@article {pmid39814846,
year = {2025},
author = {Shin, SW and Kim, SH and Gasselin, A and Lee, GM and Lee, JS},
title = {Comprehensive genome-scale CRISPR knockout screening of CHO cells.},
journal = {Scientific data},
volume = {12},
number = {1},
pages = {71},
pmid = {39814846},
issn = {2052-4463},
support = {RS-2024-00397714//National Research Foundation of Korea (NRF)/ ; RS-2024-00397714//National Research Foundation of Korea (NRF)/ ; RS-2024-00397714//National Research Foundation of Korea (NRF)/ ; RS-2024-00397714//National Research Foundation of Korea (NRF)/ ; },
mesh = {CHO Cells ; Animals ; *Cricetulus ; *CRISPR-Cas Systems ; Gene Knockout Techniques ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome ; High-Throughput Nucleotide Sequencing ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Chinese hamster ovary (CHO) cells play a pivotal role in the production of recombinant therapeutics. In the present study, we conducted a genome-scale pooled CRISPR knockout (KO) screening using a virus-free, recombinase-mediated cassette exchange-based platform in CHO-K1 host and CHO-K1 derived recombinant cells. Genome-wide guide RNA (gRNA) amplicon sequencing data were generated from cell libraries, as well as short- and long-term KO libraries, and validated through phenotypic assessment and gRNA read count distribution. Additionally, we obtained gRNA amplicon sequencing data from the highly productive recombinant cell populations. By analyzing these datasets, essential genes involved in cell fitness as well as functional target genes associated with therapeutic protein production can be identified. Collectively, our next-generation sequencing datasets, derived from a robust and reliable CRISPR screening method, provide valuable insights into CHO genomic functions, advancing the development of next-generation CHO factories.},
}

CHO Cells
Animals
*Cricetulus
*CRISPR-Cas Systems
Gene Knockout Techniques
RNA, Guide, CRISPR-Cas Systems/genetics
Genome
High-Throughput Nucleotide Sequencing
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid39812375,
year = {2025},
author = {Wang, C and Xu, X and Yao, W and Wang, L and Pang, X and Xu, S and Luo, X},
title = {Programmable DNA Nanoswitch-Regulated Plasmonic CRISPR/Cas12a-Gold Nanostars Reporter Platform for Nucleic Acid and Non-Nucleic Acid Biomarker Analysis Assisted by a Spatial Confinement Effect.},
journal = {Nano letters},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.nanolett.4c05829},
pmid = {39812375},
issn = {1530-6992},
abstract = {CRISPR/Cas 12a system based nucleic acid and non-nucleic acid targets detection faces two challenges including (1) multiple crRNAs are needed for multiple biomarkers detection and (2) insufficient sensitivity resulted from photobleaching of fluorescent dyes and the low kinetic cleavage rate for a traditional single-strand (ssDNA) reporter. To address these limitations, we developed a programmable DNA nanoswitch (NS)-regulated plasmonic CRISPR/Cas12a-gold nanostars (Au NSTs) reporter platform for detection of nucleic acid and non-nucleic acid biomarkers with the assistance of the spatial confinement effect. Through simply programming the target recognition sequence in NS, only one crRNA is required to detect both nucleic acid and non-nucleic acid biomarkers. The detection limit decreased by ∼196-fold for miRNA-375 and 122-fold for prostate-specific antigen (PSA), respectively. Moreover, versatile evaluation of miRNA-375 and PSA in clinical urine samples can also be achieved, according to which prostate cancer and healthy groups can be well identified.},
}

@article {pmid39810563,
year = {2025},
author = {Wang, W and Du, H and Dai, C and Ma, H and Luo, S and Wang, X and Guo, M and Kong, D and Wei, D},
title = {Amplification-free detection of Mycobacterium tuberculosis using CRISPR-Cas12a and graphene field-effect transistors.},
journal = {Nanoscale},
volume = {},
number = {},
pages = {},
doi = {10.1039/d4nr03852e},
pmid = {39810563},
issn = {2040-3372},
abstract = {Current molecular tests for tuberculosis (TB), such as whole genome sequencing and Xpert Mycobacterium tuberculosis/rifampicin resistance assay, exhibit limited sensitivity and necessitate the pre-amplification step of target DNA. This limitation greatly increases detection time and poses an increased risk of infection. Here, we present a graphene field-effect transistor (GFET) based on the CRISPR/Cas system for detecting Mycobacterium tuberculosis. The CRISPR/Cas12a system has the ability to specifically recognize and cleave target DNA. By integrating the system onto the FET platform and utilizing its electrical amplification capability, we achieve rapid and sensitive detection without requiring sample pre-amplification, with a limit of detection (LoD) as low as 2.42 × 10[-18] M. Cas12a-GFET devices can differentiate 30 positive cases from 56 serum samples within 5 minutes. These findings highlight its immense potential in future biological analysis and clinical diagnosis.},
}

@article {pmid39774325,
year = {2025},
author = {Funk, JS and Klimovich, M and Drangenstein, D and Pielhoop, O and Hunold, P and Borowek, A and Noeparast, M and Pavlakis, E and Neumann, M and Balourdas, DI and Kochhan, K and Merle, N and Bullwinkel, I and Wanzel, M and Elmshäuser, S and Teply-Szymanski, J and Nist, A and Procida, T and Bartkuhn, M and Humpert, K and Mernberger, M and Savai, R and Soussi, T and Joerger, AC and Stiewe, T},
title = {Deep CRISPR mutagenesis characterizes the functional diversity of TP53 mutations.},
journal = {Nature genetics},
volume = {57},
number = {1},
pages = {140-153},
pmid = {39774325},
issn = {1546-1718},
support = {STI 182/15-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; STI 182/13-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; GRK2573//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 109546710 Project A10//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; SA 1923/7-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC1213 A10N//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; JO 1473/1-3//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 031L0063//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/ ; DZL//Bundesministerium für Bildung und Forschung (Federal Ministry of Education and Research)/ ; LOEWE iCANx//Hessisches Ministerium für Wissenschaft und Kunst (Hessen State Ministry of Higher Education, Research and the Arts)/ ; 65-0004//Von-Behring-Röntgen-Stiftung (Von Behring-Röntgen-Foundation)/ ; 66-LV06//Von-Behring-Röntgen-Stiftung (Von Behring-Röntgen-Foundation)/ ; 71-0012//Von-Behring-Röntgen-Stiftung (Von Behring-Röntgen-Foundation)/ ; 09 R/2018//José Carreras Leukämie-Stiftung (Deutsche José Carreras Leukämie-Stiftung)/ ; TACTIC//Deutsche Krebshilfe (German Cancer Aid)/ ; },
mesh = {Humans ; *Tumor Suppressor Protein p53/genetics ; *Gene Editing/methods ; *Mutagenesis ; *CRISPR-Cas Systems ; *Neoplasms/genetics ; Mutation, Missense/genetics ; Cell Line, Tumor ; Mutation ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Nonsense Mediated mRNA Decay/genetics ; },
abstract = {The mutational landscape of TP53, a tumor suppressor mutated in about half of all cancers, includes over 2,000 known missense mutations. To fully leverage TP53 mutation status for personalized medicine, a thorough understanding of the functional diversity of these mutations is essential. We conducted a deep mutational scan using saturation genome editing with CRISPR-mediated homology-directed repair to engineer 9,225 TP53 variants in cancer cells. This high-resolution approach, covering 94.5% of all cancer-associated TP53 missense mutations, precisely mapped the impact of individual mutations on tumor cell fitness, surpassing previous deep mutational scan studies in distinguishing benign from pathogenic variants. Our results revealed even subtle loss-of-function phenotypes and identified promising mutants for pharmacological reactivation. Moreover, we uncovered the roles of splicing alterations and nonsense-mediated messenger RNA decay in mutation-driven TP53 dysfunction. These findings underscore the power of saturation genome editing in advancing clinical TP53 variant interpretation for genetic counseling and personalized cancer therapy.},
}

