2165-0497},
mesh = {*Mycoplasma pneumoniae/genetics/isolation & purification ; *CRISPR-Cas Systems ; Humans ; *Pneumonia, Mycoplasma/diagnosis/microbiology ; *Sensitivity and Specificity ; Bacterial Proteins/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Limit of Detection ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {UNLABELLED: Mycoplasma pneumoniae (MP) is a significant pathogen associated with community-acquired pneumonia, with considerable infectious risks posed, particularly to children and immunocompromised individuals. The current methods for detecting MP in research and clinical settings are recognized as time-consuming, instrument-dependent, and prone to non-specific cross-reactivity. Therefore, the creation of a rapid and sensitive detection method is urgently required. In this study, the MP-ERA-Cas12a system, integrating enzyme restriction amplification (ERA) with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a technology, was introduced. Three detection methods were evaluated: the two-pot system, a modified one-pot system, and a lateral flow assay (LFA) strip-based system. In the one-pot system, the amplification and detection steps were consolidated within a single reaction vessel, effectively minimizing the risk of contamination and false positives that may arise from the handling of multiple tubes. It was observed that the one-pot system generated a fluorescent signal within 1 h and produced 1.6 times higher fluorescence signal intensity compared to the two-pot system, achieving a detection limit of 1 copy/μL. In contrast, the LFA system facilitated rapid on-site screening, with visible band results appearing on the strip within 5 min of the reaction, and a detection limit of 10[2] copies/μL was achieved. High specificity for MP was demonstrated by all methods. Significant advantages, including rapid processing, the absence of complex instrumentation, and ease of use are offered by this detection system, making it particularly suitable for resource-limited clinical settings. The system is seen as an efficient tool for the early diagnosis of MP, with substantial public health and clinical relevance.

IMPORTANCE: This study successfully combined enzyme restriction amplification (ERA) with the specific detection capabilities of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a. Based on the two-pot system established before, the one-pot system and lateral flow assay (LFA) system were developed for Mycoplasma pneumoniae (MP) detection. The MP-ERA-Cas12a system eliminates the need to open the lid during the reaction, reducing aerosol contamination, and minimizing the risk of false positives. The method does not require the use of advanced instruments or equipment and shows strong specificity while not being affected by other pathogens. As a new method of MP detection, the MP-ERA-Cas12a system has an important practical application prospect.},
}

*Mycoplasma pneumoniae/genetics/isolation & purification
*CRISPR-Cas Systems
Humans
*Pneumonia, Mycoplasma/diagnosis/microbiology
*Sensitivity and Specificity
Bacterial Proteins/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Limit of Detection
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid39868490,
year = {2025},
author = {Martires, LCM and Ahronian, LG and Pratt, CB and Das, NM and Zhang, X and Whittington, DA and Zhang, H and Shen, B and Come, J and McCarren, P and Liu, MS and Min, C and Feng, T and Jahic, H and Ali, JA and Aird, DR and Li, F and Andersen, JN and Huang, A and Mallender, WD and Nicholson, HE},
title = {LIG1 Is a Synthetic Lethal Target in BRCA1 Mutant Cancers.},
journal = {Molecular cancer therapeutics},
volume = {24},
number = {4},
pages = {618-627},
doi = {10.1158/1535-7163.MCT-24-0598},
pmid = {39868490},
issn = {1538-8514},
mesh = {Humans ; *DNA Ligase ATP/genetics/metabolism ; *BRCA1 Protein/genetics ; Animals ; Mice ; Female ; *Synthetic Lethal Mutations ; Cell Line, Tumor ; Mutation ; Xenograft Model Antitumor Assays ; Breast Neoplasms/genetics/pathology/drug therapy/metabolism ; Ovarian Neoplasms/genetics/pathology/drug therapy/metabolism ; CRISPR-Cas Systems ; },
abstract = {Synthetic lethality approaches in BRCA1/2-mutated cancers have focused on PARP inhibitors, which are subject to high rates of innate or acquired resistance in patients. In this study, we used CRISPR/Cas9-based screening to identify DNA ligase I (LIG1) as a novel target for synthetic lethality in BRCA1-mutated cancers. Publicly available data supported LIG1 hyperdependence of BRCA1 mutant cells across a variety of breast and ovarian cancer cell lines. We used CRISPRn, CRISPRi, RNAi, and protein degradation to confirm the lethal effect of LIG1 inactivation at the DNA, RNA, and protein level in BRCA1 mutant cells in vitro. LIG1 inactivation resulted in viability loss across multiple BRCA1-mutated cell lines, whereas no effect was observed in BRCA1/2 wild-type cell lines, demonstrating target selectivity for the BRCA1 mutant context. On-target nature of the phenotype was demonstrated through rescue of viability with exogenous wild-type LIG1 cDNA. Next, we demonstrated a concentration-dependent relationship of LIG1 protein expression and BRCA1 mutant cell viability using a titratable, degradable LIG1 fusion protein. BRCA1 mutant viability required LIG1 catalytic activity, as catalytically dead mutant LIG1K568A failed to rescue viability loss caused by endogenous LIG1 depletion. LIG1 perturbation produced proportional increases in PAR staining in BRCA1 mutant cells, indicating a mechanism consistent with the function of LIG1 in sealing ssDNA nicks. Finally, we confirmed LIG1 hyperdependence in vivo using a xenograft model in which LIG1 loss resulted in tumor stasis in all mice. Our cumulative findings demonstrate that LIG1 is a promising synthetic lethal target for development in patients with BRCA1-mutant cancers.},
}

Humans
*DNA Ligase ATP/genetics/metabolism
*BRCA1 Protein/genetics
Animals
Mice
Female
*Synthetic Lethal Mutations
Cell Line, Tumor
Mutation
Xenograft Model Antitumor Assays
Breast Neoplasms/genetics/pathology/drug therapy/metabolism
Ovarian Neoplasms/genetics/pathology/drug therapy/metabolism
CRISPR-Cas Systems

@article {pmid39820712,
year = {2025},
author = {Tian, G and Barragan, GA and Yu, H and Martinez-Amador, C and Adaikkalavan, A and Rios, X and Guo, L and Drabek, JM and Pardias, O and Xu, X and Montalbano, A and Zhang, C and Li, Y and Courtney, AN and Di Pierro, EJ and Metelitsa, LS},
title = {PRDM1 Is a Key Regulator of the NKT-cell Central Memory Program and Effector Function.},
journal = {Cancer immunology research},
volume = {13},
number = {4},
pages = {577-590},
doi = {10.1158/2326-6066.CIR-24-0259},
pmid = {39820712},
issn = {2326-6074},
support = {RO1 CA262250//National Institutes of Health (NIH)/ ; K12 CA090433/CA/NCI NIH HHS/United States ; RP210027//Cancer Prevention and Research Institute of Texas (CPRIT)/ ; 983124//Hyundai Hope On Wheels (Hope On Wheels)/ ; //Kate Amato Foundation/ ; },
mesh = {*Natural Killer T-Cells/immunology ; Humans ; Animals ; *Positive Regulatory Domain I-Binding Factor 1/genetics/metabolism/immunology ; Mice ; *Immunologic Memory ; Cell Line, Tumor ; CRISPR-Cas Systems ; Neuroblastoma/immunology/genetics/therapy ; Immunotherapy, Adoptive/methods ; Xenograft Model Antitumor Assays ; Receptors, Chimeric Antigen/immunology/genetics ; },
abstract = {Natural killer T cells (NKTs) are a promising platform for cancer immunotherapy, but few genes involved in the regulation of NKT therapeutic activity have been identified. To find regulators of NKT functional fitness, we developed a CRISPR/Cas9-based mutagenesis screen that uses a guide RNA (gRNA) library targeting 1,118 immune-related genes. Unmodified NKTs and NKTs expressing a GD2-specific chimeric antigen receptor (GD2.CAR) were transduced with the gRNA library and exposed to CD1d+ leukemia or CD1d-GD2+ neuroblastoma cells, respectively, over six challenge cycles in vitro. Quantification of gRNA abundance revealed enrichment of PRDM1-specific gRNAs in both NKTs and GD2.CAR NKTs, a result that was validated through targeted PRDM1 knockout. Transcriptional, phenotypic, and functional analyses demonstrated that CAR NKTs with PRDM1 knockout underwent central memory-like differentiation and resisted exhaustion. However, these cells downregulated the cytotoxic mediator granzyme B and showed reduced in vitro cytotoxicity and only moderate in vivo antitumor activity in a xenogeneic neuroblastoma model. In contrast, short hairpin RNA-mediated PRDM1 knockdown preserved effector function while promoting central memory differentiation, resulting in GD2.CAR NKTs with potent in vivo antitumor activity. Thus, we have identified PRDM1 as a regulator of NKT memory differentiation and effector function that can be exploited to improve the efficacy of NKT-based cancer immunotherapies.},
}

*Natural Killer T-Cells/immunology
Humans
Animals
*Positive Regulatory Domain I-Binding Factor 1/genetics/metabolism/immunology
Mice
*Immunologic Memory
Cell Line, Tumor
CRISPR-Cas Systems
Neuroblastoma/immunology/genetics/therapy
Immunotherapy, Adoptive/methods
Xenograft Model Antitumor Assays
Receptors, Chimeric Antigen/immunology/genetics

@article {pmid39754619,
year = {2025},
author = {Raina, K and Modak, K and Premkumar, C and Joshi, G and Palani, D and Nandy, K and Sivamani, Y and Velayudhan, SR and Thummer, RP},
title = {UTF1 Expression is Important for the Generation and Maintenance of Human iPSCs.},
journal = {Stem cell reviews and reports},
volume = {21},
number = {3},
pages = {859-871},
pmid = {39754619},
issn = {2629-3277},
support = {CRG/2021/004414//SERB-DST Govt of India/ ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Cell Differentiation/genetics ; *Cellular Reprogramming/genetics ; Fibroblasts/metabolism/cytology ; CRISPR-Cas Systems/genetics ; Octamer Transcription Factor-3/metabolism/genetics ; Gene Knockout Techniques ; Gene Editing ; Nuclear Proteins/genetics/metabolism ; Trans-Activators ; },
abstract = {BACKGROUND: Undifferentiated embryonic cell transcription factor 1 (UTF1) is predominantly expressed in pluripotent stem cells and plays a vital role in embryonic development and pluripotency maintenance. Despite its established importance in murine models, the role of UTF1 on human induced pluripotent stem cells (iPSCs) has not been comprehensively studied.

METHODS: This study utilized CRISPR/Cas9 gene editing to create UTF1 knockout in human fibroblasts and iPSCs. We employed episomal vectors for reprogramming UTF1 knockout fibroblasts into iPSCs and analyzed the effects of UTF1 depletion on cellular morphology, pluripotency, and viability through Western blotting, PCR, and flow cytometry. In addition, we integrated an shRNA that downregulated the expression of UTF1 for mechanistic studies to understand the impact of UTF1 depletion in iPSC pluripotency and differentiation.

RESULTS: UTF1 knockout resulted in significantly reduced reprogramming efficiency and increased spontaneous differentiation, indicating its crucial role in maintaining human iPSC identity and stability. In knockdown experiments, gradual loss of UTF1 led to change in cellular morphologies and decreased expression of core pluripotency markers OCT4 and SOX2. Interestingly, unlike complete UTF1 knockout, the gradual downregulation of UTF1 in iPSCs did not result in apoptosis, suggesting that the loss of pluripotency can occur independently of the apoptotic pathways.

CONCLUSIONS: UTF1 is essential for maintaining the pluripotency and viability of human iPSCs. Its depletion affects the fundamental properties of stem cells, underscoring the potential challenges in using UTF1-deficient cells for therapeutic applications. Future studies should explore the mechanistic pathways through which UTF1 controls pluripotency and differentiation, which could provide insights into improving iPSC stability for clinical applications.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Cell Differentiation/genetics
*Cellular Reprogramming/genetics
Fibroblasts/metabolism/cytology
CRISPR-Cas Systems/genetics
Octamer Transcription Factor-3/metabolism/genetics
Gene Knockout Techniques
Gene Editing
Nuclear Proteins/genetics/metabolism
Trans-Activators

@article {pmid39692072,
year = {2025},
author = {Deng, YJ and Duan, AQ and Li, T and Tan, SS and Liu, SS and Wang, YH and Ma, J and Li, JW and Liu, H and Xu, ZS and Liang, Y and Zhou, JH and Xiong, AS},
title = {Altering Carotene Hydroxylase Activity of DcCYP97C1 Affects Carotenoid Flux and Changes Taproot Colour in Carrot.},
journal = {Plant, cell & environment},
volume = {48},
number = {5},
pages = {3118-3135},
doi = {10.1111/pce.15331},
pmid = {39692072},
issn = {1365-3040},
support = {//The research was supported by the National Natural Science Foundation of China (32072563) and the Priority Academic Program Development of Jiangsu Higher Education Institutions Project (PAPD)./ ; },
mesh = {*Daucus carota/metabolism/genetics/enzymology ; *Carotenoids/metabolism ; *Plant Proteins/metabolism/genetics ; *Plant Roots/metabolism/genetics ; *Cytochrome P-450 Enzyme System/metabolism/genetics ; Pigmentation/genetics ; Plants, Genetically Modified ; Color ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; Lutein/metabolism ; Gene Knockout Techniques ; Mixed Function Oxygenases/metabolism/genetics ; },
abstract = {CYP97C1 as a haem-containing cytochrome P450 hydroxylase (P450-type) is important for carotene hydroxylation and xanthophyll biosynthesis. Research about this type of hydroxylase was mainly reported in several model plant species which have no specialized tissues accumulating massive carotenoids. The function of CYP97C1 in the horticultural plant, like carrots, was not fully studied. In this study, we focused on the role of DcCYP97C1 in carotenoid flux and colour formation in carrot. DcCYP97C1 was found highly expressed in the 'turning stage' of carrot taproot. Using stable transformation and CRISPR/Cas9-mediated gene knockout technology, DcCYP97C1 was confirmed the rate-limiting enzyme for lutein biosynthesis and important for taproot colour formation. Overexpression of DcCYP97C1 in an orange carrot KRD (Kurodagosun) resulted in five times overproduction of lutein accompanied by dramatic reduction of carotenes. Knockout of DcCYP97C1 in orange KRD and yellow carrot QTH (Qitouhuang) reduced all kinds of carotenoids including lutein, α-carotene and β-carotene reflecting the key role of DcCYP97C1 for total carotenoid accumulation in taproot 'turning stage'. Our study demonstrated that manipulation of DcCYP97C1 was sufficient to influence carotenoid flux, change carrot colour and for high lutein production. The uncovered role of DcCYP97C1 may be helpful for understanding plant carotenoid metabolism and breeding colourful carrot cultivars.},
}

*Daucus carota/metabolism/genetics/enzymology
*Carotenoids/metabolism
*Plant Proteins/metabolism/genetics
*Plant Roots/metabolism/genetics
*Cytochrome P-450 Enzyme System/metabolism/genetics
Pigmentation/genetics
Plants, Genetically Modified
Color
Gene Expression Regulation, Plant
CRISPR-Cas Systems
Lutein/metabolism
Gene Knockout Techniques
Mixed Function Oxygenases/metabolism/genetics

@article {pmid39641183,
year = {2025},
author = {Li, Y and Yang, J and Zhang, Q and Zhang, K and Xue, Q and Liu, W and Ding, X and Niu, Z},
title = {CRISPR-Cas9 Mediated Gene Editing Platform Through Callus-to-Plant Regeneration and Functional Analysis of DoALA4─DoALA6 in Dendrobium officinale.},
journal = {Plant, cell & environment},
volume = {48},
number = {5},
pages = {2923-2936},
doi = {10.1111/pce.15312},
pmid = {39641183},
issn = {1365-3040},
support = {//This work was supported by grants from the National Natural Science Foundation of China (Grant No. 32470384, 31900268 and 32070353) and Agricultural Science and Technology Independent Innovation Fund Project of Jiangsu Province (CX (22)3147)./ ; },
mesh = {*Dendrobium/genetics/physiology ; *Gene Editing ; *CRISPR-Cas Systems ; *Regeneration/genetics ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Genes, Plant ; },
abstract = {Dendrobium orchids are well known for their great horticultural and medicinal values; however, the CRISPR/Cas9 gene editing system for Dendrobium species still needs to be improved. Therefore, this study aims to establish a CRISPR/Cas9-based functional validation system using Dendrobium officinale as a model species for the Dendrobium genus and to validate the DoALA4─DoALA6 genes, which may relate to growth and disease resistance. We first conducted a bioinformatics analysis of the P-type ATPase gene family in D. officinale, revealing the evolutionary diversity of P-type ATPase genes in orchids. Second, we inserted the GFP gene into the vector of CRISPR/Cas9 gene editing system to enhance the selection efficiency of genome-edited plants. Comparative analysis showed that different explants exhibited varying transformation efficiencies, ranging from 5% to 46.2%. Considering the regeneration capability, survival rate and gene editing efficiency, we selected callus as the transformation explant. Third, we used this editing system to generate DoALA4─DoALA6 mutants. Phenotypic observations of the mutants and inoculation of D. officinale with Sclerotium rolfsii indicated that DoALA4─DoALA6 are crucial for the growth of D. officinale and its resistance to southern blight disease. This efficient and stable CRISPR/Cas9 platform offers a foundation for further gene studies and Dendrobium breeding.},
}

*Dendrobium/genetics/physiology
*Gene Editing
*CRISPR-Cas Systems
*Regeneration/genetics
Plant Proteins/genetics/metabolism
Plants, Genetically Modified
Genes, Plant

@article {pmid40161755,
year = {2025},
author = {Ratna, TA and Sharon, BM and Velin, CAB and Buttaro, BA and Palmer, KL},
title = {Factors affecting CRISPR-Cas defense against antibiotic resistance plasmids harbored by Enterococcus faecalis laboratory model strains and clinical isolates.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.03.10.642232},
pmid = {40161755},
issn = {2692-8205},
abstract = {UNLABELLED: Enterococcus faecalis is a Gram-positive bacterium and opportunistic pathogen that acquires resistance to a wide range of antibiotics by horizontal gene transfer (HGT). The rapid increase of multidrug-resistant (MDR) bacteria including MDR E. faecalis necessitates the development of alternative therapies and a deeper understanding of the factors that impact HGT. CRISPR-Cas systems provide sequence-specific defense against HGT. From previous studies, we know that E. faecalis CRISPR-Cas provides sequence-specific anti-plasmid defense during agar plate biofilm mating and in the murine intestine. Those studies were mainly conducted using laboratory model strains with a single, CRISPR-targeted plasmid in the donor. MDR E. faecalis typically possess multiple plasmids that are diverse in sequence and may interact with each other to impact plasmid transfer and CRISPR-Cas efficacy. Here, we altered multiple parameters of our standard in vitro conjugation assays to assess CRISPR-Cas efficacy, including the number and genotype of plasmids in the donor; laboratory model strains as donor versus recent human isolates as donor; and the biofilm substrate utilized during conjugation. We found that the plasmids pTEF2 and pCF10, which are not targeted by CRISPR-Cas in our recipient, enhance the conjugative transfer of the CRISPR-targeted plasmid pTEF1 into both wild-type and CRISPR-Cas-deficient (via deletion of cas9) recipient cells. However, the effect of pTEF2 on pTEF1 transfer is much more pronounced, with a striking 6-log increase in pTEF1 conjugation frequency when pTEF2 is also present in the donor and recipients are deficient for CRISPR-Cas (compared to 4-log for pCF10). We also identified that E. faecalis Δ cas9 has altered biofilm structure and thickness relative to the wild-type strain when cultured on a plastic substrate, but equivalent growth in the agar plate biofilms widely used for conjugation studies. Overall, this study provides insight about the interplay between plasmids and CRISPR-Cas defense, opening avenues for developing novel therapeutic strategies to curb HGT among bacterial pathogens, and highlighting pTEF2 as a plasmid for additional mechanistic study.

IMPORTANCE: The emergence of MDR bacteria, including MDR E. faecalis, limits treatment options and necessitates development of alternative therapeutics. In these circumstances, bacterial CRISPR-Cas systems are being explored by the field to develop CRISPR-based antimicrobials. However, in many cases CRISPR-Cas efficacy has only been assessed using laboratory model strains. More studies are required that investigate clinical isolates, as those are the intended targets for CRISPR antimicrobials. Here, we demonstrated how the number of plasmids harbored by an E. faecalis donor strain can affect the apparent efficacy of CRISPR-Cas anti-plasmid defense in a recipient strain. Overall, our research is important to develop improved CRISPR-based antimicrobials to combat the spread and accumulation of antibiotic resistance determinants.},
}

@article {pmid40160815,
year = {2025},
author = {Schönberg, PY and Muñoz-Ovalle, Á and Paszkowski-Rogacz, M and Crespo, E and Sürün, D and Feldmann, A and Buchholz, F},
title = {A pooled CRISPR screen identifies the Tα2 enhancer element as a driver of TRA expression in a subset of mature human T lymphocytes.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1536003},
pmid = {40160815},
issn = {1664-3224},
mesh = {Humans ; *Enhancer Elements, Genetic ; *CRISPR-Cas Systems ; Jurkat Cells ; Receptors, Antigen, T-Cell, alpha-beta/genetics ; T-Lymphocytes/immunology/metabolism ; Gene Expression Regulation ; },
abstract = {The T cell receptor (TCR) is crucial for immune responses and represents a pivotal therapeutic target for CAR T cell therapies. However, which enhancer elements drive the constitutive expression of the TCRα chain in mature, peripheral T cells has not been well defined. Earlier work has suggested that enhancer alpha is inactive in mature peripheral T cells and that an alternative enhancer element in the 5' J region was driving TRA expression, while more recent findings indicated the opposite. Here, we applied a pooled CRISPR screen to probe a large genomic region proximal to the human TRA gene for the presence of regulatory elements. Interestingly, no sgRNA targeting the 5' J region was identified that influenced TRA expression. In contrast, several sgRNAs targeting enhancer alpha element Tα2, were identified that compromised the expression of the TCRα chain in Jurkat E6.1, as well as in a subset of human primary T cells. Our results provide new insights into the regulation of TRA in human peripheral T cells, advancing our understanding of how constitutive TRA expression is driven and regulated.},
}

Humans
*Enhancer Elements, Genetic
*CRISPR-Cas Systems
Jurkat Cells
Receptors, Antigen, T-Cell, alpha-beta/genetics
T-Lymphocytes/immunology/metabolism
Gene Expression Regulation

@article {pmid40158491,
year = {2025},
author = {Khosla, NK and Lesinski, JM and Haywood-Alexander, M and deMello, AJ and Richards, DA},
title = {Machine learning and statistical classification in CRISPR-Cas12a diagnostic assays.},
journal = {Biosensors & bioelectronics},
volume = {279},
number = {},
pages = {117402},
doi = {10.1016/j.bios.2025.117402},
pmid = {40158491},
issn = {1873-4235},
abstract = {CRISPR-based diagnostics have gained increasing attention as biosensing tools able to address limitations in contemporary molecular diagnostic tests. To maximize the performance of CRISPR-based assays, much effort has focused on optimizing the chemistry and biology of the biosensing reaction. However, less attention has been paid to improving the techniques used to analyze CRISPR-based diagnostic data. To date, diagnostic decisions typically involve various forms of slope-based classification. Such methods are superior to traditional methods based on assessing absolute signals, but still have limitations. Herein, we establish performance benchmarks (total accuracy, sensitivity, and specificity) using common slope-based methods. We compare the performance of these benchmark methods with three different quadratic empirical distribution function statistical tests, finding significant improvements in diagnostic speed and accuracy when applied to a clinical data set. Two of the three statistical techniques, the Kolmogorov-Smirnov and Anderson-Darling tests, report the lowest time-to-result and highest total test accuracy. Furthermore, we developed a long short-term memory recurrent neural network to classify CRISPR-biosensing data, achieving 100 % specificity on our model data set. Finally, we provide guidelines on choosing the classification method and classification method parameters that best suit a diagnostic assay's needs.},
}

@article {pmid40157940,
year = {2025},
author = {Fang, L and Hao, X and Fan, J and Liu, X and Chen, Y and Wang, L and Huang, X and Song, H and Cao, Y},
title = {Genome-scale CRISPRi screen identifies pcnB repression conferring improved physiology for overproduction of free fatty acids in Escherichia coli.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3060},
pmid = {40157940},
issn = {2041-1723},
support = {22478294//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22308256//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2024T170643//China Postdoctoral Science Foundation/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; *Escherichia coli Proteins/genetics/metabolism ; *Fatty Acids, Nonesterified/biosynthesis/metabolism ; Genome, Bacterial ; Metabolic Engineering/methods ; Fermentation ; Gene Expression Regulation, Bacterial ; CRISPR-Cas Systems ; Repressor Proteins/genetics/metabolism ; High-Throughput Nucleotide Sequencing ; Flow Cytometry ; },
abstract = {Microbial physiology plays a pivotal role in construction of superior microbial cell factories for efficient biosynthesis of desired products. Here we identify that pcnB repression confers improved physiology for overproduction of free fatty acids (FFAs) in Escherichia coli through genome-scale CRISPRi modulation combining fluorescence-activated cell sorting (FACS) and next-generation sequencing (NGS). The repression of pcnB can enhance the stability and abundance of the transcripts of genes involved in the proton-consuming system, thereby supporting global improvements in membrane properties, redox state, and energy level. Based on pcnB repression, further repression of acrD increases FFAs biosynthesis by enhancing FFAs efflux. The engineered strain pcnB[i]-acrD[i]-fadR[+] achieves 35.1 g L[-1] FFAs production in fed-batch fermentation, which is the maximum titer reported to date in E. coli. This study highlights the significance of uncovering hidden genetic determinants that confer improved microbial physiology for enhancing the biosynthesis of desired products.},
}

*Escherichia coli/genetics/metabolism
*Escherichia coli Proteins/genetics/metabolism
*Fatty Acids, Nonesterified/biosynthesis/metabolism
Genome, Bacterial
Metabolic Engineering/methods
Fermentation
Gene Expression Regulation, Bacterial
CRISPR-Cas Systems
Repressor Proteins/genetics/metabolism
High-Throughput Nucleotide Sequencing
Flow Cytometry

@article {pmid40157676,
year = {2025},
author = {Li, Y and Sun, C and Yao, D and Gao, X and Wei, X and Qi, Y and Liang, Y and Ye, J},
title = {A review of MicroRNAs and flavonoids: New insights into plant secondary metabolism.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {142518},
doi = {10.1016/j.ijbiomac.2025.142518},
pmid = {40157676},
issn = {1879-0003},
abstract = {Flavonoids, essential plant secondary metabolites, play crucial roles in growth regulation, stress responses, and applications in medicine, agriculture, and industry. However, the complexity of their biosynthetic pathways and regulatory networks poses challenges for industrial-scale production. MicroRNAs (miRNAs), as pivotal post-transcriptional regulators, play significant roles in fine-tuning flavonoid metabolism by targeting key enzyme genes and transcription factors. This review provides a comprehensive analysis of miRNA biogenesis and their molecular mechanisms, emphasizing miRNA-mediated regulation of flavonoid biosynthesis. We introduce the concept of "miRNA-multifactorial synergistic networks", which elucidates the collaborative interactions between miRNAs, non-coding RNAs, transcription factors, and epigenetic regulators. The review explores emerging strategies, including artificial miRNA design and CRISPR/Cas technologies, to precisely manipulate miRNA activity for enhancing flavonoid production. Additionally, integrating CRISPR/Cas13, synthetic biology, and multi-omics technologies offers new opportunities to construct efficient flavonoid metabolic systems. Artificial intelligence (AI) is proposed as a powerful tool to analyze omics data, identify regulatory nodes, and simulate environmental impacts on miRNA networks, thereby optimizing metabolic pathways. By integrating these multidisciplinary approaches, this review provides a novel theoretical framework and technical roadmap for understanding and improving flavonoid metabolism. The insights presented here aim to facilitate breakthroughs in metabolic engineering, offering significant potential for practical applications in plant breeding, functional food production, and pharmaceutical development.},
}

@article {pmid40106763,
year = {2025},
author = {Xiao, X and Yang, X and Xu, K and Huang, F and Zhang, Y and Jiang, Y and Shi, Y and Zhou, Q and Wang, L and Lu, J and Gao, Z and Lou, Y},
title = {DNA Fragment Fusion and Nucleic Acid Detection by Fusion Recombinase-Aided Amplification.},
journal = {Analytical chemistry},
volume = {97},
number = {12},
pages = {6538-6547},
doi = {10.1021/acs.analchem.4c05991},
pmid = {40106763},
issn = {1520-6882},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism ; *Methicillin-Resistant Staphylococcus aureus/genetics/isolation & purification ; DNA, Bacterial/genetics/analysis ; CRISPR-Cas Systems/genetics ; DNA/genetics/chemistry ; Humans ; },
abstract = {Constructing fusion DNA fragments is frequently used for genetic engineering purposes. To date, fusion PCR is one of the most popular approaches for generating fusion DNA fragments. Here, we describe a novel method for DNA fusion based on the isothermal DNA amplification technique, recombinase-aided amplification (RAA). We demonstrate that this method, termed "fusion RAA", can assemble two to three DNA fragments to generate a fusion fragment of up to ∼1 kb in a one-pot reaction within 40 min at 37 °C. We further demonstrate that fusion RAA can realize fragment insertion, deletion, and base mutation. Moreover, we show that fusion RAA can be harnessed to facilitate pathogen detection by simultaneously targeting two genes in one RAA assay, as demonstrated by the rapid and simplified detection of methicillin-resistant Staphylococcus aureus (MRSA). Based on fusion RAA, we establish two novel pathogen detection platforms, FREAC (Fusion REcombinase-aided Amplification combined with CRISPR/Cas13a) and FREAL (Fusion REcombinase-aided Amplification combined with Lateral flow assay). Using these two platforms, we can detect clinical MRSA strains within 55 min with high specificity and a limit of detection of 150 copies/μL of genomic DNA, highlighting their potential as user-friendly platforms for nucleic acid detection.},
}

*Nucleic Acid Amplification Techniques/methods
*Recombinases/metabolism
*Methicillin-Resistant Staphylococcus aureus/genetics/isolation & purification
DNA, Bacterial/genetics/analysis
CRISPR-Cas Systems/genetics
DNA/genetics/chemistry
Humans

@article {pmid39720847,
year = {2025},
author = {Tang, A and Yokota, T},
title = {Is Duchenne gene therapy a suitable treatment despite its immunogenic class effect?.},
journal = {Expert opinion on drug safety},
volume = {24},
number = {4},
pages = {395-411},
doi = {10.1080/14740338.2024.2447072},
pmid = {39720847},
issn = {1744-764X},
mesh = {*Muscular Dystrophy, Duchenne/therapy/genetics/immunology ; Humans ; *Genetic Therapy/methods/adverse effects ; *Gene Editing/methods ; *Dystrophin/genetics ; Animals ; *CRISPR-Cas Systems ; Utrophin/genetics ; Genetic Vectors/administration & dosage ; },
abstract = {INTRODUCTION: Duchenne muscular dystrophy (DMD) is a severe X-linked disorder characterized by progressive muscle weakness and eventual death due to cardiomyopathy or respiratory complications. Currently, there is no cure for DMD, with standard treatments primarily focusing on symptom management. Using immunosuppressive measures and optimized vector designs allows for gene therapies to better address the genetic cause of the disease.

AREAS COVERED: This review evaluates the efficacy and safety of emerging DMD gene therapies as of 2024. It also discusses the potential of utrophin upregulation, gene editing, and truncated dystrophin as therapeutic strategies. It highlights safety concerns associated with these therapies, including adverse events and patient deaths. A comprehensive overview of developments covers topics such as CRISPR-Cas9 therapies, micro-dystrophin, and the potential delivery of full-length dystrophin.