Humans
*Tumor Suppressor Protein p53/genetics
*Gene Editing/methods
*Mutagenesis
*CRISPR-Cas Systems
*Neoplasms/genetics
Mutation, Missense/genetics
Cell Line, Tumor
Mutation
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Nonsense Mediated mRNA Decay/genetics

@article {pmid39754554,
year = {2025},
author = {Hu, F and Liu, K and Zhang, Y and Zhao, S and Zhang, T and Yao, C and Lv, X and Wang, J and Liu, X and Peng, N},
title = {Short-Time Preamplification-Assisted One-Pot CRISPR Nucleic Acid Detection Method with Portable Self-Heating Equipment for Point-of-Care Diagnosis.},
journal = {Analytical chemistry},
volume = {97},
number = {1},
pages = {658-666},
doi = {10.1021/acs.analchem.4c05026},
pmid = {39754554},
issn = {1520-6882},
mesh = {*SARS-CoV-2/genetics/isolation & purification ; Humans ; *Nucleic Acid Amplification Techniques ; *COVID-19/diagnosis/virology ; *RNA, Viral/analysis/genetics ; *CRISPR-Cas Systems/genetics ; Point-of-Care Testing ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Point-of-Care Systems ; COVID-19 Nucleic Acid Testing/methods/instrumentation ; Limit of Detection ; },
abstract = {Infectious diseases, especially respiratory infections, have been significant threats to human health. Therefore, it is essential to develop rapid, portable, and highly sensitive diagnostic methods for their control. Herein, a short-time preamplified, one-pot clustered regularly interspaced short palindromic repeats (CRISPR) nucleic acid detection method (SPOC) is developed by combining the rapid recombinase polymerase amplification (RPA) with CRISPR-Cas12a to reduce the mutual interference and achieve facile and rapid molecular diagnosis. SPOC can reduce the detection time and stably detect up to 1 copy/μL of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA without affecting the detection sensitivity. A highly sensitive one-pot assay integrated with reverse transcription RPA is achieved by wrapping paraffin with a specific melting point on the lyophilized CRISPR reagent surface. A self-heating pack is designed based on thermodynamic principles to melt the paraffin and release CRISPR reagents, enabling low-cost and time-saving detection. Notably, the designed system, coupled with RNA extraction-free technology, can achieve "sample-in-answer-out" detection of the SARS-CoV-2 Orf1ab gene within 22 min using smartphone imaging. The developed assay is validated on 12 clinical samples, and the results 100% correlate with real-time polymerase chain reaction. SPOC is time-saving, is easy to operate, and can eliminate centrifugal and complex hardware devices, satisfying the demand for point-of-care diagnostics in resource-constrained settings.},
}

*SARS-CoV-2/genetics/isolation & purification
Humans
*Nucleic Acid Amplification Techniques
*COVID-19/diagnosis/virology
*RNA, Viral/analysis/genetics
*CRISPR-Cas Systems/genetics
Point-of-Care Testing
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Point-of-Care Systems
COVID-19 Nucleic Acid Testing/methods/instrumentation
Limit of Detection

@article {pmid39725131,
year = {2025},
author = {Compiro, P and Chomta, N and Nimnual, J and Sunantawanit, S and Payungporn, S and Rotcheewaphan, S and Keawsapsak, P},
title = {CRISPR-Cas12a-based detection and differentiation of Mycobacterium spp.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {567},
number = {},
pages = {120101},
doi = {10.1016/j.cca.2024.120101},
pmid = {39725131},
issn = {1873-3492},
mesh = {*Mycobacterium/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Mycobacterium species cause several vital human diseases, including tuberculosis and non-tuberculous mycobacterial infections, which are treated with different drug regimens Therefore, accurate and rapid diagnosis is essential for effective treatment and controlling the spread of these pathogens. This study aims to develop an isothermal method combining RPA and CRISPR-Cas12a techniques, named as MyTRACK, to detect and differentiate major clinical mycobacteria at the species level. The assay has no cross-reactivity with limit of detection of 1 to 100 copies/reaction for various targeted mycobacteria. The results demonstrated 100 % specificity and 92.59 % to 100 % sensitivity in clinical isolates and were consistent with the culture technique with LPA for clinical samples. The MyTRACK assay is an effective, portable, rapid, and accurate screening method for mycobacterial detection and identification, especially in low-resource clinical settings.},
}

*Mycobacterium/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
Humans
Nucleic Acid Amplification Techniques/methods

@article {pmid39720925,
year = {2025},
author = {Xu, Y and Chen, B and He, M and Yuan, G and Hu, B},
title = {Dual-Amplification Single-Particle ICP-MS Strategy Based on Strand Displacement Amplification-CRISPR/Cas12a Amplification for Homogeneous Detection of miRNA.},
journal = {Analytical chemistry},
volume = {97},
number = {1},
pages = {811-817},
doi = {10.1021/acs.analchem.4c05385},
pmid = {39720925},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Gold/chemistry ; *Nucleic Acid Amplification Techniques/methods ; *Metal Nanoparticles/chemistry ; *Mass Spectrometry ; MCF-7 Cells ; Limit of Detection ; },
abstract = {MicroRNAs (miRNAs) regulate a myriad of biological processes and thus have been regarded as useful biomarkers in biomedical research and clinical diagnosis. The specific and highly sensitive detection of miRNAs is of significant importance. Herein, a sensitive and rapid dual-amplification elemental labeling single-particle inductively coupled plasma-mass spectrometry (spICP-MS) analytical method based on strand displacement amplification (SDA) and CRISPR/Cas12a was developed for miRNA-21 detection. Taking gold nanoparticles (AuNPs) as the elemental labels, the Au NP probe initially hybridized with linker DNA, forming large aggregates. In the absence of target miRNA-21, large aggregates of AuNPs will produce high pulse signals in spICP-MS detection. In the presence of the target miRNA-21, it triggered the SDA reaction, and the SDA products activated CRISPR/Cas12a's trans-cleavage activity to cleave the linker DNA, resulting in disassembly of the AuNP aggregates. The AuNP aggregates with smaller size displayed lower pulse signals in spICP-MS detection. Under the optimal conditions, a good relationship between the average pulse signal intensity of AuNP aggregates and the concentration of miRNA-21 was obtained in the range of 0.5 fmol L[-1]-100 pmol L[-1] with a quantification limit as low as 0.5 fmol L[-1]. The developed method was successfully used for determination of miRNA-21 in human breast cancer cell lines (SK-BR-3 and MCF-7) and real blood samples from breast cancer patients. It is versatile, can be adapted to detect other targets by modifying the specific sequence of the SDA template chain that is complementary to the analytes, and offers a promising strategy for detecting various biomarkers with high sensitivity and specificity.},
}

*MicroRNAs/analysis/genetics
Humans
*CRISPR-Cas Systems/genetics
*Gold/chemistry
*Nucleic Acid Amplification Techniques/methods
*Metal Nanoparticles/chemistry
*Mass Spectrometry
MCF-7 Cells
Limit of Detection

@article {pmid39716527,
year = {2025},
author = {Liu, R and Ji, W and Jiang, M and Shen, J},
title = {CRISPR technology combined with isothermal amplification methods for the diagnosis of Candida albicans infection.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {567},
number = {},
pages = {120106},
doi = {10.1016/j.cca.2024.120106},
pmid = {39716527},
issn = {1873-3492},
mesh = {*Candida albicans/genetics/isolation & purification ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Candidiasis/diagnosis/microbiology ; CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Since Candida albicans, a type of fungus, causes severe infections that pose a significant threat to human health, its rapid detection is critical in clinical antifungal therapy. Traditional fungal diagnostic approaches are largely based on the culture method. This method is time-consuming and laborious, taking about 48-72 h, and cannot identify emerging species, making it unsuitable for critically ill patients with bloodstream infections, sepsis, and so on. Other antigen or nucleic acid amplification-based methods were also found to be unsuitable for Point-of-Care Testing (POCT) diagnosis due to various limitations. Therefore, establishing a new approach for the rapid diagnosis of Candida spp is imperative. Herein, we proposed a novel diagnostic method for invasive fungi detection. Specifically, we created a new CRISPR diagnostic platform for Candida albicans-specific Internal Transcriptional Spacer 2 (ITS2) gene by combining the DNase cleavage activity of Cas12a with Recombinase Polymerase Amplification (RPA). Furthermore, to achieve rapid on-site detection under low-resource conditions, we used a transverse lateral flow strip with a single target to visualize the Cas12a single enzyme digestion product. We designated the platform as a rapid molecular detection tool that integrates RPA and the CRISPR-Cas12a technology. The entire platform can accurately identify Candida albicans within 50 minwhile remaining unaffected by other fungi or bacteria. Furthermore, the detection limit of the platform could reach 10[2] CFU/ml. Moreover, this approach offers additional benefits, including easy operation, low set-up cost, and broad applicability for Candida albicans detection across medical institutions at all levels, especially in township health centers in resource-poor regions.},
}

*Candida albicans/genetics/isolation & purification
Humans
*Nucleic Acid Amplification Techniques/methods
*Candidiasis/diagnosis/microbiology
CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid39711046,
year = {2025},
author = {Yu, H and Xu, PF and Liu, Y and Jia, ZS and Li, YY and Tang, HW},
title = {LRET-Based Simultaneous Detection of Dual miRNAs via Multitrap Optical Tweezers Assisted Suspension Array Tagged by Two Different Luminescent Quenchable UCNPs Combining CRISPR/Cas12a Amplification.},
journal = {Analytical chemistry},
volume = {97},
number = {1},
pages = {602-612},
doi = {10.1021/acs.analchem.4c04895},
pmid = {39711046},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics ; Humans ; *Optical Tweezers ; *CRISPR-Cas Systems/genetics ; Nanoparticles/chemistry ; Fluorescence Resonance Energy Transfer ; Luminescence ; Nucleic Acid Amplification Techniques ; Limit of Detection ; MCF-7 Cells ; },
abstract = {Nowadays, optical tweezers play a vital role not only in optical manipulation but also in bioassay. As principal optical trapping objects, microbeads can combine optical tweezers with suspension array technology, with amply focused laser beams and adequately concentrated tags contributing to highly sensitive detection. In view of the inefficiency of conventional single-trap optical tweezers, multitrap systems are developed. Here, green- and blue-emitting core-shell-shell upconversion nanoparticles (UCNPs) are adopted to encode microbeads and determine dual miRNAs, with the internal shells leading the luminescence process to facilitate quenching through luminescence resonance energy transfer (LRET). Utilizing the trans cleavage of CRISPR/Cas12a, quenched luminescence signals are recovered and amplified, causing further enhanced detection sensitivity. Ultimately, limits of detection (LOD) of 17 and 22 aM are obtained with excellent specificities verified. Furthermore, dual miRNAs from MCF-7, A549, and MCF-10A cells are extracted and detected, with results consistent with those obtained by PCR. Notably, miR-155 in MCF-7 and A549 cells is detectable at the single-cell level. Thus, the differences in the measured miRNA levels between MCF-7 and MCF-10A cells imply the potential of this method to discriminate breast cancer cells from epithelial cells despite the difficulty in distinguishing different cancer cells due to similar miRNA levels.},
}

*MicroRNAs/analysis/genetics
Humans
*Optical Tweezers
*CRISPR-Cas Systems/genetics
Nanoparticles/chemistry
Fluorescence Resonance Energy Transfer
Luminescence
Nucleic Acid Amplification Techniques
Limit of Detection
MCF-7 Cells

@article {pmid39704707,
year = {2025},
author = {Deng, R and Sheng, J and Xie, Z and Yang, H and Yang, S and Xie, S and Tang, X and Zhao, S and Dong, H and Chen, M and Chang, K},
title = {miR-Cabiner: A Universal microRNA Sensing Platform Based on Self-Stacking Cascaded Bicyclic DNA Circuit-Mediated CRISPR/Cas12a.},
journal = {Analytical chemistry},
volume = {97},
number = {1},
pages = {799-810},
doi = {10.1021/acs.analchem.4c05370},
pmid = {39704707},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis ; Humans ; *CRISPR-Cas Systems/genetics ; *DNA/chemistry ; Biosensing Techniques/methods ; },
abstract = {CRISPR/Cas12a-based diagnostics have great potential for sensing nucleic acids, but their application is limited by the sequence-dependent property. A platform termed miR-Cabiner (a universal miRNA sensing platform based on self-stacking cascaded bicyclic DNA circuit-mediated CRISPR/Cas12a) is demonstrated herein that is sensitive and universal for analyzing miRNAs. This platform combines catalytic hairpin assembly (CHA) and hybrid chain reaction (HCR) into a unified circuit and finally cascades to CRISPR/Cas12a. Compared with the CHA-Cas12a and HCR-Cas12a systems, miR-Cabiner exhibits a significantly higher reaction rate. Panels of miRNAs (miR-130a, miR-10b, miR-21, and miR-1285), which are associated with diagnosis, staging, and prognosis of breast cancer, are designed to demonstrate the universality of miR-Cabiner. Four miRNAs can be detected to the fM-level by simply tuning the sequence in CHA components. Additionally, miRNA panel analysis also shows high accuracy in practical samples. This universally applicable platform for detecting miRNA may serve as an excellent tool for clinical diagnosis.},
}

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

@article {pmid39701943,
year = {2025},
author = {Zhao, Z and Xiong, Q and Zhu, Y and Zhang, C and Li, Z and Chen, Z and Zhang, Y and Deng, X and Tao, Y and Xu, S},
title = {CRISPR/Cas12a-Enabled Amplification-Free Colorimetric Visual Sensing Strategy for Point-of-Care Diagnostics of Biomarkers.},
journal = {Analytical chemistry},
volume = {97},
number = {1},
pages = {1019-1027},
doi = {10.1021/acs.analchem.4c06196},
pmid = {39701943},
issn = {1520-6882},
mesh = {Humans ; *Colorimetry ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques ; *Gold/chemistry ; BRCA1 Protein/genetics ; Metal Nanoparticles/chemistry ; Point-of-Care Testing ; Biomarkers, Tumor/blood/analysis ; Benzidines/chemistry ; Point-of-Care Systems ; Horseradish Peroxidase/chemistry/metabolism ; Biomarkers/blood/analysis ; Hydrogen Peroxide/chemistry ; },
abstract = {CRISPR/Cas12a-based biosensors have garnered significant attention in the field of point-of-care testing (POCT), yet the majority of the CRISPR-based POCT methods employ fluorescent systems as report probes. Herein, we report a new CRISPR/Cas12a-enabled multicolor visual biosensing strategy for the rapid detection of disease biomarkers. The proposed assay provided vivid color responses to enhance the accuracy of visual detection. In the existence of the target, the trans-cleavage activity of CRISPR-Cas12a was activated. The report probe modified with magnetic beads (MBs) and horseradish peroxidase (HRP) was cleaved, and HRP was released in the supernatant. As a result, HRP mediated the etching of gold nanobipyramids (AuNBPs) under hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine and generated a vivid color response. The proposed method has been verified by the detection of the breast cancer 1 gene (BRCA1) as a proof-of-principle target. According to the different colors of AuNBPs, our experimental results have demonstrated that as low as 30 pM BRCA1 can be detected with no more than 60 min. Additionally, the proposed sensor has been successfully applied in the analysis of BRCA1 in human serum samples with satisfactory results, which indicates great potential for the sensitive determination of biomarkers and the POCT area.},
}