EXPERT OPINION: The FDA's recent approval of delandistrogene moxeparvovec (Elevidys) underscores the promise of gene replacement therapies for DMD patients. Understanding the mechanisms behind the adverse effects and excluding patients with specific pathogenic variants may enhance the safety profiles of these therapies. CRISPR/Cas9 therapies, while promising, face significant regulatory and safety challenges that hinder their clinical application. Optimal DMD therapies should target both skeletal and cardiac muscles to be effective.},
}

*Muscular Dystrophy, Duchenne/therapy/genetics/immunology
Humans
*Genetic Therapy/methods/adverse effects
*Gene Editing/methods
*Dystrophin/genetics
Animals
*CRISPR-Cas Systems
Utrophin/genetics
Genetic Vectors/administration & dosage

@article {pmid39497550,
year = {2025},
author = {Zhu, Z and Fu, B and Lu, J and Wang, P and Yan, C and Guan, F and Huang, J and Yu, P},
title = {Engineered production of 5-aminolevulinic acid in recombinant Escherichia coli BL21.},
journal = {Preparative biochemistry & biotechnology},
volume = {55},
number = {4},
pages = {446-456},
doi = {10.1080/10826068.2024.2423644},
pmid = {39497550},
issn = {1532-2297},
mesh = {*Aminolevulinic Acid/metabolism ; *Escherichia coli/genetics/metabolism ; *Metabolic Engineering/methods ; CRISPR-Cas Systems ; Fermentation ; Escherichia coli Proteins/genetics/metabolism ; },
abstract = {5-aminolevulinic acid (ALA) is a non-protein amino acid that has been widely used in the fields of medicine and agriculture. This study aims to engineer the C5 pathway of the ALA biosynthesis in Escherichia coli BL21 to enhance ALA production. The ALA synthase genes gltX, hemA, and hemL were overexpressed in E. coli BL21 to lead to the increase in the production of ALA. The sRNA RyhB was also overexpressed to downregulate the expression of ALA dehydratase to reduce the downstream bioconversion of ALA to porphobilinogen. Next, the gene arcA was knocked out by CRISPR-Cas9 technology to open the TCA cycle to promote the respiratory metabolism of the strain to reduce the feedback inhibition of heme to ALA. The fermentation conditions of the engineered strain were optimized by response surface experiments. The time-course analysis of the ALA production was carried out in a 1 L shake flask. Through these efforts, the production of ALA in engineered strain reached 2953 mg/L in a 1 L shake flask. This study contributes to the industrial production of ALA by the engineered E. coli in the future.},
}

*Aminolevulinic Acid/metabolism
*Escherichia coli/genetics/metabolism
*Metabolic Engineering/methods
CRISPR-Cas Systems
Fermentation
Escherichia coli Proteins/genetics/metabolism

@article {pmid40157335,
year = {2025},
author = {Paranthaman, S and Uthaiah, CA and Md, S and Alkreathy, HM},
title = {Comprehensive strategies for constructing efficient CRISPR/Cas based cancer therapy: Target gene selection, sgRNA optimization, delivery methods and evaluation.},
journal = {Advances in colloid and interface science},
volume = {341},
number = {},
pages = {103497},
doi = {10.1016/j.cis.2025.103497},
pmid = {40157335},
issn = {1873-3727},
abstract = {Cancer is a complicated disease that results from the interplay between specific changes in cellular genetics and diverse microenvironments. The application of high-performance and customizable clustered regularly interspaced palindromic repeats/associated protein (CRISPR/Cas) nuclease systems has significantly enhanced genome editing for accurate cancer modeling and facilitated simultaneous genetic modification for cancer therapy and mutation identification. Achieving an effective CRISPR/Cas platform for cancer treatment depends on the identification, selection, and optimization of specific mutated genes in targeted cancer tissues. However, overcoming the off-target effects, specificity, and immunogenicity are additional challenges that must be addressed while developing a gene editing system for cancer therapy. From this perspective, we briefly covered the pipeline of CRISPR/Cas cancer therapy, identified target genes to optimize gRNAs and sgRNAs, and explored alternative delivery modalities, including viral, non-viral, and extracellular vesicles. In addition, the list of patents and current clinical trials related to this unique cancer therapy method is discussed. In summary, we have discussed comprehensive start-to-end pipeline strategies for CRISPR/Cas development to advance the precision, effectiveness, and safety of clinical applications for cancer therapy.},
}

@article {pmid40157150,
year = {2025},
author = {Song, Y and Long, J and Wang, H and Tang, W and Yang, W and Zheng, Y and Yuan, R and Zhang, D and Gu, B and Nian, W},
title = {High-efficiency detection of APE1 using a defective PAM-driven CRISPR-Cas12a self-catalytic biosensor.},
journal = {Biosensors & bioelectronics},
volume = {279},
number = {},
pages = {117410},
doi = {10.1016/j.bios.2025.117410},
pmid = {40157150},
issn = {1873-4235},
abstract = {The trans-cleavage activity of the CRISPR-Cas system offers tremendous potential for developing highly sensitive and selective molecular diagnostic tools. However, conventional methods often face challenges such as limited catalytic efficiency of single Cas proteins and the necessity of complex multi-enzyme preamplification steps. To address these limitations, we present a novel defective PAM-mediated CRISPR-Cas12a self-catalytic signal amplification strategy, termed DEP-Cas-APE, for the rapid, sensitive, and specific detection of apurinic/apyrimidinic endonuclease 1 (APE1) activity. This approach integrates defective PAM-modified DNA probes to synergize Cas12a trans-cleavage with self-catalytic circuit, achieving efficient signal transformation and amplification under isothermal, one-step conditions. We systematically investigated the influence of defective PAM sequences containing apurinic/apyrimidinic (AP) sites on Cas12a activation and validated the feasibility of the DEP-Cas-APE strategy in detecting APE1. Under optimized conditions, DEP-Cas-APE achieved a detection limit as low as 7.66 × 10[-8] U μL[-1] within 30 min using a simple isothermal reaction. Additionally, we developed a point-of-care testing (POCT) platform by integrating DEP-Cas-APE with a colorimetric assay based on gold nanoparticles (AuNPs), enabling portable, equipment-free detection. This sensitive and selective strategy successfully detected APE1 in complex biological samples, including serum from lung cancer patients, and demonstrated the ability to distinguish cancerous from normal samples. DEP-Cas-APE represents a robust and versatile platform for advancing CRISPR-Cas12a biosensing technologies, offering new opportunities for molecular diagnostics and clinical research.},
}

@article {pmid40156858,
year = {2025},
author = {Schmidt, T and Wiesbeck, M and Egert, L and Truong, TT and Danese, A and Voshagen, L and Imhof, S and Iraci Borgia, M and Deeksha, and Neuner, AM and Köferle, A and Geerlof, A and Santos Dias Mourão, A and Stricker, SH},
title = {Efficient DNA- and virus-free engineering of cellular transcriptomic states using dCas9 ribonucleoprotein (dRNP) complexes.},
journal = {Nucleic acids research},
volume = {53},
number = {6},
pages = {},
doi = {10.1093/nar/gkaf235},
pmid = {40156858},
issn = {1362-4962},
support = {//EpiCrossBorders/ ; //International Helmholtz-Edinburgh Research School for Epigenetics/ ; //Helmholtz Zentrum München/ ; STR 1385/5-1//DFG/ ; INST 86/2110-1//SFB/ ; //REGENERAR/ ; /CAPMC/CIHR/Canada ; //Natural Sciences and Engineering Research Council of Canada/ ; //Humanities Research Council of Canada/ ; },
mesh = {Humans ; *Ribonucleoproteins/metabolism/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Transcriptome ; CRISPR-Associated Protein 9/metabolism/genetics ; HEK293 Cells ; DNA/metabolism/genetics ; Promoter Regions, Genetic ; Transcriptional Activation ; },
abstract = {For genome editing, the use of CRISPR ribonucleoprotein (RNP) complexes is well established and often the superior choice over plasmid-based or viral strategies. RNPs containing dCas9 fusion proteins, which enable the targeted manipulation of transcriptomes and epigenomes, remain significantly less accessible. Here, we describe the production, delivery, and optimization of second generation CRISPRa RNPs (dRNPs). We characterize the transcriptional and cellular consequences of dRNP treatments in a variety of human target cells and show that the uptake is very efficient. The targeted activation of genes demonstrates remarkable potency, even for genes that are strongly silenced, such as developmental master transcription factors. In contrast to DNA-based CRISPRa strategies, gene activation is immediate and characterized by a sharp temporal precision. We also show that dRNPs allow very high-target multiplexing, enabling undiminished gene activation of multiple genes simultaneously. Applying these insights, we find that intensive target multiplexing at single promoters synergistically elevates gene transcription. Finally, we demonstrate in human stem and differentiated cells that the preferable features of dRNPs allow to instruct and convert cell fates efficiently without the need for DNA delivery or viral vectors.},
}

Humans
*Ribonucleoproteins/metabolism/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Transcriptome
CRISPR-Associated Protein 9/metabolism/genetics
HEK293 Cells
DNA/metabolism/genetics
Promoter Regions, Genetic
Transcriptional Activation

@article {pmid40156857,
year = {2025},
author = {Vasquez, CA and Osgood, NRB and Zepeda, MU and Sandel, DK and Cowan, QT and Peiris, MN and Donoghue, DJ and Komor, AC},
title = {Precision genome editing and in-cell measurements of oxidative DNA damage repair enable functional and mechanistic characterization of cancer-associated MUTYH variants.},
journal = {Nucleic acids research},
volume = {53},
number = {6},
pages = {},
doi = {10.1093/nar/gkaf037},
pmid = {40156857},
issn = {1362-4962},
support = {//University of California, San Diego/ ; 27502//Research Corporation for Science Advancement/ ; /HHMI/Howard Hughes Medical Institute/United States ; //National Academies of Sciences, Engineering, and Medicine/ ; //Cancer Cell Signaling and Communication Training Program/ ; T32 CA009523/GF/NIH HHS/United States ; //Molecular Biophysics Training/ ; },
mesh = {*DNA Glycosylases/genetics/metabolism ; Humans ; *Gene Editing/methods ; *DNA Repair/genetics ; *DNA Damage/genetics ; Mutation ; Oxidative Stress/genetics ; Colorectal Neoplasms/genetics ; Guanine/metabolism/analogs & derivatives ; CRISPR-Cas Systems ; Cell Line, Tumor ; },
abstract = {Functional characterization of genetic variants has the potential to advance the field of precision medicine by enhancing the efficacy of current therapies and accelerating the development of new approaches to combat genetic diseases. MUTYH is a DNA repair enzyme that recognizes and repairs oxidatively damaged guanines [8-oxoguanine (8-oxoG)] mispaired with adenines (8-oxoG·A). While some mutations in the MUTYH gene are associated with colorectal cancer, most MUTYH variants identified in sequencing databases are classified as variants of uncertain significance. Convoluting clinical classification is the absence of data directly comparing homozygous versus heterozygous MUTYH mutations. In this study, we present the first effort to functionally characterize MUTYH variants using precision genome editing to generate heterozygous and homozygous isogenic cell lines. Using a MUTYH-specific lesion reporter in which we site-specifically incorporate an 8-oxoG·A lesion in a fluorescent protein gene, we measure endogenous MUTYH enzymatic activity and classify them as pathogenic or benign. Further, we modify this reporter to incorporate the MUTYH repair intermediate (8-oxoG across from an abasic site) and validate it with co-immunoprecipitation experiments to demonstrate its ability to characterize the mechanism by which MUTYH mutants are defective at DNA repair.},
}

*DNA Glycosylases/genetics/metabolism
Humans
*Gene Editing/methods
*DNA Repair/genetics
*DNA Damage/genetics
Mutation
Oxidative Stress/genetics
Colorectal Neoplasms/genetics
Guanine/metabolism/analogs & derivatives
CRISPR-Cas Systems
Cell Line, Tumor

@article {pmid40156855,
year = {2025},
author = {Miceli, F and Bracaglia, S and Sorrentino, D and Porchetta, A and Ranallo, S and Ricci, F},
title = {MAIGRET: a CRISPR-based immunoassay that employs antibody-induced cell-free transcription of CRISPR guide RNA strands.},
journal = {Nucleic acids research},
volume = {53},
number = {6},
pages = {},
doi = {10.1093/nar/gkaf238},
pmid = {40156855},
issn = {1362-4962},
support = {819160/ERC_/European Research Council/International ; 21965//Associazione Italiana per la Ricerca sul Cancro/ ; 2022ANCEK//Italian Ministry of University and Research/ ; 101165168/ERC_/European Research Council/International ; //Associazione Italiana per la Ricerca sul Cancro/ ; 2022FPYZ2N//Italian Ministry of University and Research/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; *Transcription, Genetic ; Immunoassay/methods ; CRISPR-Associated Proteins/metabolism ; Antibodies/immunology ; Humans ; Bacterial Proteins/genetics/metabolism ; Cell-Free System ; Endodeoxyribonucleases/metabolism ; Antigens/immunology/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Here we report on the development of a CRISPR-based assay for the sensitive and specific detection of antibodies and antigens directly in complex sample matrices. The assay, called Molecular Assay based on antibody-Induced Guide-RNA Enzymatic Transcription (MAIGRET), is based on the use of a responsive synthetic DNA template that triggers the cell-free in vitro transcription of a guide RNA strand upon recognition of a specific target antibody. Such transcribed guide RNA activates the DNA collateral activity of the Cas12a enzyme, leading to the downstream cleavage of a fluorophore/quencher-labeled reporter and thus resulting in an increase in the measured fluorescence signal. We have used MAIGRET for the detection of six different antibodies with high sensitivity (detection limit in the picomolar range) and specificity (no signal in the presence of non-target antibodies). MAIGRET can also be adapted to a competitive approach for the detection of specific antigens. With MAIGRET, we significantly expand the scope and applicability of CRISPR-based sensing approaches to potentially enable the measurement of any molecular target for which an antibody is available.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems
*Transcription, Genetic
Immunoassay/methods
CRISPR-Associated Proteins/metabolism
Antibodies/immunology
Humans
Bacterial Proteins/genetics/metabolism
Cell-Free System
Endodeoxyribonucleases/metabolism
Antigens/immunology/genetics
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40156749,
year = {2025},
author = {Reķēna, A and Pals, K and Gavrilović, S and Lahtvee, PJ},
title = {The role of ATP citrate lyase, phosphoketolase, and malic enzyme in oleaginous Rhodotorula toruloides.},
journal = {Applied microbiology and biotechnology},
volume = {109},
number = {1},
pages = {77},
pmid = {40156749},
issn = {1432-0614},
mesh = {*Rhodotorula/genetics/enzymology/metabolism/growth & development ; *ATP Citrate (pro-S)-Lyase/metabolism/genetics ; *Gene Knockout Techniques ; *Malate Dehydrogenase/metabolism/genetics ; Xylose/metabolism ; Aldehyde-Lyases/metabolism/genetics ; Glucose/metabolism ; Lipid Metabolism ; CRISPR-Cas Systems ; Culture Media/chemistry ; Acetates/metabolism ; },
abstract = {Rhodotorula toruloides is an oleaginous yeast recognized for its robustness and the production of high content of neutral lipids. Early biochemical studies have linked ATP citrate lyase (ACL), phosphoketolase (PK), and cytosolic malic enzyme (cMAE) with de novo lipid synthesis. In this study, we discovered that upon a CRISPR/Cas9-mediated knockout of the ACL gene, lipid content in R. toruloides IFO0880 decreased from 50 to 9% of its dry cell weight (DCW) in glucose medium and caused severe growth defects (reduced specific growth rate, changes in cell morphology). In xylose medium, the lipid content decreased from 43 to 38% of DCW. However, when grown on acetate as the sole carbon source, the lipid content decreased from 45 to 20% of DCW. Significant growth defects as a result of ACL knockout were observed on all substrates. In contrast, PK knockout resulted in no change in growth or lipid synthesis. Knocking out cMAE gene resulted in lipid increase of 2.9% of DCW and 23% increase in specific growth rate on glucose. In xylose or acetate medium, no change in lipid production as a result of cMAE gene knockout was observed. These results demonstrated that ACL plays a crucial role in lipid synthesis in R. toruloides IFO0880, as opposed to PK pathway or cMAE, whose presence in some conditions even disfavors lipid production. These results provided valuable information for future metabolic engineering of R. toruloides. KEY POINTS: • ACL is crucial for the fatty acid synthesis and growth in R. toruloides IFO0880. • Lipid production and cell growth is are unchanged as a result of PK knockout. • Cytosolic malic enzyme does not play a significant role in lipogenesis.},
}

*Rhodotorula/genetics/enzymology/metabolism/growth & development
*ATP Citrate (pro-S)-Lyase/metabolism/genetics
*Gene Knockout Techniques
*Malate Dehydrogenase/metabolism/genetics
Xylose/metabolism
Aldehyde-Lyases/metabolism/genetics
Glucose/metabolism
Lipid Metabolism
CRISPR-Cas Systems
Culture Media/chemistry
Acetates/metabolism

@article {pmid40156192,
year = {2025},
author = {Georgiadis, C and Preece, R and Waseem Qasim, },
title = {Clinical development of allogeneic chimeric antigen receptor αβ-T Cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2025.03.040},
pmid = {40156192},
issn = {1525-0024},
abstract = {Ready-made banks of allogeneic chimeric antigen receptor (CAR) T cells, produced to be available at the time of need, offer the prospect of accessible and cost-effective cellular therapies. Various strategies have been developed to overcome allogeneic barriers, drawing on cell engineering platforms including RNA interference, protein-based restriction and genome editing, including RNA-guided CRISPR-Cas and base editing tools. Alloreactivity and the risk of graft versus host disease from non-matched donor cells have been mitigated by disruption of αβ-T cell receptor expression on the surface of T cells, and stringent removal of any residual αβ-T cell populations. In addition, host mediated rejection has been tackled through a combination of augmented lymphodepletion and cell engineering strategies that have allowed infused cells to evade immune recognition or conferred resistance to lymphodepleting agents to promote persistence and expansion of effector populations. Early phase studies using 'off-the shelf' universal donor CAR T cells have been undertaken mainly in the context of blood malignancies, where emerging data of clinical responses have supported wider adoption and further applications. These developments offer the prospect of alternatives to current autologous approaches through the emerging application of genome engineering solutions to improve safety, persistence and function of universal donor products.},
}

@article {pmid40156040,
year = {2025},
author = {Zhang, Y and Zou, W and Zhou, Y and Chen, J and Hu, Y and Wu, F},
title = {Pamoic acid and carbenoxolone specifically inhibit CRISPR/Cas9 in bacteria, mammalian cells, and mice in a DNA topology-specific manner.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {75},
pmid = {40156040},
issn = {1474-760X},
support = {YG2025QNB55//Research Fund of Medicine and Engineering of Shanghai Jiao Tong University/ ; 32271304//National Natural Science Foundation of China/ ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *Staphylococcus aureus/drug effects/genetics ; *DNA/metabolism ; *Streptococcus pyogenes/drug effects/enzymology ; Humans ; *Carbenoxolone/pharmacology ; CRISPR-Associated Protein 9/metabolism ; DNA Cleavage/drug effects ; Gene Editing ; HEK293 Cells ; },
abstract = {BACKGROUND: Regulation of the target DNA cleavage activity of CRISPR/Cas has naturally evolved in a few bacteria or bacteriophages but is lacking in higher species. Thus, identification of bioactive agents and mechanisms that can suppress the activity of Cas9 is urgently needed to rebalance this new genetic pressure.