Humans
*Colorimetry
*CRISPR-Cas Systems/genetics
*Biosensing Techniques
*Gold/chemistry
BRCA1 Protein/genetics
Metal Nanoparticles/chemistry
Point-of-Care Testing
Biomarkers, Tumor/blood/analysis
Benzidines/chemistry
Point-of-Care Systems
Horseradish Peroxidase/chemistry/metabolism
Biomarkers/blood/analysis
Hydrogen Peroxide/chemistry

@article {pmid39670529,
year = {2025},
author = {Chang, G and Li, Y and Peng, L and Shen, C and Lu, Y and Teng, W and Liu, Y and Wang, Y and Zhu, W and Liu, C and He, X and Tong, Y and Zhao, X},
title = {TavWA1 is critical for wheat growth by modulating cell morphology and arrangement.},
journal = {Journal of integrative plant biology},
volume = {67},
number = {1},
pages = {71-86},
doi = {10.1111/jipb.13807},
pmid = {39670529},
issn = {1744-7909},
support = {2023ZD0406802//Biological Breeding-National Science and Technology Major Project/ ; XDA24010202//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; },
mesh = {*Triticum/genetics/growth & development/metabolism ; *Plant Proteins/metabolism/genetics ; *Mutation/genetics ; Phenotype ; Gene Expression Regulation, Plant ; Gene Editing ; CRISPR-Cas Systems/genetics ; },
abstract = {Plant growth is determined by the production of cells and initiation of new organs. Exploring genes that control cell number and cell size is of great significance for understanding plant growth regulation. In this study, we characterized two wheat mutants, ah and dl, with abnormal growth. The ah mutant is a naturally occurring variant characterized by severe dwarfism, increased tiller number, and reduced grain length, while the dl mutant is derived from an ethyl methane sulfonate (EMS)-mutagenized population and exhibits smaller grain size and slightly reduced plant height. Cytological analyses revealed abnormal cell number, cell morphology and arrangement in the stems and leaves of the ah mutant, along with reduced cell length in the grains of the dl mutant. Map-based cloning identified that both mutants carry mutations in the same gene TavWA1-7D, which encodes a protein with a von Willebrand factor A (vWA) domain. The ah mutant harbors a 174-bp insertion in the 1,402-bp coding sequence (CDS) of TavWA1-7D, causing premature termination of protein translation, while the dl mutant contains a Glu420Lys substitution. Mimicking the TavWA1-7D[ah] through clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9-mediated genome editing leads to a severe dwarfism phenotype. The C-terminus of the protein is crucial for its correct subcellular localization and interaction, supporting its critical role for TavWA1-7D function. Proteomic analysis showed that the dwarf phenotype of the ah mutant is associated with impaired photosynthesis, ribosome function, and nucleosome formation. Additionally, TavWA1-7D interacts with an E3 ligase, TaVIP1-3B, the expression levels of which are elevated in both mutants. Overexpression and knockout studies of TaVIP1-3B demonstrated its negative regulatory role in cell length and grain size. Together, our findings suggest that TavWA1-7D plays a vital role in regulating wheat growth and yield-related traits, with the dl mutant's short grain phenotype being associated with TaVIP1-3B expression levels.},
}

*Triticum/genetics/growth & development/metabolism
*Plant Proteins/metabolism/genetics
*Mutation/genetics
Phenotype
Gene Expression Regulation, Plant
Gene Editing
CRISPR-Cas Systems/genetics

@article {pmid39642875,
year = {2025},
author = {Rottner, AK and Lundin, A and Li, S and Firth, M and Maresca, M and Sienski, G},
title = {Optimized prime editing of the Alzheimer's disease-associated APOE4 mutation.},
journal = {Stem cell reports},
volume = {20},
number = {1},
pages = {102372},
doi = {10.1016/j.stemcr.2024.11.002},
pmid = {39642875},
issn = {2213-6711},
mesh = {Humans ; *Alzheimer Disease/genetics ; *Gene Editing/methods ; *Apolipoprotein E4/genetics ; *Induced Pluripotent Stem Cells/metabolism ; *Mutation ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Gene editing strategies to safely and robustly modify the Alzheimer's disease-associated APOE4 isoform are still lacking. Prime editing (PE) enables the precise introduction of genetic variants with minimal unintended editing and without donor templates. However, it requires optimization for each target site and has not yet been applied to APOE4 gene editing. Here, we screened PE guide RNA (pegRNA) parameters and PE systems for introducing the APOE4 variant and applied the optimized PE strategy to generate disease-relevant human induced pluripotent stem cell models. We show that introducing a single-nucleotide difference required for APOE4 correction inhibits PE activity. To advance efficient and robust genome engineering of precise genetic variants, we further present a reliable PE enrichment strategy based on diphtheria toxin co-selection. Our work provides an optimized and reproducible genome engineering pipeline to generate APOE4 disease models and outlines novel strategies to accelerate genome editing in cellular disease model generation.},
}

Humans
*Alzheimer Disease/genetics
*Gene Editing/methods
*Apolipoprotein E4/genetics
*Induced Pluripotent Stem Cells/metabolism
*Mutation
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
HEK293 Cells

@article {pmid39642091,
year = {2025},
author = {Basol, M and Ersoz-Gulseven, E and Ozaktas, H and Kalyoncu, S and Utine, CA and Cakan-Akdogan, G},
title = {Loss of carbohydrate sulfotransferase 6 function leads to macular corneal dystrophy phenotypes and skeletal defects in zebrafish.},
journal = {The FEBS journal},
volume = {292},
number = {2},
pages = {373-390},
doi = {10.1111/febs.17337},
pmid = {39642091},
issn = {1742-4658},
support = {219S943//Türkiye Bilimsel ve Teknolojik Araştırma Kurumu/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Zebrafish Proteins/genetics/metabolism ; *Carbohydrate Sulfotransferases ; *Sulfotransferases/genetics/metabolism ; *Corneal Dystrophies, Hereditary/genetics/pathology/metabolism ; *Phenotype ; *Disease Models, Animal ; Cornea/pathology/metabolism ; CRISPR-Cas Systems ; Keratan Sulfate/genetics/metabolism ; Humans ; Gene Editing ; },
abstract = {The carbohydrate sulfotransferase 6 (chst6) gene is linked to macular corneal dystrophy (MCD), a rare disease that leads to bilateral blindness due to the accumulation of opaque aggregates in the corneal stroma. chst6 encodes for a keratan sulfate proteoglycan (KSPG) specific sulfotransferase. MCD patients lose sulfated KSPGs (cKS) in the cornea and the serum. The significance of serum cKS loss has not been understood. Zebrafish cornea structure is similar to that of humans and it contains high levels of sulfated cKS in the stroma. Here, zebrafish chst6 is shown to be expressed in the cornea and head structures of the embryos. An animal model of MCD is developed by generating chst6 mutant animals with CRISPR/Cas9-mediated gene editing. The dramatic decrease in cKS epitopes in the mutants was shown with ELISA and immunofluorescence. Morphological defects or alterations of jaw cartilage were detected in a minor fraction of the mutant larvae. Loss of cKS epitopes and morphological defects was fully rescued with wild-type chst6. Mutant adult zebrafish displayed all clinical manifestations of MCD, while a fraction also displayed jaw and skeleton defects. Opaque accumulations formed in the eye, which were alcian blue positive. Loss of cKS in the corneal stroma and a decrease in corneal thickness were shown. Interestingly, alteration of transforming growth factor beta-induced (BIGH3) expression which was not described in patients was also observed. This is the first report of an MCD model in a genetically tractable organism, providing a preclinical model and insight into the importance of KSPG sulfation for proper skeletal morphogenesis.},
}

Animals
*Zebrafish/genetics
*Zebrafish Proteins/genetics/metabolism
*Carbohydrate Sulfotransferases
*Sulfotransferases/genetics/metabolism
*Corneal Dystrophies, Hereditary/genetics/pathology/metabolism
*Phenotype
*Disease Models, Animal
Cornea/pathology/metabolism
CRISPR-Cas Systems
Keratan Sulfate/genetics/metabolism
Humans
Gene Editing

@article {pmid39471459,
year = {2025},
author = {Hesami, M and Pepe, M and Spitzer-Rimon, B and Eskandari, M and Jones, AMP},
title = {Epigenetic factors related to recalcitrance in plant biotechnology.},
journal = {Genome},
volume = {68},
number = {},
pages = {1-11},
doi = {10.1139/gen-2024-0098},
pmid = {39471459},
issn = {1480-3321},
mesh = {*Epigenesis, Genetic ; *Biotechnology/methods ; *Plant Somatic Embryogenesis Techniques/methods ; *Gene Editing/methods ; Plants/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {This review explores the challenges and potential solutions in plant micropropagation and biotechnology. While these techniques have proven successful for many species, certain plants or tissues are recalcitrant and do not respond as desired, limiting the application of these technologies due to unattainable or minimal in vitro regeneration rates. Indeed, traditional in vitro culture techniques may fail to induce organogenesis or somatic embryogenesis in some plants, leading to classification as in vitro recalcitrance. This paper focuses on recalcitrance to somatic embryogenesis due to its promise for regenerating juvenile propagules and applications in biotechnology. Specifically, this paper will focus on epigenetic factors that regulate recalcitrance as understanding them may help overcome these barriers. Transformation recalcitrance is also addressed, with strategies proposed to improve transformation frequency. The paper concludes with a review of CRISPR-mediated genome editing's potential in modifying somatic embryogenesis-related epigenetic status and strategies for addressing transformation recalcitrance.},
}

*Epigenesis, Genetic
*Biotechnology/methods
*Plant Somatic Embryogenesis Techniques/methods
*Gene Editing/methods
Plants/genetics
CRISPR-Cas Systems
Plants, Genetically Modified

@article {pmid39451161,
year = {2025},
author = {Pan, W and Gao, C and Niu, D and Cheng, J and Zhang, J and Yan, X and Long, Q and Zhu, Y and Sun, W and Xie, Q and He, Y and Deng, XW and Zhang, H and Li, J},
title = {Efficient gene disruption in polyploid genome by Cas9-Trex2 fusion protein.},
journal = {Journal of integrative plant biology},
volume = {67},
number = {1},
pages = {7-10},
doi = {10.1111/jipb.13797},
pmid = {39451161},
issn = {1744-7909},
support = {TSQN202103160//Taishan Scholar Foundation of Shandong Province/ ; ZR202211070163//the Key R&D Program of Shandong Province/ ; ZR2021ZD30//Natural Science Foundation of Shandong Province/ ; ZR2022ZD22//Natural Science Foundation of Shandong Province/ ; ZR202103010168//the Excellent Youth Foundation of Shandong Scientific Committee/ ; },
mesh = {*Polyploidy ; *Gene Editing/methods ; *Genome, Plant/genetics ; Triticum/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; CRISPR-Cas Systems/genetics ; Exodeoxyribonucleases/genetics/metabolism ; Plant Proteins/genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; },
abstract = {The fusion of the exonuclease Trex2 with the Cas9 protein significantly enhanced the efficiency of genome editing in hexaploid common wheat, particularly for the simultaneous editing of multiple favorable alleles within a single generation, thereby facilitating genome editing-assisted breeding in polyploid crops.},
}

*Polyploidy
*Gene Editing/methods
*Genome, Plant/genetics
Triticum/genetics
CRISPR-Associated Protein 9/metabolism/genetics
CRISPR-Cas Systems/genetics
Exodeoxyribonucleases/genetics/metabolism
Plant Proteins/genetics/metabolism
Recombinant Fusion Proteins/genetics/metabolism

@article {pmid39240004,
year = {2025},
author = {Zhao, Y and Cheng, P and Liu, Y and Liu, C and Hu, Z and Xin, D and Wu, X and Yang, M and Chen, Q},
title = {A highly efficient soybean transformation system using GRF3-GIF1 chimeric protein.},
journal = {Journal of integrative plant biology},
volume = {67},
number = {1},
pages = {3-6},
doi = {10.1111/jipb.13767},
pmid = {39240004},
issn = {1744-7909},
support = {TD2022C003//Natural Science Foundation of Heilongjiang Province/ ; YQ2022C010//Natural Science Foundation of Heilongjiang Province/ ; 31971899//National Natural Science Foundation of China/ ; 32272072//National Natural Science Foundation of China/ ; 32272093//National Natural Science Foundation of China/ ; U20A2027//National Natural Science Foundation of China/ ; 2021YFF1001202//National Key R&D Program of China/ ; 2023YFD2300101-02//National Key R&D Program of China/ ; },
mesh = {*Glycine max/genetics ; *Plants, Genetically Modified/genetics ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Plant Proteins/genetics/metabolism ; Transformation, Genetic ; Recombinant Fusion Proteins/genetics/metabolism ; },
abstract = {Expression of GRF3-GIF1 chimera significantly enhanced regeneration and transformation efficiency in soybean, increasing the number of transformable cultivars. Moreover, GmGRF3-GIF1 can be combined with CRISPR/Cas9 for highly effective gene editing.},
}

*Glycine max/genetics
*Plants, Genetically Modified/genetics
Gene Editing/methods
CRISPR-Cas Systems/genetics
Plant Proteins/genetics/metabolism
Transformation, Genetic
Recombinant Fusion Proteins/genetics/metabolism

@article {pmid39810095,
year = {2025},
author = {Liu, W and Wang, D and He, Q and Cao, S and Cao, J and Zhao, H and Cui, J and Yang, F},
title = {A strategy for controlling Hypervirulent Klebsiella pneumoniae: inhibition of ClpV expression.},
journal = {BMC microbiology},
volume = {25},
number = {1},
pages = {22},
pmid = {39810095},
issn = {1471-2180},
mesh = {*Klebsiella pneumoniae/genetics/pathogenicity/drug effects ; Virulence/genetics ; *Virulence Factors/genetics ; *Klebsiella Infections/microbiology/prevention & control ; Animals ; *Biofilms/growth & development/drug effects ; *Bacterial Proteins/genetics/metabolism ; *Gene Deletion ; Gene Expression Regulation, Bacterial ; Moths/microbiology ; Larva/microbiology ; CRISPR-Cas Systems ; },
abstract = {The emergence and prevalence of hypervirulent Klebsiella pneumoniae (hvKP) have proposed a great challenge to control this infection. Therefore, exploring some new drugs or strategies for treating hvKP infection is an urgent issue for scientific researchers. In the present study, the clpV gene deletion strain of hvKP (ΔclpV-hvKP) was constructed using CRISPR-Cas9 technology, and the biological characteristics of ΔclpV-hvKP were investigated to explore the new targets for controlling this pathogen. The results showed that clpV gene deletion did not affect the growth ability of hvKP. However, knocking out the clpV gene markedly decreased the mucoid phenotype and the biofilm formation ability of hvKP. It reduced the interspecific competition of hvKP with Escherichia coli, Salmonella, Pseudomonas aeruginosa, and Staphylococcus aureus. The clpV deletion significantly changed the transcriptome profile of hvKP, inhibited the expression of virulence factors, and decreased the lethality of hvKP against Galleria mellonella larvae. In vitro experiments showed that lithocholic acid could inhibit the expression of the clpV gene and reduce the virulence of hvKP. Our data suggested that the clpV gene may be a potential target for decreasing hvKP infection risk.},
}