RESULTS: Here, we identify four specific inhibitors of Cas9 by screening 4607 compounds that could inhibit the endonuclease activity of Cas9 via three distinct mechanisms: substrate-competitive and protospacer adjacent motif (PAM)-binding site-occupation; substrate-targeting; and sgRNA-targeting mechanisms. These inhibitors inhibit, in a dose-dependent manner, the activity of Streptococcus pyogenes Cas9 (SpyCas9), Staphylococcus aureus Cas9 (SauCas9), and SpyCas9 nickase-based BE4 base editors in in vitro purified enzyme assays, bacteria, mammalian cells, and mice. Importantly, pamoic acid and carbenoxolone show DNA-topology selectivity and preferentially inhibit the cleavage of linear DNA compared with a supercoiled plasmid.

CONCLUSIONS: These pharmacologically selective inhibitors and new mechanisms offer new tools for controlling the DNA-topology selective activity of Cas9.},
}

Animals
Mice
*CRISPR-Cas Systems
*Staphylococcus aureus/drug effects/genetics
*DNA/metabolism
*Streptococcus pyogenes/drug effects/enzymology
Humans
*Carbenoxolone/pharmacology
CRISPR-Associated Protein 9/metabolism
DNA Cleavage/drug effects
Gene Editing
HEK293 Cells

@article {pmid40155371,
year = {2025},
author = {Chen, P and Wu, Y and Wang, H and Liu, H and Zhou, J and Chen, J and Lei, J and Sun, Z and Paek, C and Yin, L},
title = {Highly parallel profiling of the activities and specificities of Cas12a variants in human cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3022},
pmid = {40155371},
issn = {2041-1723},
support = {32371271//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32171210//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32101196//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2021TQ0253//China Postdoctoral Science Foundation/ ; 2022M712468//China Postdoctoral Science Foundation/ ; 2022M722473//China Postdoctoral Science Foundation/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; HEK293 Cells ; *CRISPR-Associated Proteins/metabolism/genetics ; Bacterial Proteins/genetics/metabolism ; Deep Learning ; Endodeoxyribonucleases/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; High-Throughput Nucleotide Sequencing ; },
abstract = {Several Cas12a variants have been developed to broaden its targeting range, improve the gene editing specificity or the efficiency. However, selecting the appropriate Cas12a among the many orthologs for a given target sequence remains difficult. Here, we perform high-throughput analyses to evaluate the activity and compatibility with specific PAMs of 24 Cas12a variants and develop deep learning models for these Cas12a variants to predict gene editing activities at target sequences of interest. Furthermore, we reveal and enhance the truncation in the integrated tag sequence that may hinder off-targeting detection for Cas12a by GUIDE-seq. This enhanced system, which we term enGUIDE-seq, is used to evaluate and compare the off-targeting and translocations of these Cas12a variants.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems
HEK293 Cells
*CRISPR-Associated Proteins/metabolism/genetics
Bacterial Proteins/genetics/metabolism
Deep Learning
Endodeoxyribonucleases/metabolism/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
High-Throughput Nucleotide Sequencing

@article {pmid40154762,
year = {2025},
author = {Awan, MJA and Farooq, MA and Buzdar, MI and Zia, A and Ehsan, A and Waqas, MAB and Hensel, G and Amin, I and Mansoor, S},
title = {Advances in gene editing-led route for hybrid breeding in crops.},
journal = {Biotechnology advances},
volume = {81},
number = {},
pages = {108569},
doi = {10.1016/j.biotechadv.2025.108569},
pmid = {40154762},
issn = {1873-1899},
abstract = {With the global demand for sustainable agriculture on the rise, RNA-guided nuclease technology offers transformative applications in crop breeding. Traditional hybrid breeding methods, like three-line and two-line systems, are often labor-intensive, transgenic, and economically burdensome. While chemical mutagens facilitate these systems, they not only generate weak alleles but also produce strong alleles that induce permanent sterility through random mutagenesis. In contrast, RNA-guided nuclease system, such as clustered regularly interspaced short palindromic repeats (CRISPR)- associated protein (Cas) system, facilitates more efficient hybrid production by inducing male sterility through targeted genome modifications in male sterility genes, such as MS8, MS10, MS26, and MS45 which allows precise manipulation of pollen development or pollen abortion in various crops. Moreover, this approach allows haploid induction for the rapid generation of recombinant and homozygous lines from hybrid parents by editing essential genes, like CENH3, MTL/NLD/PLA, and DMP, resulting in high-yield, transgene-free hybrids. Additionally, this system supports synthetic apomixis induction by employing the MiMe (Mitosis instead of Meiosis) strategy, coupled with parthenogenesis in hybrid plants, to create heterozygous lines and retain hybrid vigor in subsequent generations. RNA-guided nuclease-induced synthetic apomixis also enables genome stacking for autopolyploid progressive heterosis via clonal gamete production for trait maintenance to enhance crop adaptability without compromising yield. Additionally, CRISPR-Cas-mediated de novo domestication of wild relatives, along with recent advances to circumvent tissue culture- recalcitrance and -dependency through heterologous expression of morphogenic regulators, holds great promise for incorporating diversity-enriched germplasm into the breeding programs. These approaches aim to generate elite hybrids adapted to dynamic environments and address the anticipated challenges of food insecurity.},
}

@article {pmid40154215,
year = {2025},
author = {Zhang, Z and Ji, Q and Zhang, Z and Lyu, B and Li, P and Zhang, L and Chen, K and Fang, M and Song, J},
title = {Ultra-sensitive detection of melanoma NRAS mutant ctDNA based on programmable endonucleases.},
journal = {Cancer genetics},
volume = {294-295},
number = {},
pages = {47-56},
doi = {10.1016/j.cancergen.2025.02.008},
pmid = {40154215},
issn = {2210-7762},
abstract = {BACKGROUND: Melanoma is a complex and often fatal disease, with NRAS being one of the most frequently mutated genes in this type of cancer. Liquid biopsies, specifically tests for circulating tumor DNA (ctDNA), represent a promising and less invasive approach to diagnosis. This study aims to develop an ultra-sensitive assay for detecting melanoma NRAS mutant ctDNA.

METHODS: To detect rare NRAS mutant ctDNA, we developed the NRAS PASEA assay by screening CRISPR-Cas proteins that recognize the PAM sequence 5'-TTN-3'. This method employs CRISPR-Cas proteins to continuously shear wild-type alleles during isothermal amplification, resulting in exponential amplification of mutant alleles to a detectable level by Sanger sequencing.

RESULTS: The developed NRAS Q61R/L/K mutation detection method can detect simulated ctDNA samples with mutant allele fractions (MAF) as low as 0.01 % with 30 mins of PASEA treatment. Notably, the NRAS Q61 K mutation was accurately identified by FnCas12a-based NRAS PASEA, even with the nucleotide at the "N" position in the PAM site "TTN." The method successfully detected ctDNA in patients with malignant melanoma. All patients (5/5) from 15 melanoma blood samples with NRAS Q61R (4/4) and NRAS Q61 K (1/1) mutations were accurately identified, with no false positives among patients with wildtype NRAS Q61.

CONCLUSION: Detecting ctDNA from peripheral blood samples is highly significant for melanomas in areas where imaging evaluation is challenging. Our assay demonstrated 100 % consistency with tumor tissue NGS, providing a new analytical strategy for companion diagnosis and dynamic assessment of therapeutic efficacy and disease progression in melanoma.},
}

@article {pmid40153308,
year = {2025},
author = {Hayhurst, M and Vink, JNA and Remerand, M and Gerth, ML},
title = {Transient expression of fluorescent proteins and Cas nucleases in Phytophthora agathidicida via PEG-mediated protoplast transformation.},
journal = {Microbiology (Reading, England)},
volume = {171},
number = {3},
pages = {},
pmid = {40153308},
issn = {1465-2080},
mesh = {*Phytophthora/genetics/enzymology ; *Protoplasts/metabolism ; *Transformation, Genetic ; *Polyethylene Glycols/pharmacology ; Luminescent Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Plant Diseases/microbiology/parasitology ; Green Fluorescent Proteins/genetics/metabolism ; Plasmids/genetics ; Calcium Chloride/metabolism/pharmacology ; },
abstract = {Phytophthora species are eukaryotic plant pathogens that cause root rot and dieback diseases in thousands of plant species worldwide. Despite their significant economic and ecological impacts, fundamental molecular tools such as DNA transformation methods are not yet established for many Phytophthora species. In this study, we have established a PEG/calcium chloride (CaCl2)-mediated protoplast transformation method for Phytophthora agathidicida, the causal agent of kauri dieback disease. Adapting a protocol from Phytophthora sojae, we systematically optimized the protoplast digesting enzymes, recovery media composition and pH. Our findings reveal that chitinases are essential for P. agathidicida protoplast formation, and the optimum pH of the recovery medium is 5. The media type did not significantly impact protoplast regeneration. Using this protocol, we generated transformants using three plasmids (i.e. pTdTomatoN, pYF2-PsNLS-Cas9-GFP and pYF2-PsNLS-Cas12a-GFP), which expressed fluorescent proteins and/or Cas nucleases. The transformants were unstable unless maintained under antibiotic selective pressure; however, under selection, fluorescence was maintained across multiple generations and life cycle stages, including the production of fluorescent zoospores from transformed mycelia. Notably, we observed the expression of GFP-tagged Cas nucleases, which is promising for future CRISPR-Cas genome editing applications. This study demonstrates that P. agathidicida is amenable to PEG/CaCl2-mediated protoplast transformation. Although the resulting transformants require antibiotic selective pressure to remain stable, this transient expression system can be valuable for applications such as cell tracking, chemotaxis studies and CRISPR-Cas genome editing. The protocol also provides a foundation for further optimization of transformation methods. It serves as a valuable tool for exploring the molecular biology of P. agathidicida and potentially other closely related Phytophthora species.},
}

*Phytophthora/genetics/enzymology
*Protoplasts/metabolism
*Transformation, Genetic
*Polyethylene Glycols/pharmacology
Luminescent Proteins/genetics/metabolism
CRISPR-Cas Systems
Gene Editing
Plant Diseases/microbiology/parasitology
Green Fluorescent Proteins/genetics/metabolism
Plasmids/genetics
Calcium Chloride/metabolism/pharmacology

@article {pmid40153053,
year = {2025},
author = {Kiraz, D and Özcan, A},
title = {Comparative genome analysis of 15 Streptococcus thermophilus strains isolated from Turkish traditional yogurt.},
journal = {Antonie van Leeuwenhoek},
volume = {118},
number = {4},
pages = {64},
pmid = {40153053},
issn = {1572-9699},
mesh = {*Yogurt/microbiology ; *Streptococcus thermophilus/genetics/classification/isolation & purification ; *Genome, Bacterial ; *Phylogeny ; Turkey ; Fermentation ; Whole Genome Sequencing ; Food Microbiology ; Genomics ; },
abstract = {Streptococcus thermophilus plays a pivotal role in yogurt fermentation, yet strains from traditional fermented products remain largely unexplored compared to their industrial counterparts. This study aimed to characterize the genomic diversity and functional potential of 15 S. thermophilus strains isolated from Turkish traditional yogurts, and to compare them with industrial strains. Through whole-genome sequencing and advanced bioinformatics analyses, we revealed distinct phylogenetic patterns and genetic features that differentiate these traditional strains from industrial isolates. The genomes (1.68-1.86 Mb) exhibited high genetic homogeneity (ANI > 98.69%) while maintaining significant functional diversity. Pan-genome analysis identified 1160 core genes and 5694 accessory genes, highlighting substantial genomic plasticity that enables niche adaptation. Our analysis uncovered several distinctive features: (1) unique phylogenetic clustering patterns based on both housekeeping genes and whole-genome SNPs, suggesting geographical isolation effects; (2) an extensive repertoire of carbohydrate-active enzymes (CAZymes), comprising 111 Glycoside Hydrolases, 227 Glycosyl Transferases, and 44 Carbohydrate Esterases and 13 Carbohydrate-Binding Modules, demonstrating sophisticated carbohydrate metabolism adaptation significantly enriched compared to industrial strains; (3) widespread GABA biosynthesis pathways in 8 strains, including complete gadB gene, indicating potential health-promoting properties; (4) multiple genomic islands containing genes for galactose utilization and stress response, suggesting specific adaptation to traditional fermentation environments; (5) diverse exopolysaccharide biosynthesis and bacteriocin gene clusters; and (6) widespread CRISPR-Cas systems with variable spacer content. Notably, we identified vanY glycopeptide resistance genes across all strains, with two strains additionally harboring vanT. These results reveal the genetic mechanisms behind S. thermophilus adaptation to traditional yogurt environments, offering valuable insights for developing starter cultures and preserving the unique qualities and potential health benefits of traditional dairy products.},
}

*Yogurt/microbiology
*Streptococcus thermophilus/genetics/classification/isolation & purification
*Genome, Bacterial
*Phylogeny
Turkey
Fermentation
Whole Genome Sequencing
Food Microbiology
Genomics

@article {pmid40152981,
year = {2025},
author = {Feng, B and Wang, Y and Zhang, X and Mu, T and Zhang, B and Li, Y and Zhang, H and Hua, W and Yuan, W and Li, H},
title = {Targeted mutagenesis and functional marker development of two Bna.TAC1s conferring novel rapeseed germplasm with compact architecture.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {138},
number = {4},
pages = {86},
pmid = {40152981},
issn = {1432-2242},
support = {32172028//National Natural Science Foundation of China/ ; U22A20477//National Natural Science Foundation of China/ ; },
mesh = {*Brassica napus/genetics/growth & development ; Plant Proteins/genetics/metabolism ; Phenotype ; Genetic Markers ; Plant Breeding ; Mutagenesis ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; Gene Editing ; Plants, Genetically Modified/genetics/growth & development ; Genes, Plant ; },
abstract = {Simultaneous disruption of two Bna.TAC1s, redundantly controlling the branch angle, generates a compact architecture in rapeseed, and two functional markers are developed to facilitate breeding rapeseed cultivars with compact architecture. Shoot branch angle is a key factor in determining the aerial plant architecture. A narrow branch angle can increase yields by facilitating mechanized harvest and high-density planting in rapeseed, a globally important oil crop. However, the available rapeseed varieties with narrow branch angle are very limited. In this study, two Bna.TAC1 members named BnaA5.TAC1 and BnaC4.TAC1 were found to have the four canonical domains of TAC1-like members, including domains I, II, III and IV in rapeseed. Each Bna.TAC1 exhibits dominant expression in the lateral branch with gradual dynamic response to light and encodes a protein localized in the plasma membrane. CRISPR/Cas9-mediated editing system was used to simultaneously knock out the two Bna.TAC1s to obtain two different Bna.tac1 double mutants, designed as CR-Bna.tac1-1 and CR-Bna.tac1-2. These two mutants displayed different degrees of compact architecture without affecting plant height and yield-related traits. The two Bna.TAC1s were also shown to play a redundant role in controlling branch angle by regulating the gravitropic response. In addition, we developed two specific gel-based functional markers in each Bna.TAC1 for the transgene-free mutant CR-Bna.tac1-1, which co-segregate with narrower branch angle and could help to identify the mutant alleles in a segregating population. We also found that the genomic variation of the two Bna.TAC1s is not associated with branch angle variation in the natural rapeseed population. Overall, these results reveal the key roles of Bna.TAC1s in regulation of rapeseed branch angle and provide a novel germplasm and functional markers for breeding superior varieties with compact architecture in rapeseed.},
}