*Klebsiella pneumoniae/genetics/pathogenicity/drug effects
Virulence/genetics
*Virulence Factors/genetics
*Klebsiella Infections/microbiology/prevention & control
Animals
*Biofilms/growth & development/drug effects
*Bacterial Proteins/genetics/metabolism
*Gene Deletion
Gene Expression Regulation, Bacterial
Moths/microbiology
Larva/microbiology
CRISPR-Cas Systems

@article {pmid39810027,
year = {2025},
author = {Weng, Y and Wang, Y and Wang, K and Wu, F and Wei, Y and Jiang, J and Zhu, Y and Wang, F and Xie, H and Xiao, Y and Cai, Q and Xie, H and Zhang, J},
title = {OsLOX1 positively regulates seed vigor and drought tolerance in rice.},
journal = {Plant molecular biology},
volume = {115},
number = {1},
pages = {16},
pmid = {39810027},
issn = {1573-5028},
support = {CARS-01-20//the National Rice Industry Technology System of Modern Agriculture for China/ ; XTCXGC2021001//he "5511" Collaborative Innovation Project for High-quality Development and Surpasses of Agriculture between the Government of Fujian and Chinese Academy of Agricultural Sciences/ ; 2020R1023008//the Special Foundation of Non-Profit Research Institutes of Fujian Province/ ; },
mesh = {*Oryza/genetics/physiology/enzymology ; *Seeds/genetics/physiology/growth & development ; *Droughts ; *Gene Expression Regulation, Plant ; *Germination/genetics ; *Plant Proteins/genetics/metabolism ; *Lipoxygenase/genetics/metabolism ; *Stress, Physiological ; Hydrogen Peroxide/metabolism ; Malondialdehyde/metabolism ; Plants, Genetically Modified ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Antioxidants/metabolism ; Superoxide Dismutase/metabolism/genetics ; Catalase/metabolism/genetics ; Drought Resistance ; },
abstract = {The lipoxygenase (LOX) gene family is widely distributed in plants, and its activity is closely associated with seed viability and stress tolerance. In this study, we cloned the rice(Oryza sativa)lipoxygenase gene OsLOX1, a key participant in the 13-lipoxygenase metabolic pathway. Our primary focus was to investigate its role in mediating responses to drought stress and seed germination in rice. Histochemical staining and qPCR analysis indicated that the expression level of OsLOX1 was relatively high in leaves and early germinating seeds. Our findings revealed that mutant lines with CRISPR/Cas9-induced knockout of OsLOX1 exhibited reduced tolerance to drought stress compared with the wild-type. This was accompanied by elevated levels of H2O2 and malondialdehyde, and a decrease in the expression levels of genes associated with antioxidant enzymes. Furthermore, knockout of OsLOX1 reduced the longevity of rice seeds increased H2O2 and MDA levels, and decreased the activities of the antioxidant enzymes superoxide dismutase and catalase, compared with the wild-type. These findings demonstrated that OsLOX1 positively regulated rice seed vigor and drought stress.},
}

*Oryza/genetics/physiology/enzymology
*Seeds/genetics/physiology/growth & development
*Droughts
*Gene Expression Regulation, Plant
*Germination/genetics
*Plant Proteins/genetics/metabolism
*Lipoxygenase/genetics/metabolism
*Stress, Physiological
Hydrogen Peroxide/metabolism
Malondialdehyde/metabolism
Plants, Genetically Modified
Gene Knockout Techniques
CRISPR-Cas Systems
Antioxidants/metabolism
Superoxide Dismutase/metabolism/genetics
Catalase/metabolism/genetics
Drought Resistance

@article {pmid39809780,
year = {2025},
author = {Park, SJ and Ju, S and Jung, WJ and Jeong, TY and Yoon, DE and Lee, JH and Yang, J and Lee, H and Choi, J and Kim, HS and Kim, K},
title = {Robust genome editing activity and the applications of enhanced miniature CRISPR-Cas12f1.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {677},
pmid = {39809780},
issn = {2041-1723},
support = {RS-2023-00220894//National Research Foundation of Korea (NRF)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; HEK293 Cells ; Apoptosis/genetics ; Genetic Therapy/methods ; Cell Line, Tumor ; CRISPR-Associated Proteins/metabolism/genetics ; },
abstract = {With recent advancements in gene editing technology using the CRISPR/Cas system, there is a demand for more effective gene editors. A key factor facilitating efficient gene editing is effective CRISPR delivery into cells, which is known to be associated with the size of the CRISPR system. Accordingly, compact CRISPR-Cas systems derived from various strains are discovered, among which Un1Cas12f1 is 2.6 times smaller than SpCas9, providing advantages for gene therapy research. Despite extensive engineering efforts to improve Un1Cas12f1, the editing efficiency of Un1Cas12f1 is still shown to be low depending on the target site. To overcome this limitation, we develop enhanced Cas12f1 (eCas12f1), which exhibits gene editing activity similar to SpCas9 and AsCpf1, even in gene targets where previously improved Un1Cas12f1 variants showed low gene editing efficiency. Furthermore, we demonstrate that eCas12f1 efficiently induces apoptosis in cancer cells and is compatible with base editing and regulation of gene expression, verifying its high utility and applicability in gene therapy research.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
Humans
HEK293 Cells
Apoptosis/genetics
Genetic Therapy/methods
Cell Line, Tumor
CRISPR-Associated Proteins/metabolism/genetics

@article {pmid39809757,
year = {2025},
author = {Zou, J and Jiang, M and Xiao, R and Sun, H and Liu, H and Peacock, T and Tu, S and Chen, T and Guo, J and Zhao, Y and Barclay, W and Xie, S and Zhou, H},
title = {GGCX promotes Eurasian avian-like H1N1 swine influenza virus adaption to interspecies receptor binding.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {670},
pmid = {39809757},
issn = {2041-1723},
support = {32025036//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32430104//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2021CFA016//Natural Science Foundation of Hebei Province (Hebei Provincial Natural Science Foundation)/ ; },
mesh = {Animals ; *Influenza A Virus, H1N1 Subtype/genetics/metabolism ; Swine ; Humans ; *Virus Replication ; *Receptors, Virus/metabolism/genetics ; CRISPR-Cas Systems ; Hemagglutinin Glycoproteins, Influenza Virus/metabolism/genetics ; Orthomyxoviridae Infections/virology/metabolism ; Madin Darby Canine Kidney Cells ; Dogs ; HEK293 Cells ; Cell Line ; Receptors, Cell Surface/metabolism/genetics ; Mice ; },
abstract = {The Eurasian avian-like (EA) H1N1 swine influenza virus (SIV) possesses the capacity to instigate the next influenza pandemic, owing to its heightened affinity for the human-type α-2,6 sialic acid (SA) receptor. Nevertheless, the molecular mechanisms underlying the switch in receptor binding preferences of EA H1N1 SIV remain elusive. In this study, we conduct a comprehensive genome-wide CRISPR/Cas9 knockout screen utilizing EA H1N1 SIV in porcine kidney cells. Knocking out the enzyme gamma glutamyl carboxylase (GGCX) reduces virus replication in vitro and in vivo by inhibiting the carboxylation modification of viral haemagglutinin (HA) and the adhesion of progeny viruses, ultimately impeding the replication of EA H1N1 SIV. Furthermore, GGCX is revealed to be the determinant of the D225E substitution of EA H1N1 SIV, and GGCX-medicated carboxylation modification of HA 225E contributes to the receptor binding adaption of EA H1N1 SIV to the α-2,6 SA receptor. Taken together, our CRISPR screen has elucidated a novel function of GGCX in the support of EA H1N1 SIV adaption for binding to α-2,6 SA receptor. Consequently, GGCX emerges as a prospective antiviral target against the infection and transmission of EA H1N1 SIV.},
}