*Brassica napus/genetics/growth & development
Plant Proteins/genetics/metabolism
Phenotype
Genetic Markers
Plant Breeding
Mutagenesis
CRISPR-Cas Systems
Gene Expression Regulation, Plant
Gene Editing
Plants, Genetically Modified/genetics/growth & development
Genes, Plant

@article {pmid40152921,
year = {2025},
author = {Prithiviraj, S and Garcia Garcia, A and Linderfalk, K and Yiguang, B and Ferveur, S and Falck, LN and Subramaniam, A and Mohlin, S and Hidalgo Gil, D and Dupard, SJ and Zacharaki, D and Raina, DB and Bourgine, PE},
title = {Compositional editing of extracellular matrices by CRISPR/Cas9 engineering of human mesenchymal stem cell lines.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {40152921},
issn = {2050-084X},
support = {2019-01864_3//Vetenskapsrådet/ ; 948588/ERC_/European Research Council/International ; },
mesh = {Humans ; *Mesenchymal Stem Cells/metabolism ; *Extracellular Matrix/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; Rats ; Tissue Engineering/methods ; Core Binding Factor Alpha 1 Subunit/metabolism/genetics ; Cell Line ; Vascular Endothelial Growth Factor A/metabolism/genetics ; },
abstract = {Tissue engineering strategies predominantly rely on the production of living substitutes, whereby implanted cells actively participate in the regenerative process. Beyond cost and delayed graft availability, the patient-specific performance of engineered tissues poses serious concerns on their clinical translation ability. A more exciting paradigm consists in exploiting cell-laid, engineered extracellular matrices (eECMs), which can be used as off-the-shelf materials. Here, the regenerative capacity solely relies on the preservation of the eECM structure and embedded signals to instruct an endogenous repair. We recently described the possibility to exploit custom human stem cell lines for eECM manufacturing. In addition to the conferred standardization, the availability of such cell lines opened avenues for the design of tailored eECMs by applying dedicated genetic tools. In this study, we demonstrated the exploitation of CRISPR/Cas9 as a high precision system for editing the composition and function of eECMs. Human mesenchymal stromal/stem cell (hMSC) lines were modified to knock out vascular endothelial growth factor (VEGF) and Runt-related transcription factor 2 (RUNX2) and assessed for their capacity to generate osteoinductive cartilage matrices. We report the successful editing of hMSCs, subsequently leading to targeted VEGF and RUNX2-knockout cartilage eECMs. Despite the absence of VEGF, eECMs retained full capacity to instruct ectopic endochondral ossification. Conversely, RUNX2-edited eECMs exhibited impaired hypertrophy, reduced ectopic ossification, and superior cartilage repair in a rat osteochondral defect. In summary, our approach can be harnessed to identify the necessary eECM factors driving endogenous repair. Our work paves the road toward the compositional eECMs editing and their exploitation in broad regenerative contexts.},
}

Humans
*Mesenchymal Stem Cells/metabolism
*Extracellular Matrix/metabolism
*CRISPR-Cas Systems
*Gene Editing/methods
Animals
Rats
Tissue Engineering/methods
Core Binding Factor Alpha 1 Subunit/metabolism/genetics
Cell Line
Vascular Endothelial Growth Factor A/metabolism/genetics

@article {pmid40152384,
year = {2025},
author = {Fragale, N and Divvela, SSK and Williams-Ward, VC and Brand-Saberi, B},
title = {Loss of Atoh8 Affects Neurocranial and Axial Skeleton Development in Zebrafish.},
journal = {Frontiers in bioscience (Landmark edition)},
volume = {30},
number = {3},
pages = {26806},
doi = {10.31083/FBL26806},
pmid = {40152384},
issn = {2768-6698},
support = {G1001029//MRC/ ; MR/N021231/1//MRC/ ; },
mesh = {Animals ; *Zebrafish/genetics/embryology ; *Zebrafish Proteins/genetics/metabolism ; *Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; Bone Development/genetics ; Gene Knockout Techniques ; Skull/embryology/metabolism/growth & development ; },
abstract = {BACKGROUND: The basic helix-loop-helix (bHLH) transcription factor atonal homologue 8 (Atoh8) has been implicated in various developmental and physiological processes by means of transient knockdown and conditional knockout approaches in zebrafish, chick and mouse. Despite its demonstrated involvement in multiple tissues, the role of Atoh8 remains elusive in zebrafish. A recent permanent knockout study in zebrafish investigated the role of Atoh8 on the background of previous morpholino studies which demonstrated various developmental defects but could not find any of the morpholino-based effects in the mutant. In mice, a knockout study demonstrated involvement of the transcription factor in skeletal development, showing that disruption of the atoh8 gene results in reduction of skeletal size. We investigated a mutant fish line generated using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9)-technology for possible phenotypic effects on zebrafish skeletogenesis.

METHODS: Here, we present a CRISPR/Cas9-generated atoh8 permanent zebrafish mutant and investigate the phenotypic effects of the knockout on the developing zebrafish craniofacial and axial skeleton. We investigated the expression pattern of the gene in wildtype and conducted detailed morphometric analysis for a variety of bone and cartilage elements of the developing skeleton at 12 days post fertilisation (dpf) in zebrafish siblings from a heterozygous mating using detailed morphometric measurements and statistical analysis of the results.

RESULTS: Homozygous mutants are viable into late adulthood and show no overt morphological phenotype. Despite the prominent appearance of atoh8 signal in various embryonic and larval craniofacial and axial skeletal structures, detailed morphometric analysis revealed only subtle phenotypic effects of the mutation on skeletal development in zebrafish. We found the formation of the orbital cartilages of the developing neurocranium and the progress of chordacentra mineralisation to be negatively affected by loss of the transcription factor.

CONCLUSIONS: Despite the very subtle phenotypic effect of our mutation, we were able to show involvement of atoh8 in the skeletal development of zebrafish. We attribute the mild phenotype to a compensatory mechanism induced by nonsense-mediated degradation of messenger ribonucleic acid (mRNA) as suggested in the recent literature. The effect of atoh8-disruption on zebrafish skeletal development suggests that the loss of atoh8 cannot be compensated for at interfaces where more than one embryonic cell lineage contributes to bone and cartilage formation.},
}

Animals
*Zebrafish/genetics/embryology
*Zebrafish Proteins/genetics/metabolism
*Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism
CRISPR-Cas Systems
Gene Expression Regulation, Developmental
Bone Development/genetics
Gene Knockout Techniques
Skull/embryology/metabolism/growth & development

@article {pmid40119192,
year = {2025},
author = {Rea, A and Santana-Hernández, S and Villanueva, J and Sanvicente-García, M and Cabo, M and Suarez-Olmos, J and Quimis, F and Qin, M and Llorens, E and Blasco-Benito, S and Torralba-Raga, L and Perez, L and Bhattarai, B and Alari-Pahissa, E and Georgoudaki, AM and Balaguer, F and Juan, M and Pardo, J and Celià-Terrassa, T and Rovira, A and Möker, N and Zhang, C and Colonna, M and Spanholtz, J and Malmberg, KJ and Montagut, C and Albanell, J and Güell, M and López-Botet, M and Muntasell, A},
title = {Enhancing human NK cell antitumor function by knocking out SMAD4 to counteract TGFβ and activin A suppression.},
journal = {Nature immunology},
volume = {26},
number = {4},
pages = {582-594},
pmid = {40119192},
issn = {1529-2916},
support = {PI22/00040//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; PI21/00002//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; ICI24/00041//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; FI23/00075//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; SGR863//Generalitat de Catalunya (Government of Catalonia)/ ; 2024PROD00089//Departament d'Innovació, Universitats i Empresa, Generalitat de Catalunya (Department of Innovation, Education and Enterprise, Government of Catalonia)/ ; 765104//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 Marie Sklodowska-Curie Actions (H2020 Excellent Science - Marie Sklodowska-Curie Actions)/ ; 765104//EC | EC Seventh Framework Programm | FP7 People: Marie-Curie Actions (FP7-PEOPLE - Specific Programme "People" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013))/ ; },
mesh = {Humans ; *Killer Cells, Natural/immunology/metabolism ; *Smad4 Protein/metabolism/genetics ; *Activins/metabolism ; *Transforming Growth Factor beta/metabolism ; Mice ; Animals ; Cell Line, Tumor ; CRISPR-Cas Systems ; Immunotherapy, Adoptive/methods ; Gene Knockout Techniques ; Cytotoxicity, Immunologic ; Neoplasms/immunology/therapy/metabolism ; Signal Transduction ; Cell Proliferation ; Xenograft Model Antitumor Assays ; },
abstract = {Transforming growth factor beta (TGFβ) and activin A suppress natural killer (NK) cell function and proliferation, limiting the efficacy of adoptive NK cell therapies. Inspired by the partial resistance to TGFβ of NK cells with SMAD4 haploinsufficiency, we used CRISPR-Cas9 for knockout of SMAD4 in human NK cells. Here we show that SMAD4[KO] NK cells were resistant to TGFβ and activin A inhibition, retaining their cytotoxicity, cytokine secretion and interleukin-2/interleukin-15-driven proliferation. They showed enhanced tumor penetration and tumor growth control, both as monotherapy and in combination with tumor-targeted therapeutic antibodies. Notably, SMAD4[KO] NK cells outperformed control NK cells treated with a TGFβ inhibitor, underscoring the benefit of maintaining SMAD4-independent TGFβ signaling. SMAD4[KO] conferred TGFβ resistance across diverse NK cell platforms, including CD19-CAR NK cells, stem cell-derived NK cells and ADAPT-NK cells. These findings position SMAD4 knockout as a versatile and compelling strategy to enhance NK cell antitumor activity, providing a new avenue for improving NK cell-based cancer immunotherapies.},
}

Humans
*Killer Cells, Natural/immunology/metabolism
*Smad4 Protein/metabolism/genetics
*Activins/metabolism
*Transforming Growth Factor beta/metabolism
Mice
Animals
Cell Line, Tumor
CRISPR-Cas Systems
Immunotherapy, Adoptive/methods
Gene Knockout Techniques
Cytotoxicity, Immunologic
Neoplasms/immunology/therapy/metabolism
Signal Transduction
Cell Proliferation
Xenograft Model Antitumor Assays

@article {pmid40087529,
year = {2025},
author = {Kim, J and Nam, Y and Jeon, D and Choi, Y and Choi, S and Hong, CP and Kim, S and Jung, H and Park, N and Sohn, Y and Rim, YA and Ju, JH},
title = {Generation of hypoimmunogenic universal iPS cells through HLA-type gene knockout.},
journal = {Experimental & molecular medicine},
volume = {57},
number = {3},
pages = {686-699},
pmid = {40087529},
issn = {2092-6413},
support = {HI23C1234//Korea Health Industry Development Institute (KHIDI)/ ; 20024297//Ministry of Trade, Industry and Energy (Ministry of Trade, Industry and Energy, Korea)/ ; NRF-2023M3A9E4009811//Ministry of Education (Ministry of Education of the Republic of Korea)/ ; },
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Gene Knockout Techniques ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; HLA Antigens/genetics/immunology ; Cell Line ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Hypoimmunogenic universal induced pluripotent stemn (iPS) cells were generated through the targeted disruption of key genes, including human leukocyte antigen (HLA)-A, HLA-B and HLA-DR alpha (DRA), using the CRISPR-Cas9 system. This approach aimed to minimize immune recognition and enhance the potential of iPS cells for allogeneic therapy. Heterozygous iPS cells were used for guide RNA design and validation to facilitate the knockout (KO) of the HLA-A, HLA-B and HLA-DRA genes. The electroporation of iPS cells using the selected guide RNAs enabled the generation of triple-KO iPS cells, followed by single-cell cloning for clone selection. Clone A7, an iPS cell with targeted KOs of the HLA-A, HLA-B and HLA-DRA genes, was identified as the final candidate. Messenger RNA analysis revealed robust expression of pluripotency markers, such as octamer-binding transcription factor 4, sex-determining region Y box 2, Krüppel-like factor 4, Lin-28 homolog A and Nanog homeobox, while protein expression assays confirmed the presence of octamer-binding transcription factor 4, stage-specific embryonic antigen 4, Nanog homeobox and tumor rejection antigen 1-60. A karyotype examination revealed no anomalies, and three-germ layer differentiation assays confirmed the differentiation potential. After interferon gamma stimulation, the gene-corrected clone A7 lacked HLA-A, HLA-B and HLA-DR protein expression. Immunogenicity testing further confirmed the hypoimmunogenicity of clone A7, which was evidenced by the absence of proliferation in central memory T cells and effector memory T cells. In conclusion, clone A7, a triple-KO iPS cell clone that demonstrates immune evasion properties, retained its intrinsic iPS cell characteristics and exhibited no immunogenicity.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology
Humans
*Gene Knockout Techniques
CRISPR-Cas Systems
Cell Differentiation/genetics
HLA Antigens/genetics/immunology
Cell Line
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid39786412,
year = {2025},
author = {Uchida, K and Fuji, Y and Tabeta, H and Akashi, T and Hirai, MY},
title = {Omics-based identification of the broader effects of 2-hydroxyisoflavanone synthase gene editing on a gene regulatory network beyond isoflavonoid loss in soybean hairy roots.},
journal = {Plant & cell physiology},
volume = {66},
number = {3},
pages = {304-317},
doi = {10.1093/pcp/pcae151},
pmid = {39786412},
issn = {1471-9053},
support = {JP19K15821//Japan Society for the Promotion of Science/ ; JPJ012287//Cross-ministerial Strategic Innovation Promotion Program/ ; //soybean protein research/ ; },
mesh = {*Glycine max/genetics/metabolism ; *Gene Editing/methods ; *Plant Roots/genetics/metabolism ; *Isoflavones/metabolism ; *Gene Regulatory Networks ; *Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Flavonoids/metabolism ; },
abstract = {Soybean (Glycine max) is a leguminous crop cultivated worldwide that accumulates high levels of isoflavones. Although previous research has often focused on increasing the soybean isoflavone content because of the estrogen-like activity of dietary soy in humans, the rapidly increasing demand for soybean as a plant-based meat substitute has raised concerns about excessive isoflavone intake. Therefore, the production of isoflavone-free soybean has been anticipated. However, there have been no reports of an isoflavone-free soybean until now. Here, 2-hydroxyisoflavanone synthase (IFS), which is essential for isoflavone biosynthesis, was targeted for genome editing in soybean. A novel CRISPR/Cas9 system using Staphylococcus aureus Cas9 instead of the commonly used Streptococcus pyogenes Cas9 was established and customized. Through Agrobacterium rhizogenes-mediated transformation, IFS-edited hairy roots were generated in which all three IFS genes contained deletion mutations. Metabolome analyses of IFS-edited hairy roots revealed that isoflavone content significantly decreased, whereas levels of flavonoids, including a novel chalcone derivative, increased. A transcriptome analysis revealed changes in the expression levels of a large number of genes, including jasmonic acid-inducible genes. In addition, the functions of selected transcription factor genes (MYB14-L, GmbHLH112, and GmbHLH113), which were dramatically upregulated by IFS editing, were investigated by multiomics analyses of their over-expressing hairy root lines. They appear to be involved in flavonoid and triterpene saponin biosynthesis, salicylic acid metabolism, and central carbon metabolism. Overall, the results indicated that editing IFS genes caused the redirection of the metabolic flux from isoflavonoid biosynthesis to flavonoid accumulation, as well as dynamic changes in gene regulatory networks.},
}

*Glycine max/genetics/metabolism
*Gene Editing/methods
*Plant Roots/genetics/metabolism
*Isoflavones/metabolism
*Gene Regulatory Networks
*Gene Expression Regulation, Plant
CRISPR-Cas Systems
Plant Proteins/genetics/metabolism
Plants, Genetically Modified
Flavonoids/metabolism