Animals
*Influenza A Virus, H1N1 Subtype/genetics/metabolism
Swine
Humans
*Virus Replication
*Receptors, Virus/metabolism/genetics
CRISPR-Cas Systems
Hemagglutinin Glycoproteins, Influenza Virus/metabolism/genetics
Orthomyxoviridae Infections/virology/metabolism
Madin Darby Canine Kidney Cells
Dogs
HEK293 Cells
Cell Line
Receptors, Cell Surface/metabolism/genetics
Mice

@article {pmid39809734,
year = {2025},
author = {Mormile, BW and Yan, Y and Bauer, T and Wang, L and Rivero, RC and Carpenter, SCD and Danmaigona Clement, C and Cox, KL and Zhang, L and Ma, X and Wheeler, TA and Dever, JK and He, P and Bogdanove, AJ and Shan, L},
title = {Activation of three targets by a TAL effector confers susceptibility to bacterial blight of cotton.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {644},
pmid = {39809734},
issn = {2041-1723},
support = {2018-67013-28513//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2020-67013-41537//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 18-123TX//Cotton Incorporated (Cotton Inc.)/ ; IOS-2421016//National Science Foundation (NSF)/ ; },
mesh = {*Xanthomonas/pathogenicity/genetics/metabolism ; *Plant Diseases/microbiology ; *Transcription Activator-Like Effectors/metabolism/genetics ; *Gossypium/microbiology ; Virulence/genetics ; *CRISPR-Cas Systems ; *Bacterial Proteins/metabolism/genetics ; Gene Editing ; Host-Pathogen Interactions ; Genome, Bacterial ; Gene Expression Profiling ; },
abstract = {Bacterial transcription activator-like effectors (TALEs) promote pathogenicity by activating host susceptibility (S) genes. To understand the pathogenicity and host adaptation of Xanthomonas citri pv. malvacearum (Xcm), we assemble the genome and the TALE repertoire of three recent Xcm Texas isolates. A newly evolved TALE, Tal7b, activates GhSWEET14a and GhSWEET14b, different from GhSWEET10 targeted by a TALE in an early Xcm isolate. Activation of GhSWEET14a and GhSWEET14b results in water-soaked lesions. Transcriptome profiling coupled with TALE-binding element prediction identify a pectin lyase gene as an additional Tal7b target, quantitatively contributing to Xcm virulence alongside GhSWEET14a/b. CRISPR-Cas9 gene editing supports the function of GhSWEETs in cotton bacterial blight and the promise of disrupting the TALE-binding site in S genes for disease management. Collectively, our findings elucidate the rapid evolution of TALEs in Xanthomonas field isolates and highlight the virulence mechanism wherein TALEs induce multiple S genes to promote pathogenicity.},
}

*Xanthomonas/pathogenicity/genetics/metabolism
*Plant Diseases/microbiology
*Transcription Activator-Like Effectors/metabolism/genetics
*Gossypium/microbiology
Virulence/genetics
*CRISPR-Cas Systems
*Bacterial Proteins/metabolism/genetics
Gene Editing
Host-Pathogen Interactions
Genome, Bacterial
Gene Expression Profiling

@article {pmid39807869,
year = {2025},
author = {Sáenz, JS and Rios-Galicia, B and Seifert, J},
title = {Antiviral defense systems in the rumen microbiome.},
journal = {mSystems},
volume = {},
number = {},
pages = {e0152124},
doi = {10.1128/msystems.01521-24},
pmid = {39807869},
issn = {2379-5077},
abstract = {The continuous interaction between phages and their respective hosts has resulted in the evolution of multiple bacterial immune mechanisms. However, the diversity and prevalence of antiviral defense systems in complex communities are still unknown. We therefore investigated the diversity and abundance of viral defense systems in 3,038 high-quality bacterial and archaeal genomes from the rumen. In total, 14,241 defense systems and 31,948 antiviral-related genes were identified. Those genes represented 114 unique system types grouped into 49 families. We observed a high prevalence of defense systems in the genomes. However, the number of defense systems, defense system families, and system density varied widely from genome to genome. Additionally, the number of defense system per genome correlated positively with the number of defense system families and the genome size. Restriction modification, Abi, and cas system families were the most common, but many rare systems were present in only 1% of the genomes. Antiviral defense systems are prevalent and diverse in the rumen, but only a few are dominant, indicating that most systems are rarely present. However, the collection of systems throughout the rumen may represent a pool of mechanisms that can be shared by different members of the community and modulate the phage-host interaction.IMPORTANCEPhages may act antagonistically at the cell level but have a mutualistic interaction at the microbiome level. This interaction shapes the structure of microbial communities and is mainly driven by the defense mechanism. However, the diversity of such mechanism is larger than previously thought. Because of that, we described the abundance and diversity of the antiviral defense system of a collection of genomes, metagenome-assembled genomes (MAGs) and isolates, from the rumen. While defense mechanisms seem to be prevalent among bacteria and archaea, only a few were common. This suggests that most of these defense mechanisms are not present in many rumen microbes but could be shared among different members of the microbial community. This is consistent with the "pan-immune system" model, which appears to be common across different environments.},
}

@article {pmid39807514,
year = {2025},
author = {Saha, D and Panda, AK and Datta, S},
title = {Critical considerations and computational tools in plant genome editing.},
journal = {Heliyon},
volume = {11},
number = {1},
pages = {e41135},
pmid = {39807514},
issn = {2405-8440},
abstract = {Recent advances in genome editing tools and CRISPR-Cas technologies have enabled plant genome engineering reach new heights. The current regulatory exemptions for certain categories of genome edited products, such as those derived from SDN-1 and SDN-2, which are free of any transgene, have significantly accelerated genome editing research in a number of agricultural crop plants in different countries. Although CRISPR-Cas technology is becoming increasingly popular, it is still important to carefully consider a number of factors before planning and carrying conducting CRISPR-Cas studies. To attempt genome editing in a plant, a high-quality genome sequence and a repeatable tissue culture protocol for in vitro regeneration are essential. One of the most important steps in plant genome editing is the designing of a CRISPR construct, which involves selecting the appropriate Cas protein, sgRNA sequence, and appropriate regulatory sequence to trigger expression. Computational tools and algorithms play a crucial role in construct design and gRNA selection to minimize off-target effects and also to optimize their delivery techniques. Researchers may need to select appropriate software tools capable of analyzing post-editing detection of mutation events and other DNA sequence abnormalities to identify off-target effects. To fully fulfill the potential of plant genome editing, continued advances in computational biology are essential to meet the challenges it faces today.},
}