@article {pmid40151969,
year = {2025},
author = {Tsubota, T and Takasu, Y and Yonemura, N and Sezutsu, H},
title = {Enhancements of the CRISPR-Cas System in the Silkworm Bombyx mori.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2024.0089},
pmid = {40151969},
issn = {2573-1602},
abstract = {The silkworm (Bombyx mori) is a lepidopteran model insect that has been utilized for basic research and industrial applications. In this species, transcription activator-like effector nucleases (TALENs) have been found to function efficiently, and we previously developed a TALEN-mediated genome editing system for knockout and knock-in experiments using plasmids and single-stranded oligodeoxynucleotides (ssODNs) as donors. By contrast, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated genome editing, especially for gene integration, remains limited. In this study, we attempted to improve CRISPR-Cas systems to expand the utility of genome editing in the silkworm. Codon optimization of Cas9 improved genome editing efficiency, and single-guide RNA utilization also resulted in a higher genome editing efficiency than crRNA/tracrRNA when Cas9 messenger RNA (mRNA) was used. CRISPR-Cas12a-mediated genome editing and targeted sequence integration using ssODNs were both successfully performed. Overall, our study provides a robust technical platform that can facilitate basic and applied silkworm studies.},
}

@article {pmid40151342,
year = {2025},
author = {Singh, A and Yasheshwar, and Kaushik, NK and Kala, D and Nagraik, R and Gupta, S and Kaushal, A and Walia, Y and Dhir, S and Noorani, MS},
title = {Conventional and cutting-edge advances in plant virus detection: emerging trends and techniques.},
journal = {3 Biotech},
volume = {15},
number = {4},
pages = {100},
pmid = {40151342},
issn = {2190-572X},
abstract = {Plant viruses pose a significant threat to global agriculture. For a long time, conventional methods including detection based on visual symptoms, host range investigations, electron microscopy, serological assays (e.g., ELISA, Western blotting), and nucleic acid-based techniques (PCR, RT-PCR) have been used for virus identification. With increased sensitivity, speed, and specificity, new technologies like loop-mediated isothermal amplification (LAMP), high-throughput sequencing (HTS), nanotechnology-based biosensors, and CRISPR diagnostics have completely changed the way plant viruses are detected. Recent advances in detection techniques integrate artificial intelligence (AI), machine learning (ML), and the Internet of Things (IoT) for real-time monitoring. Innovations like hyperspectral imaging, deep learning, and cloud-based IoT platforms further support disease identification and surveillance. Nanotechnology-based lateral flow assays and CRISPR-Cas systems provide rapid, field-deployable solutions. Despite these advancements, challenges such as sequence limitations, multiplexing constraints, and environmental concerns remain. Future research should focus on refining portable on-site diagnostic kits, optimizing nanotechnology applications, and enhancing global surveillance systems. Interdisciplinary collaboration across molecular biology, bioinformatics, and engineering is essential to developing scalable, cost-effective solutions for plant virus detection, ensuring agricultural sustainability and ecosystem protection.},
}

@article {pmid40151107,
year = {2025},
author = {Zou, G and Si, P and Wang, J and Yang, M and Chen, J and Liu, C and Luo, Z},
title = {Chemical Modification Coupled with Isothermal CRISPR-Based Assay for Sensitive Detection of DNA Hydroxymethylation.},
journal = {ACS sensors},
volume = {10},
number = {3},
pages = {2073-2079},
doi = {10.1021/acssensors.4c03312},
pmid = {40151107},
issn = {2379-3694},
mesh = {*5-Methylcytosine/analogs & derivatives/analysis ; Humans ; *DNA Methylation ; *DNA/chemistry/genetics ; CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; },
abstract = {5-Hydroxymethylcytosine (5hmC) plays a key role in the DNA demethylation process and serves as a stable epigenetic marker in the human genome which is closely associated with disease progression, particularly in diabetes, colorectal cancer, and liver cancer. However, convenient and sensitive methods for detecting and quantifying 5hmC in the genome are scarce, especially in complex biological environments. Herein, a novel attempt at hypersensitive quantitative detection of 5hmC was presented. A multifunctional photosensitive probe was therefore introduced for specific labeling, enrichment, and elution of 5hmC-DNA. Combining with isothermal assay leveraging rolling circle amplification and Cas12a for accurate recognition, we achieved quantitative detection of 5hmC DNA in trace amounts at a level of 11 fM. Global 5hmC was measured in various biological samples using as little as 10 ng of input DNA by a real-time PCR instrument. The reported approach imposed no sequence restrictions, demonstrating promising potential for detecting modified bases in trace amounts of nucleic acids within complex environments, such as blood, urine, and saliva samples.},
}

*5-Methylcytosine/analogs & derivatives/analysis
Humans
*DNA Methylation
*DNA/chemistry/genetics
CRISPR-Cas Systems/genetics
Nucleic Acid Amplification Techniques/methods
Limit of Detection

@article {pmid40149851,
year = {2025},
author = {Pan, D and Mijit, M and Wang, H and Sun, C and Pingcuo, B and Yu, Z and Xiong, B and Tang, X},
title = {Rapid Genotyping of FecB Mutation in Sheep Using CRISPR-Cas12a Integrated with DNA Nanotree Biosensing Platform.},
journal = {Biomolecules},
volume = {15},
number = {3},
pages = {},
doi = {10.3390/biom15030315},
pmid = {40149851},
issn = {2218-273X},
support = {2022YFD1301102//Xiangfang Tang/ ; 2022BBF02020//Xiangfang Tang，Benhai Xiong/ ; Guike AA22068099//Xiangfang Tang/ ; 2004DA125184G2405//Hui Wang/ ; jc-cxgc-ias-09-1//Xiangfang Tang/ ; 2024-YWF-ZYSQ-10//Hui Wang/ ; CAAS-CSSAE-202402//Hui Wang/ ; },
mesh = {Animals ; Sheep/genetics ; *CRISPR-Cas Systems/genetics ; *Mutation ; *Biosensing Techniques/methods ; Bone Morphogenetic Protein Receptors, Type I/genetics ; DNA/genetics ; Genotyping Techniques/methods ; Genotype ; },
abstract = {The A-to-G mutation (FecB) in the BMPR1B gene is strongly linked to fertility in sheep, significantly increasing ovulation rates and litter sizes compared to wild-type populations. The rapid and reliable screening of the FecB gene is therefore critical for advancing sheep breeding programs. This study aimed to develop a fast and accurate method for detecting the FecB mutation and genotyping the gene to enhance sheep reproduction and productivity. To achieve this, we integrated the CRISPR-Cas12a system with an optimized amplification refractory mutation system (ARMS). A similar DNA origami technique-based fluorescence reporter nanotree structure was synthesized using gold nanomagnetic beads as carriers to amplify the fluorescence signal further. The resulting biosensing platform, termed CRISPR-ARMS, demonstrated excellent sensitivity for detecting FecB mutations, with a detection limit as low as 0.02 pmol. Therefore, this innovative approach shows great promise for single-base mutation detection and represents a pioneering tool for high-yield genetic screening.},
}

Animals
Sheep/genetics
*CRISPR-Cas Systems/genetics
*Mutation
*Biosensing Techniques/methods
Bone Morphogenetic Protein Receptors, Type I/genetics
DNA/genetics
Genotyping Techniques/methods
Genotype

@article {pmid40148961,
year = {2025},
author = {Zheng, Y and Mo, Y and Yuan, Y and Su, T and Qi, Q},
title = {A rapid and efficient strategy for combinatorial repression of multiple genes in Escherichia coli.},
journal = {Microbial cell factories},
volume = {24},
number = {1},
pages = {74},
pmid = {40148961},
issn = {1475-2859},
support = {ZR2021QC021//Natural Science Foundation of Shandong Province/ ; tsqn202312029//Young Taishan Scholars Program of Shandong Province/ ; No. 32200081//National Natural Science Foundation of China/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; *Metabolic Engineering/methods ; *Plasmids/genetics ; *Gene Expression Regulation, Bacterial ; *Promoter Regions, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics ; Escherichia coli Proteins/genetics/metabolism ; N-Acetylneuraminic Acid/metabolism ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: The regulation of multiple gene expression is pivotal for metabolic engineering. Although CRISPR interference (CRISPRi) has been extensively utilized for multi-gene regulation, the construction of numerous single-guide RNA (sgRNA) expression plasmids for combinatorial regulation remains a significant challenge.

RESULTS: In this study, we developed a combinatorial repression system for multiple genes by optimizing the expression of multi-sgRNA with various inducible promoters in Escherichia coli. We designed a modified Golden Gate Assembly method to rapidly construct the sgRNA expression plasmid p3gRNA-LTA. By optimizing both the promoter and the sgRNA handle sequence, we substantially mitigated undesired repression caused by the leaky expression of sgRNA. This method facilitates the rapid assessment of the effects of various inhibitory combinations on three genes by simply adding different inducers. Using the biosynthesis of N-acetylneuraminic acid (NeuAc) as an example, we found that the optimal combinatorial inhibition of the pta, ptsI, and pykA genes resulted in a 2.4-fold increase in NeuAc yield compared to the control.

CONCLUSION: We anticipate that our combinatorial repression system will greatly simplify the regulation of multiple genes and facilitate the fine-tuning of metabolic flow in the engineered strains.},
}

*Escherichia coli/genetics/metabolism
*Metabolic Engineering/methods
*Plasmids/genetics
*Gene Expression Regulation, Bacterial
*Promoter Regions, Genetic
RNA, Guide, CRISPR-Cas Systems/genetics
Escherichia coli Proteins/genetics/metabolism
N-Acetylneuraminic Acid/metabolism
CRISPR-Cas Systems

@article {pmid40055023,
year = {2025},
author = {Ge, H and Feng, J and Huang, L and Luo, Z and Ling, H and Ma, L and Wang, M and Chen, H and Ren, L},
title = {Development of a highly sensitive, high-throughput and automated CRISPR-based device for the contamination-free pathogen detection.},
journal = {Biosensors & bioelectronics},
volume = {278},
number = {},
pages = {117323},
doi = {10.1016/j.bios.2025.117323},
pmid = {40055023},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/instrumentation/methods ; Humans ; Lab-On-A-Chip Devices ; Equipment Design ; Bacteria/isolation & purification/genetics ; Viruses/isolation & purification/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Rapid, portable, and contamination-resistant nucleic acid detection methods are necessary due to the threat posed by emerging viruses to public health and agricultural output. We establish CARE (CRISPR-associated airtight real-time electronic diagnostic device), a novel platform that combines CRISPR-Cas12a with a hermetically sealed microfluidic chip to overcome the limitations of present technologies, which struggle to balance sensitivity, multiplexing, and field applicability. By combining isothermal amplification and CRISPR detection within a hermetically sealed microfluidic chip, CARE eliminates the risk of nucleic acid aerosol contamination while enabling simultaneous high-throughput analysis of seven pathogens. The device is complemented by a user-friendly nucleic acid quantification App, enabling rapid and precise analysis. The RPA-CRISPR/Cas12a system demonstrates exceptional sensitivity, detecting as few as 1 copy μL[-1] (single-plex) and 10-10[2] copies μL[-1] (multiplexed), with real-sample performance matching gold-standard methods. CARE represents a significant advancement in CRISPR-based diagnostics, offering a robust, portable solution for on-site pathogen detection in food and agricultural applications.},
}

*Biosensing Techniques/instrumentation
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/instrumentation/methods
Humans
Lab-On-A-Chip Devices
Equipment Design
Bacteria/isolation & purification/genetics
Viruses/isolation & purification/genetics
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40054154,
year = {2025},
author = {Liu, S and Yang, Y and Li, X and Choi, JW and Guo, J and Luo, H and Li, C},
title = {Development of a single-tube RPA/CRISPR-cas12a detection platform for monkeypox virus.},
journal = {Biosensors & bioelectronics},
volume = {278},
number = {},
pages = {117221},
doi = {10.1016/j.bios.2025.117221},
pmid = {40054154},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques/methods/instrumentation ; Humans ; *Monkeypox virus/genetics/isolation & purification ; Limit of Detection ; Mpox, Monkeypox/diagnosis/virology ; Point-of-Care Testing ; CRISPR-Associated Proteins/genetics ; Animals ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Monkeypox is a zoonotic disease caused by the monkeypox virus (MPXV), with outbreaks primarily occurring in West and Central Africa. The recent global MPXV outbreak underscores the urgent need for effective detection methods. Currently, qPCR is considered the gold standard for MPXV detection; however, it requires specialized personnel and costly equipment. This study introduces a CRISPR-Cas12a-based detection system targeting the MPXV A27L gene, achieving a detection limit as low as 10 aM. This system exhibits high specificity, with no cross-reactivity with other orthopoxviruses, and delivers results in under 40 min. To support point-of-care testing (POCT), we developed a lateral flow assay (LFA) strip for easy result visualization. The detection system was validated using six different clinical sample types, revealing that herpes fluid and saliva are the most suitable sources. The findings of this study align with qPCR results. Additionally, we lyophilized the RPA and CRISPR reagents to improve transport, storage, and field deployment. In conclusion, this study presents a reliable molecular diagnostic approach for early MPXV detection and point-of-care testing, contributing to epidemic prevention and control.},
}

*CRISPR-Cas Systems
*Biosensing Techniques/methods/instrumentation
Humans
*Monkeypox virus/genetics/isolation & purification
Limit of Detection
Mpox, Monkeypox/diagnosis/virology
Point-of-Care Testing
CRISPR-Associated Proteins/genetics
Animals
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid39342962,
year = {2025},
author = {Zheng, L and Jiang, W and Zou, X and Song, L and Xu, X and Han, Y and Lian, H and Wu, X and Fang, X and Zhang, L},
title = {Establishment of a Cas12a-Based Visual Detection Method Involving PMNT for the Colletotrichum gloeosporioides Species Complex.},
journal = {Plant disease},
volume = {109},
number = {3},
pages = {532-541},
doi = {10.1094/PDIS-07-24-1411-SR},
pmid = {39342962},
issn = {0191-2917},
mesh = {*Colletotrichum/genetics/isolation & purification ; *Fragaria/microbiology ; *Plant Diseases/microbiology ; CRISPR-Cas Systems ; Thiophenes/chemistry/pharmacology ; Seedlings/microbiology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Strawberry anthracnose, caused by Colletotrichum spp., is a devastating disease that significantly reduces strawberry yield and quality. This study aimed to develop a simple diagnostic method to detect infection by the Colletotrichum gloeosporioides species complex (CGSC), the most predominant and virulent Colletotrichum species complex causing strawberry anthracnose in China. In this study, a Cas12aVIP diagnostic method was developed for the rapid detection of the CGSC in strawberry seedlings. This method targets the β-tubulin gene and combines recombinase polymerase amplification (RPA), the CRISPR/Cas12a system, and a cationic-conjugated polythiophene derivative [poly(3-(3'-N,N,N-triethylamino-1'-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT)] mixed with single-stranded DNA. This method shows high sensitivity (10 copies per reaction) and no cross-reactivity against related pathogens. The entire procedure, from sample to result, can be completed within 50 min, including simplified DNA extraction (15 min), RPA reaction (37°C for 20 min), CRISPR/Cas12a detection (37°C for 10 min), and visual detection by the naked eye (1 to 2 min). Furthermore, the Cas12aVIP assay successfully detected the CGSC in naturally infected strawberry seedling samples in field conditions. Asymptomatic infected plants and plant residues have been identified as primary inoculum sources for the CGSC. This method enables visible detection without the need for expensive equipment or specialized technical skills, thereby offering an efficient and straightforward approach for detecting the CGSC in strawberries. The newly developed detection method can be used to promote healthier strawberry production.},
}

*Colletotrichum/genetics/isolation & purification
*Fragaria/microbiology
*Plant Diseases/microbiology
CRISPR-Cas Systems
Thiophenes/chemistry/pharmacology
Seedlings/microbiology
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40148327,
year = {2025},
author = {Zhang, X and Li, M and Chen, K and Liu, Y and Liu, J and Wang, J and Huang, H and Zhang, Y and Huang, T and Ma, S and Liao, K and Zhou, J and Wang, M and Lin, Y and Rong, Z},
title = {Engineered circular guide RNAs enhance miniature CRISPR/Cas12f-based gene activation and adenine base editing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {3016},
pmid = {40148327},
issn = {2041-1723},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; *Adenine/metabolism/chemistry ; Mice ; Humans ; CRISPR-Associated Proteins/metabolism/genetics ; HEK293 Cells ; Transcriptional Activation ; Liver/metabolism ; Genetic Therapy/methods ; },
abstract = {CRISPR system has been widely used due to its precision and versatility in gene editing. Un1Cas12f1 from uncultured archaeon (hereafter referred to as Cas12f), known for its compact size (529 aa), exhibits obvious delivery advantage for gene editing in vitro and in vivo. However, its activity remains suboptimal. In this study, we engineer circular guide RNA (cgRNA) for Cas12f and significantly improve the efficiency of gene activation about 1.9-19.2-fold. When combined with a phase separation system, the activation efficiency is further increased about 2.3-3.9-fold. In addition, cgRNA enhances the editing efficiency and narrows the editing window of adenine base editing about 1.2-2.5-fold. Importantly, this optimization strategy also boosts the Cas12f-induced gene activation efficiency in mouse liver. Therefore, we demonstrate that cgRNA is able to enhance Cas12f-based gene activation and adenine base editing, which holds great potential for gene therapy.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems
Animals
*Adenine/metabolism/chemistry
Mice
Humans
CRISPR-Associated Proteins/metabolism/genetics
HEK293 Cells
Transcriptional Activation
Liver/metabolism
Genetic Therapy/methods

@article {pmid40147084,
year = {2025},
author = {Chen, X and Zhao, D and Yu, C and Wei, J and Zhou, G},
title = {A novel photosensitive nanoprobe combined with CRISPR/Cas12a for dual signal amplification detection of ANGPTL2.},
journal = {Talanta},
volume = {292},
number = {},
pages = {128010},
doi = {10.1016/j.talanta.2025.128010},
pmid = {40147084},
issn = {1873-3573},
abstract = {The detection of specific protein biomarkers holds significant potential for the early diagnosis of colorectal cancer (CRC). However, the accurate quantification of low-abundance proteins in serum presents a major challenge due to factors such as limited sensitivity and the complexity of the required methodologies. In this work, we established a universal CRISPR/Cas biosensing platform by integrating novel photosensitive nanoprobes (DA/PL@Cu NPs) and CRISPR/Cas12 system (DPC-Cas) for the highly sensitive, specific and user-friendly detection of angiopoietin-like protein 2 (ANGPTL2). The DA/PL@Cu NPs serve as a critical component in the transduction of protein recognition information into nucleic acid amplification events to produce Cas12a activators. The DPC-Cas biosensor integrates DA/PL@Cu NPs-assisted amplification with Cas12a self-amplification, enabling ultrasensitive detection of ANGPTL2 at concentrations as low as 20.00 pg/mL. The proposed DPC-Cas biosensor successfully detected ANGPTL2 in serum, demonstrating significant potential for the early diagnosis of CRC.},
}

@article {pmid40146761,
year = {2025},
author = {Winiarczyk, D and Khodadadi, H and Leszczyński, P and Taniguchi, H},
title = {A simple validation and screening method for CRISPR/Cas9-mediated gene editing in mouse embryos to facilitate genetically modified mice production.},
journal = {PloS one},
volume = {20},
number = {3},
pages = {e0312722},
pmid = {40146761},
issn = {1932-6203},
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Mice ; Embryo, Mammalian/metabolism ; Female ; Blastocyst/metabolism ; Mice, Transgenic ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) is a genome engineering method for generating site-specific editing in target genes in a variety of species. It is a common tool for generating mouse models of different diseases. However, detecting target modifications in mouse embryos can be time-consuming and expensive. Accordingly, developing a screening method to confirm gene modification may be useful. We propose herein an evaluation method (cleavage assay - CA) for CRISPR/Cas9-mediated gene editing in preimplantation mouse embryos that allows us to detect mutants efficiently and later on initiate in vivo production without the extensive number of samples needing to be sent for Sanger sequencing and animal usage. Our method is based on the inability of the RNP complex to recognize the target sequence after CRISPR-mediated genome editing due to modification of the target locus. It allows us to establish gene edited mice in a user-friendly fashion with a limited number of mice usage by confirming each step of CRISPR-mediated gene editing of mouse embryos and, therefore, can be considered as a supportive tool to existing procedures for verification of successful CRISPR/Cas9-mediated gene alterations in mouse embryos and further mutant production.},
}

Animals
*CRISPR-Cas Systems
*Gene Editing/methods
Mice
Embryo, Mammalian/metabolism
Female
Blastocyst/metabolism
Mice, Transgenic
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40146517,
year = {2025},
author = {Tripathi, JN and Tripathi, L},
title = {Agrobacterium tumefaciens-Mediated Genome Editing in Banana.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2911},
number = {},
pages = {143-153},
pmid = {40146517},
issn = {1940-6029},
mesh = {*Agrobacterium tumefaciens/genetics ; *Gene Editing/methods ; *Musa/genetics/microbiology ; *CRISPR-Cas Systems ; *Plants, Genetically Modified/genetics ; *Transformation, Genetic ; Genome, Plant ; Plasmids/genetics ; },
abstract = {This protocol outlines the Agrobacterium tumefaciens-mediated transformation process for genome editing in banana (Musa spp.). As a crop of significant agricultural and economic importance globally, improving banana cultivars is crucial for addressing challenges such as disease resistance, climate resilience and yield enhancement. The procedure presented here involves the establishment of embryogenic cell suspensions (ECSs) from immature male flowers, followed by co-cultivation with Agrobacterium containing the desired gene construct. Key steps include callus induction, ECS generation and maintenance, transformation using plasmid construct containing genome editing reagents such as CRISPR/Cas9, and regeneration. The method ensures the editing of target genes in the banana genome, facilitating genetic improvements.},
}

*Agrobacterium tumefaciens/genetics
*Gene Editing/methods
*Musa/genetics/microbiology
*CRISPR-Cas Systems
*Plants, Genetically Modified/genetics
*Transformation, Genetic
Genome, Plant
Plasmids/genetics

@article {pmid40146515,
year = {2025},
author = {Arruabarrena, A and Vidal, S},
title = {Agrobacterium-Mediated Transformation for Gene Editing Tomato Elite Breeding Lines.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2911},
number = {},
pages = {121-131},
pmid = {40146515},
issn = {1940-6029},
mesh = {*Solanum lycopersicum/genetics ; *Gene Editing/methods ; *Transformation, Genetic ; *Plant Breeding/methods ; *CRISPR-Cas Systems ; *Plants, Genetically Modified/genetics ; Agrobacterium/genetics ; Agrobacterium tumefaciens/genetics ; },
abstract = {Genome editing success in crop species is largely dependent on the availability of highly efficient plant transformation protocols. Tomato (Solanum lycopersicum) was the first dicotyledonous crop to be successfully mutagenized using CRISPR-Cas9. Despite many efforts, no standardized, simple protocol is available for non-model tomato genotypes. With the increasing availability of gene editing tools, the transformation of elite tomato breeding lines has gained importance because mutant variants can be easily incorporated into breeding programs. This chapter describes a protocol for transforming and gene editing in elite tomato breeding lines, reaching 3.6% transformation efficiency.},
}

*Solanum lycopersicum/genetics
*Gene Editing/methods
*Transformation, Genetic
*Plant Breeding/methods
*CRISPR-Cas Systems
*Plants, Genetically Modified/genetics
Agrobacterium/genetics
Agrobacterium tumefaciens/genetics

@article {pmid40146510,
year = {2025},
author = {Castillo Castro, FM and Payacán Ortiz, C},
title = {Agrobacterium-Mediated Transformation for Commercial Wheat Varieties.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2911},
number = {},
pages = {61-70},
pmid = {40146510},
issn = {1940-6029},
mesh = {*Triticum/genetics/growth & development/microbiology ; *Transformation, Genetic ; *Agrobacterium tumefaciens/genetics ; *Plants, Genetically Modified/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Plant Breeding/methods ; },
abstract = {Wheat is a crucial crop for global food security, and new breeding techniques face limitations due to low regeneration rates and a lack of transformable genotypes. We follow and adapt a robust Agrobacterium tumefaciens-mediated transformation system in spring commercial wheat varieties from Chile and Argentina grown in speed breeding conditions. By incorporating the developmental gene fusion GRF4-GIF1, we achieved successful regeneration of plantlets in different wheat varieties through CRISPR/Cas9-based gene editing. This advancement reduces genotype dependency, allowing broader use of genome-editing tools in commercial wheat varieties. In addition to delving into technical complexities, this contribution aims to advance fundamental understanding and practical applications in wheat genetics, serving as a valuable resource for researchers expanding their expertise.},
}

*Triticum/genetics/growth & development/microbiology
*Transformation, Genetic
*Agrobacterium tumefaciens/genetics
*Plants, Genetically Modified/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
Plant Breeding/methods

@article {pmid40146509,
year = {2025},
author = {Faion-Molina, M and Molina-Risco, MD and Bellinatti-Della Gracia, MD and Ibarra, O and Kim, B and Septiningsih, EM and Thomson, MJ},
title = {Agrobacterium tumefaciens-Mediated Plant Transformation and Gene Editing in Rice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2911},
number = {},
pages = {45-59},
pmid = {40146509},
issn = {1940-6029},
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *Transformation, Genetic ; *CRISPR-Cas Systems ; *Agrobacterium tumefaciens/genetics ; *Plants, Genetically Modified/genetics ; Oxidoreductases ; },
abstract = {Bottlenecks in plant transformation and regeneration have slowed progress in applying CRISPR/Cas9-based genome editing for crop improvement. Rice (Oryza sativa L.) has highly efficient temperate japonica transformation protocols, along with reasonably efficient indica protocols using immature embryos. However, rapid and efficient protocols are not available for transformation and regeneration in tropical japonica varieties, even though they represent most of the rice production in the USA and South America, along with some regions in Asia. This chapter describes a protocol for CRISPR/Cas9 gene editing using Agrobacterium-mediated transformation for the tropical japonica rice cultivar Presidio leading to knock-out mutations in the phytoene desaturase (PDS) gene.},
}

*Oryza/genetics
*Gene Editing/methods
*Transformation, Genetic
*CRISPR-Cas Systems
*Agrobacterium tumefaciens/genetics
*Plants, Genetically Modified/genetics
Oxidoreductases

@article {pmid40146284,
year = {2025},
author = {Kaniganti, S and Palakolanu, SR and Thiombiano, B and Damarasingh, J and Bommineni, PR and Che, P and Sharma, KK and Jones, T and Bouwmeester, H and Bhatnagar-Mathur, P},
title = {Developing Striga resistance in sorghum by modulating host cues through CRISPR/Cas9 gene editing.},
journal = {Plant cell reports},
volume = {44},
number = {4},
pages = {90},
pmid = {40146284},
issn = {1432-203X},
support = {DBT/2017/ICRISAT/973//Department of Biotechnology, Minstry of Science and Technology, India (IN)/ ; },
mesh = {*Sorghum/genetics/parasitology ; *CRISPR-Cas Systems ; *Striga/physiology ; *Gene Editing/methods ; *Plant Diseases/genetics/parasitology/immunology ; *Disease Resistance/genetics ; *Plants, Genetically Modified ; Mutation ; Lactones/metabolism ; Plant Proteins/genetics/metabolism ; Plant Weeds/genetics ; },
abstract = {High transformation and gene editing efficiencies in sorghum-produced, transgene-free SDN1-edited plants exhibit precise mutations, reduced germination stimulants, and enhanced resistance to Striga infection. Sorghum (Sorghum bicolor L.) is a primary food staple grain for millions in Sub-Saharan Africa (SSA). It is mainly constrained by the parasitic weed Striga, which causes up to 100% yield losses and affects over 60% of cultivable farmlands and livelihoods. In this study, CRISPR/Cas9 technology is utilized to induce mutations in core strigolactone (SL) biosynthetic genes, i.e., CCD7, CCD8, MAX1, in addition to an uncharacterized gene (DUF) in the fine-mapped 400 kb lgs1 region in sorghum to develop durable Striga resistance. Two sorghum cultivars were delivered with the expression cassettes through immature embryo-based Agrobacterium-mediated transformation. Our study demonstrated transformation and gene editing efficiencies of ~ 70 and up to 17.5% (calculated based on the numuber of established plants), respectively, in two sorghum genotypes. Subsequent analysis of homozygous E0 lines in the E1 generation confirmed stable integration of mutations for all targeted genes. Loss-of-function mutations in the CCD7, CCD8, MAX1, and DUF genes led to a significant downregulation of the expression of associated genes in the SL biosynthetic pathway. The phenotypic analysis of edited lines revealed changes in phenotypic patterns compared to wild-type plants. Analysis of root exudates showed significant reductions in SL production in edited lines compared to wild-type plants. Striga infection experiments demonstrated delayed or reduced emergence rates of Striga in edited lines with lower SL production, highlighting the potential for genetically altering SL production to control Striga infestations. This study provides insights into the functional roles of CCD7, CCD8, MAX1, and DUF genes in sorghum towards reduced and/or altered SL production and improved resistance to Striga infestations.},
}

*Sorghum/genetics/parasitology
*CRISPR-Cas Systems
*Striga/physiology
*Gene Editing/methods
*Plant Diseases/genetics/parasitology/immunology
*Disease Resistance/genetics
*Plants, Genetically Modified
Mutation
Lactones/metabolism
Plant Proteins/genetics/metabolism
Plant Weeds/genetics

@article {pmid40144376,
year = {2025},
author = {Smirnov, AV and Korablev, AN and Serova, IA and Yunusova, AM and Muravyova, AA and Valeev, ES and Battulin, NR},
title = {Studying concatenation of the Cas9-cleaved transgenes using barcodes.},
journal = {Vavilovskii zhurnal genetiki i selektsii},
volume = {29},
number = {1},
pages = {26-34},
doi = {10.18699/vjgb-25-04},
pmid = {40144376},
issn = {2500-0462},
abstract = {In pronuclear microinjection, the Cas9 endonuclease is employed to introduce in vivo DNA double-strand breaks at the genomic target locus or within the donor vector, thereby enhancing transgene integration. The manner by which Cas9 interacts with DNA repair factors during transgene end processing and integration is a topic of considerable interest and debate. In a previous study, we developed a barcode-based genetic system for the analysis of transgene recombination following pronuclear microinjection in mice. In this approach, the plasmid library is linearized with a restriction enzyme or a Cas9 RNP complex at the site between a pair of barcodes. A pool of barcoded molecules is injected into the pronucleus, resulting in the generation of multicopy concatemers. In the present report, we compared the effects of in vivo Cas9 cleavage (RNP+ experiment) and in vitro production of Cas9- linearized transgenes (RNP- experiment) on concatenation. In the RNP+ experiment, two transgenic single-copy embryos were identified. In the RNP- experiment, six positive embryos were identified, four of which exhibited lowcopy concatemers. Next-generation sequencing (NGS) analysis of the barcodes revealed that 53 % of the barcoded ends had switched their initial library pairs, indicating the involvement of the homologous recombination pathway. Out of the 20 transgene-transgene junctions examined, 11 exhibited no mutations and were presumably generated through re-ligation of Cas9-induced blunt ends. The majority of mutated junctions harbored asymmetrical deletions of 2-4 nucleotides, which were attributed to Cas9 end trimming. These findings suggest that Cas9-bound DNA may present obstacles to concatenation. Conversely, clean DNA ends were observed to be joined in a manner similar to restriction-digested ends, albeit with distinctive asymmetry. Future experiments utilizing in vivo CRISPR/ Cas cleavage will facilitate a deeper understanding of how CRISPR-endonucleases influence DNA repair processes.},
}

@article {pmid40143364,
year = {2025},
author = {Chakraborty, M and Nielsen, L and Nash, D and Nissimov, JI and Charles, TC and Aucoin, MG},
title = {Adapting Next-Generation Sequencing to in Process CRISPR-Cas9 Genome Editing of Recombinant AcMNPV Vectors: From Shotgun to Tiled-Amplicon Sequencing.},
journal = {Viruses},
volume = {17},
number = {3},
pages = {},
pmid = {40143364},
issn = {1999-4915},
support = {RGPIN 355513-2017//Natural Sciences and Engineering Research Council/ ; RGPIN-2023-03666//Natural Sciences and Engineering Research Council/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Nucleopolyhedroviruses/genetics ; *Genetic Vectors/genetics ; Animals ; *High-Throughput Nucleotide Sequencing ; Sf9 Cells ; *Genome, Viral ; Spodoptera/genetics/virology ; Cell Line ; },
abstract = {The alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the most commonly used virus in the Baculovirus Expression Vector System (BEVS) and has been utilized for the production of many human and veterinary biologics. AcMNPV has a large dsDNA genome that remains understudied, and relatively unmodified from the wild-type, especially considering how extensively utilized it is as an expression vector. Previously, our group utilized CRISPR-Cas9 genome engineering that revealed phenotypic changes when baculovirus genes are targeted using either co-expressed sgRNA or transfected sgRNA into a stable insect cell line that produced the Cas9 protein. Here, we describe a pipeline to sequence the recombinant AcMNPV expression vectors using shotgun sequencing, provide a set of primers for tiled-amplicon sequencing, show that untargeted baculovirus vector genomes remain relatively unchanged when amplified in Sf9-Cas9 cells, and confirm that AcMNPV gp64 gene disruption can minimize baculovirus contamination in cell cultures. Our findings provide a robust baseline for analyzing in process genome editing of baculoviruses.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*Nucleopolyhedroviruses/genetics
*Genetic Vectors/genetics
Animals
*High-Throughput Nucleotide Sequencing
Sf9 Cells
*Genome, Viral
Spodoptera/genetics/virology
Cell Line

@article {pmid40143350,
year = {2025},
author = {Wu, H and Wang, Z and Zhang, Y and Hu, L and Yang, J and Zhang, C and Lou, M and Pi, N and Wang, Q and Fan, S and Huang, Z},
title = {A New Human SCARB2 Knock-In Mouse Model for Studying Coxsackievirus A16 and Its Neurotoxicity.},
journal = {Viruses},
volume = {17},
number = {3},
pages = {},
pmid = {40143350},
issn = {1999-4915},
support = {NO.202105AD160018//Technology Innovation Talents Project of Yunnan Province/ ; NO. 2021-I2M-1-043//CAMS Innovation Fund for Medical Sciences (CIFMS)/ ; U2202214//Joint Funds of the National Natural Science Foundation of China/ ; 2023ZD040630//Scientific and Technological Innovation 2030/ ; },
mesh = {Animals ; Mice ; *Disease Models, Animal ; Humans ; *Gene Knock-In Techniques ; *Lysosomal Membrane Proteins/genetics/metabolism ; *Receptors, Scavenger/genetics/metabolism ; CRISPR-Cas Systems ; Enterovirus/genetics/pathogenicity/physiology ; Hand, Foot and Mouth Disease/virology/pathology ; Coxsackievirus Infections/virology ; Neurons/virology/pathology ; Gene Editing ; },
abstract = {Hand, Foot, and Mouth Disease (HFMD) is a viral illness caused by enterovirus infections. While the introduction of the enterovirus 71 (EV71) vaccine has significantly reduced the number of EV71-related cases, the continued spread of Coxsackievirus A16 (CVA16) remains a major public health threat. Previous studies have shown that human SCARB2 (hSCARB2) knock-in (KI) mice, generated using embryonic stem cell (ESC) technology, are susceptible to CVA16. However, these models have failed to reproduce the clinical pathology and neurotoxicity after CVA16 infection. Therefore, there is an urgent need for a more reliable and effective animal model to study CVA16. In this study, we successfully created a hSCARB2 KI mouse model targeting the ROSA26 locus using CRISPR/Cas9 gene editing technology. The application of CRISPR/Cas9 enabled stable and widespread expression of hSCARB2 in the model. After infection, the KI mice exhibited a clinical pathology that closely mimics human infection, with prominent limb weakness and paralysis. The virus was detectable in multiple major organs of the mice, with peak viral load observed on day 7 post-infection, gradually clearing thereafter. Further analysis revealed widespread neuronal necrosis and infiltration of inflammatory cells in the brain and spinal cord of the KI mice. Additionally, significant activation of astrocytes (GFAP-positive) and microglia (IBA1-positive) was observed in the brain, suggesting that CVA16 infection may induce limb paralysis by attacking neuronal cells. Overall, this model effectively replicates the neuropathological changes induced by CVA16 infection and provides a potential experimental platform for studying CVA16-associated pathogenesis and neurotoxicity.},
}

Animals
Mice
*Disease Models, Animal
Humans
*Gene Knock-In Techniques
*Lysosomal Membrane Proteins/genetics/metabolism
*Receptors, Scavenger/genetics/metabolism
CRISPR-Cas Systems
Enterovirus/genetics/pathogenicity/physiology
Hand, Foot and Mouth Disease/virology/pathology
Coxsackievirus Infections/virology
Neurons/virology/pathology
Gene Editing

@article {pmid39543397,
year = {2025},
author = {Loveless, TB and Carlson, CK and Dentzel Helmy, CA and Hu, VJ and Ross, SK and Demelo, MC and Murtaza, A and Liang, G and Ficht, M and Singhai, A and Pajoh-Casco, MJ and Liu, CC},
title = {Open-ended molecular recording of sequential cellular events into DNA.},
journal = {Nature chemical biology},
volume = {21},
number = {4},
pages = {512-521},
pmid = {39543397},
issn = {1552-4469},
support = {Predoctoral Fellowship//American Heart Association (American Heart Association, Inc.)/ ; R00GM140254//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; DP2 GM119163/GM/NIGMS NIH HHS/United States ; DP2GM119163//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; GRFP//National Science Foundation (NSF)/ ; R35 GM136297/GM/NIGMS NIH HHS/United States ; K99GM140254//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R21GM126287//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R21 GM126287/GM/NIGMS NIH HHS/United States ; 1763272//National Science Foundation (NSF)/ ; },
mesh = {*DNA/genetics ; Humans ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Animals ; HEK293 Cells ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Genetically encoded DNA recorders noninvasively convert transient biological events into durable mutations in a cell's genome, allowing for the later reconstruction of cellular experiences by DNA sequencing. We present a DNA recorder, peCHYRON, that achieves high-information, durable, and temporally resolved multiplexed recording of multiple cellular signals in mammalian cells. In each step of recording, prime editor, a Cas9-reverse transcriptase fusion protein, inserts a variable triplet DNA sequence alongside a constant propagator sequence that deactivates the previous and activates the next step of insertion. Insertions accumulate sequentially in a unidirectional order, editing can continue indefinitely, and high information is achieved by coexpressing a variety of prime editing guide RNAs (pegRNAs), each harboring unique triplet DNA sequences. We demonstrate that the constitutive expression of pegRNA collections generates insertion patterns for the straightforward reconstruction of cell lineage relationships and that the inducible expression of specific pegRNAs results in the accurate recording of exposures to biological stimuli.},
}

*DNA/genetics
Humans
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems
Animals
HEK293 Cells
CRISPR-Associated Protein 9/genetics/metabolism

@article {pmid40143178,
year = {2025},
author = {Elshobary, ME and Badawy, NK and Ashraf, Y and Zatioun, AA and Masriya, HH and Ammar, MM and Mohamed, NA and Mourad, S and Assy, AM},
title = {Combating Antibiotic Resistance: Mechanisms, Multidrug-Resistant Pathogens, and Novel Therapeutic Approaches: An Updated Review.},
journal = {Pharmaceuticals (Basel, Switzerland)},
volume = {18},
number = {3},
pages = {},
doi = {10.3390/ph18030402},
pmid = {40143178},
issn = {1424-8247},
abstract = {The escalating global health crisis of antibiotic resistance, driven by the rapid emergence of multidrug-resistant (MDR) bacterial pathogens, necessitates urgent and innovative countermeasures. This review comprehensively examines the diverse mechanisms employed by bacteria to evade antibiotic action, including alterations in cell membrane permeability, efflux pump overexpression, biofilm formation, target site modifications, and the enzymatic degradation of antibiotics. Specific focus is given to membrane transport systems such as ATP-binding cassette (ABC) transporters, resistance-nodulation-division (RND) efflux pumps, major facilitator superfamily (MFS) transporters, multidrug and toxic compound extrusion (MATE) systems, small multidrug resistance (SMR) families, and proteobacterial antimicrobial compound efflux (PACE) families. Additionally, the review explores the global burden of MDR pathogens and evaluates emerging therapeutic strategies, including quorum quenching (QQ), probiotics, postbiotics, synbiotics, antimicrobial peptides (AMPs), stem cell applications, immunotherapy, antibacterial photodynamic therapy (aPDT), and bacteriophage. Furthermore, this review discusses novel antimicrobial agents, such as animal-venom-derived compounds and nanobiotics, as promising alternatives to conventional antibiotics. The interplay between clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) in bacterial adaptive immunity is analyzed, revealing opportunities for targeted genetic interventions. By synthesizing current advancements and emerging strategies, this review underscores the necessity of interdisciplinary collaboration among biomedical scientists, researchers, and the pharmaceutical industry to drive the development of novel antibacterial agents. Ultimately, this comprehensive analysis provides a roadmap for future research, emphasizing the urgent need for sustainable and cooperative approaches to combat antibiotic resistance and safeguard global health.},
}

@article {pmid40142492,
year = {2025},
author = {Sadanov, AK and Baimakhanova, BB and Orasymbet, SE and Ratnikova, IA and Turlybaeva, ZZ and Baimakhanova, GB and Amitova, AA and Omirbekova, AA and Aitkaliyeva, GS and Kossalbayev, BD and Belkozhayev, AM},
title = {Engineering Useful Microbial Species for Pharmaceutical Applications.},
journal = {Microorganisms},
volume = {13},
number = {3},
pages = {},
doi = {10.3390/microorganisms13030599},
pmid = {40142492},
issn = {2076-2607},
support = {BR21882248//Program-targeted funding of the Committee of Science of the Ministry of Science and Higher Education of the Republic of Kazakh-stan/ ; },
abstract = {Microbial engineering has made a significant breakthrough in pharmaceutical biotechnology, greatly expanding the production of biologically active compounds, therapeutic proteins, and novel drug candidates. Recent advancements in genetic engineering, synthetic biology, and adaptive evolution have contributed to the optimization of microbial strains for pharmaceutical applications, playing a crucial role in enhancing their productivity and stability. The CRISPR-Cas system is widely utilized as a precise genome modification tool, enabling the enhancement of metabolite biosynthesis and the activation of synthetic biological pathways. Additionally, synthetic biology approaches allow for the targeted design of microorganisms with improved metabolic efficiency and therapeutic potential, thereby accelerating the development of new pharmaceutical products. The integration of artificial intelligence (AI) and machine learning (ML) plays a vital role in further advancing microbial engineering by predicting metabolic network interactions, optimizing bioprocesses, and accelerating the drug discovery process. However, challenges such as the efficient optimization of metabolic pathways, ensuring sustainable industrial-scale production, and meeting international regulatory requirements remain critical barriers in the field. Furthermore, to mitigate potential risks, it is essential to develop stringent biocontainment strategies and implement appropriate regulatory oversight. This review comprehensively examines recent innovations in microbial engineering, analyzing key technological advancements, regulatory challenges, and future development perspectives.},
}

@article {pmid40141287,
year = {2025},
author = {Li, J and Zhang, L and Li, C and Chen, W and Wang, T and Tan, L and Qiu, Y and Song, S and Li, B and Li, L},
title = {The Pentatricopeptide Repeat Protein OsPPR674 Regulates Rice Growth and Drought Sensitivity by Modulating RNA Editing of the Mitochondrial Transcript ccmC.},
journal = {International journal of molecular sciences},
volume = {26},
number = {6},
pages = {},
doi = {10.3390/ijms26062646},
pmid = {40141287},
issn = {1422-0067},
support = {U24A20396//National Natural Science Foundation of China/ ; 2024CX119//Hunan agricultural science and technology innovation fund project/ ; },
mesh = {*Oryza/genetics/growth & development/metabolism ; *RNA Editing ; *Plant Proteins/genetics/metabolism ; *Droughts ; *Gene Expression Regulation, Plant ; *Mitochondria/metabolism/genetics ; RNA, Mitochondrial/metabolism/genetics ; Mitochondrial Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified ; Mutation ; },
abstract = {The P-type pentatricopeptide repeat (PPR) proteins are crucial for RNA editing and post-transcriptional regulation in plant organelles, particularly mitochondria. This study investigates the role of OsPPR674 in rice, focusing on its function in mitochondrial RNA editing. Using CRISPR/Cas9 technology, we generated ppr674 mutant and examined its phenotypic and molecular characteristics. The results indicate that ppr674 exhibits reduced plant height, decreased seed-setting rate, and poor drought tolerance. Further analysis revealed that in the ppr674 mutant, RNA editing at the 299th nucleotide position of the mitochondrial ccmC gene (C-to-U conversion) was abolished. REMSAs showed that GST-PPR674 specifically binds to RNA probes targeting this ccmC-299 site, confirming its role in this editing process. In summary, these results suggest that OsPPR674 plays a pivotal role in mitochondrial RNA editing, emphasizing the significance of PPR proteins in organelle function and plant development.},
}

*Oryza/genetics/growth & development/metabolism
*RNA Editing
*Plant Proteins/genetics/metabolism
*Droughts
*Gene Expression Regulation, Plant
*Mitochondria/metabolism/genetics
RNA, Mitochondrial/metabolism/genetics
Mitochondrial Proteins/metabolism/genetics
CRISPR-Cas Systems
Plants, Genetically Modified
Mutation

@article {pmid40141236,
year = {2025},
author = {Livneh, Y and Agmon, D and Leor-Librach, E and Vainstein, A},
title = {Viral-Based Gene Editing System for Nutritional Improvement of Fructan Content in Lettuce.},
journal = {International journal of molecular sciences},
volume = {26},
number = {6},
pages = {},
doi = {10.3390/ijms26062594},
pmid = {40141236},
issn = {1422-0067},
support = {12-01-0037//Office of the Chief Scientist/ ; 20-01-0209//The National Center for Genome Editing in Agriculture, Israel/ ; 7500158//Ministry of Science and Technology, Israel/ ; },
mesh = {*Lactuca/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Fructans/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Plant Viruses/genetics ; },
abstract = {Lettuce is a globally cultivated and consumed leafy crop. Here we developed an efficient tobacco rattle virus (TRV)-based guide RNA (gRNA) delivery system for CRISPR/Cas editing in the commercial lettuce cultivar 'Noga'. Plants stably expressing Cas9 were inoculated with TRV vectors carrying gRNAs targeting five nutrient-associated genes. The system achieved an average editing efficiency of 48.7%, with up to 78.9% of regenerated plantlets showing independent mutations. This approach eliminates the need for antibiotic selection, simplifying tissue culture processes. The system supports diverse applications, including Cas12a editing and large-fragment deletions using dual gRNA sets. Targeting the fructan 1-exohydrolase 2 (1-FEH2) gene produced knockout lines with significant increases in prebiotic dietary fibre fructan content, up to 5.2-fold, and an average rise in the degree of polymerisation by 2.15 units compared with controls. Combining 1-FEH1 and 1-FEH2 knockouts did not further increase fructan levels, revealing 1-FEH2 as the predominant isozyme in lettuce. RT-qPCR analysis showed reduced expression of the upstream biosynthetic enzyme sucrose:sucrose 1-fructosyl transferase (1-SST), suggesting potential feedback inhibition in fructan metabolism. This TRV-based gene editing approach, utilised here to increase fructan content, could be applied to improve other valuable traits in lettuce, and may inspire similar systems to enhance nutritional content of crops.},
}

*Lactuca/genetics/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems
*Fructans/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
Plant Viruses/genetics

@article {pmid40141178,
year = {2025},
author = {Tan, Q and Wang, J and Hao, Y and Yang, S and Cao, B and Pan, W and Cao, M},
title = {Elf1 Deficiency Impairs Macrophage Development in Zebrafish Model Organism.},
journal = {International journal of molecular sciences},
volume = {26},
number = {6},
pages = {},
doi = {10.3390/ijms26062537},
pmid = {40141178},
issn = {1422-0067},
support = {2023YFA1802000//National Key R&D Program of China/ ; ZDBS-LY-SM010//Key Research Program of Frontier Sciences, Chinese Academy of Sciences/ ; YSBR-077//CAS Project for Young Scientists in Basic Research/ ; 21JC1406300//Shanghai Science and Technology In-novation Action Plan for Basic Research Program/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Macrophages/metabolism ; *Zebrafish Proteins/genetics/metabolism/deficiency ; Transcription Factors/genetics/metabolism/deficiency ; Receptors, CXCR4/genetics/metabolism ; Cell Differentiation/genetics ; Cell Movement/genetics ; CRISPR-Cas Systems ; Gene Editing ; Gene Knockdown Techniques ; },
abstract = {The Ets (E-twenty-six) family of transcription factors plays a critical role in hematopoiesis and myeloid differentiation. However, the specific functions of many family members in these processes remain largely underexplored and poorly understood. Here, we identify Elf1 (E74-like factor 1), an Ets family member, as a critical regulator of macrophage development in the zebrafish model organism, with minimal impact on neutrophil differentiation. Through morpholino knockdown screening and CRISPR/Cas9-mediated gene editing, we demonstrate that Elf1 is critical for macrophage development and tissue injury responses. Specific overexpression of dominant-negative Elf1 (DN-Elf1) in macrophages demonstrated a cell-autonomous effect on macrophage infiltration. Furthermore, the overexpression of cxcr4b, a gene downstream of Elf1 regulation and essential for cell migration and injury response, significantly rescued this defect, indicating Elf1 as a key regulator of macrophage function. Our findings shed light on the roles of Elf1 in macrophage development and injury response and also highlight zebrafish as a powerful model for immunity research.},
}

Animals
*Zebrafish/genetics
*Macrophages/metabolism
*Zebrafish Proteins/genetics/metabolism/deficiency
Transcription Factors/genetics/metabolism/deficiency
Receptors, CXCR4/genetics/metabolism
Cell Differentiation/genetics
Cell Movement/genetics
CRISPR-Cas Systems
Gene Editing
Gene Knockdown Techniques