@article {pmid39806509,
year = {2025},
author = {Allais-Bonnet, A and Richard, C and André, M and Gelin, V and Deloche, MC and Lamadon, A and Morin, G and Mandon-Pépin, B and Canon, E and Thépot, D and Laubier, J and Moazami-Goudarzi, K and Laffont, L and Dubois, O and Fassier, T and Congar, P and Lasserre, O and Aguirre-Lavin, T and Vilotte, JL and Pailhoux, E},
title = {CRISPR/Cas9-editing of PRNP in Alpine goats.},
journal = {Veterinary research},
volume = {56},
number = {1},
pages = {11},
pmid = {39806509},
issn = {1297-9716},
support = {101000226//Horizon 2020 Framework Programme/ ; },
mesh = {Animals ; *Goats/genetics ; *CRISPR-Cas Systems ; *Gene Editing/veterinary ; Female ; Goat Diseases/genetics ; Male ; Prion Proteins/genetics ; Gene Knockout Techniques/veterinary ; },
abstract = {Misfolding of the cellular PrP (PrP[c]) protein causes prion disease, leading to neurodegenerative disorders in numerous mammalian species, including goats. A lack of PrP[c] induces complete resistance to prion disease. The aim of this work was to engineer Alpine goats carrying knockout (KO) alleles of PRNP, the PrP[c]-encoding gene, using CRISPR/Cas9-ribonucleoproteins and single-stranded donor oligonucleotides. The targeted region preceded the PRNP[Ter] mutation previously described in Norwegian goats. Genome editors were injected under the zona pellucida prior to the electroporation of 565 Alpine goat embryos/oocytes. A total of 122 two-cell-stage embryos were transferred to 46 hormonally synchronized recipient goats. Six of the goats remained pregnant and naturally gave birth to 10 offspring. Among the 10 newborns, eight founder animals carrying PRNP genome-edited alleles were obtained. Eight different mutated alleles were observed, including five inducing KO mutations. Three founders carried only genome-edited alleles and were phenotypically indistinguishable from their wild-type counterparts. Among them, one male carrying a one base pair insertion leading to a KO allele is currently used to rapidly extend a PRNP-KO line of Alpine goats for future characterization. In addition to KO alleles, a PRNP[del6] genetic variant has been identified in one-third of founder animals. This new variant will be tested for its potential properties with respect to prion disease. Future studies will also evaluate the effects of genetic background on other characters associated with PRNP KO, as previously described in the Norwegian breed or other species.},
}

Animals
*Goats/genetics
*CRISPR-Cas Systems
*Gene Editing/veterinary
Female
Goat Diseases/genetics
Male
Prion Proteins/genetics
Gene Knockout Techniques/veterinary

@article {pmid39806441,
year = {2025},
author = {Lin, Y and Li, C and Chen, Y and Gao, J and Li, J and Huang, C and Liu, Z and Wang, W and Zheng, X and Song, X and Wu, J and Wu, J and Luo, OJ and Tu, Z and Li, S and Li, XJ and Lai, L and Yan, S},
title = {RNA-Targeting CRISPR/CasRx system relieves disease symptoms in Huntington's disease models.},
journal = {Molecular neurodegeneration},
volume = {20},
number = {1},
pages = {4},
pmid = {39806441},
issn = {1750-1326},
mesh = {Animals ; *Huntington Disease/genetics/therapy ; Humans ; *CRISPR-Cas Systems ; *Disease Models, Animal ; Mice ; *Huntingtin Protein/genetics ; HEK293 Cells ; Swine ; Gene Editing/methods ; Mice, Transgenic ; },
abstract = {BACKGROUND: HD is a devastating neurodegenerative disorder caused by the expansion of CAG repeats in the HTT. Silencing the expression of mutated proteins is a therapeutic direction to rescue HD patients, and recent advances in gene editing technology such as CRISPR/CasRx have opened up new avenues for therapeutic intervention.

METHODS: The CRISPR/CasRx system was employed to target human HTT exon 1, resulting in an efficient knockdown of HTT mRNA. This therapeutic effect was substantiated in various models: HEK 293 T cell, the HD 140Q-KI mouse, and the HD-KI pig model. The efficiency of the knockdown was analyzed through Western blot and RT-qPCR. Additionally, neuropathological changes were examined using Western blot, immunostaining, and RNA sequencing. The impact on motor abilities was assessed via behavioral experiments, providing a comprehensive evaluation of the treatment's effectiveness.

RESULTS: CRISPR/CasRx system can significantly reduce HTT mRNA levels across various models, including HEK 293 T cells, HD 140Q-KI mice at various disease stages, and HD-KI pigs, and resulted in decreased expression of mHTT. Utilizing the CRISPR/CasRx system to knock down HTT RNA has shown to ameliorate gliosis in HD 140Q-KI mice and delay neurodegeneration in HD pigs.

CONCLUSIONS: These findings highlight the effectiveness of the RNA-targeting CRISPR/CasRx as a potential therapeutic strategy for HD. Furthermore, the success of this approach provides valuable insights and novel avenues for the treatment of other genetic disorders caused by gene mutations.},
}

Animals
*Huntington Disease/genetics/therapy
Humans
*CRISPR-Cas Systems
*Disease Models, Animal
Mice
*Huntingtin Protein/genetics
HEK293 Cells
Swine
Gene Editing/methods
Mice, Transgenic

@article {pmid39516652,
year = {2025},
author = {Murphy, R and Martin, KR},
title = {Genetic engineering and the eye.},
journal = {Eye (London, England)},
volume = {39},
number = {1},
pages = {57-68},
pmid = {39516652},
issn = {1476-5454},
mesh = {Humans ; *Genetic Therapy/methods ; *Genetic Engineering ; *Eye Diseases/therapy/genetics ; Gene Editing/methods ; CRISPR-Cas Systems ; Genetic Vectors ; },
abstract = {The transformative potential of genetic engineering in ophthalmology is remarkable, promising new treatments for a wide range of blinding eye diseases. The eye is an attractive target organ for genetic engineering approaches, in part due to its relatively immune-privileged status, its accessibility, and the ease of monitoring of efficacy and safety. Consequently, the eye has been at the forefront of genetic engineering advances in recent years. The development of Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9), base editors, prime editors, and transposases have enabled efficient and specific gene modification. Ocular gene therapy continues to progress, with recent advances in delivery systems using viral / non-viral vectors and novel promoters and enhancers. New strategies to achieve neuroprotection and neuroregeneration are evolving, including direct in-vivo cell reprogramming and optogenetic approaches. In this review, we discuss recent advances in ocular genetic engineering, examine their current therapeutic roles, and explore their potential use in future strategies to reduce the growing burden of vision loss and blindness.},
}

Humans
*Genetic Therapy/methods
*Genetic Engineering
*Eye Diseases/therapy/genetics
Gene Editing/methods
CRISPR-Cas Systems
Genetic Vectors

@article {pmid39806115,
year = {2025},
author = {Zhang, X and Sun, R and Zheng, H and Qi, Y},
title = {Amplification-free sensitive detection of Staphylococcus aureus by spherical nucleic acid triggered CRISPR/Cas12a and Poly T-Cu reporter.},
journal = {Mikrochimica acta},
volume = {192},
number = {2},
pages = {76},
pmid = {39806115},
issn = {1436-5073},
support = {82073602//National Natural Science Foundation of China/ ; 20200201081JC//Natural Science Foundation of Jilin Province/ ; },
mesh = {*Staphylococcus aureus/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Copper/chemistry ; *Metal Nanoparticles/chemistry ; *Gold/chemistry ; *Limit of Detection ; Aptamers, Nucleotide/chemistry ; CRISPR-Associated Proteins/metabolism/genetics ; Biosensing Techniques/methods ; Bacterial Proteins/genetics ; Endodeoxyribonucleases/chemistry/metabolism ; },
abstract = {A spherical nucleic acid (SNA, AuNPs-aptamer) into CRISPR/Cas12a system combined with poly T-template copper nanoparticles as fluorescence reporter was fabricated to establish an amplification-free sensitive method for Staphylococcus aureus (S. aureus) detection. This method, named PTCas12a, utilizes the concept that the bifunction of SNA recognizes the S. aureus and triggers the Cas12a cleavage activity. Then, the Cas12a enzyme cleaves the Poly T40 to generate a signal change in Poly T-Cu fluorescence, indicating the presence or absence of the target bacteria. The PTCas12a platform demonstrated a detection limit as low as 3.0 CFU/mL (3 N/S) in a wide response range of 1.0 × 10[1]-1.0 × 10[6] CFU/mL for S. aureus detection, which holds significant potential in ensuring food safety and preventing the spread of diseases.},
}

*Staphylococcus aureus/genetics/isolation & purification
*CRISPR-Cas Systems
*Copper/chemistry
*Metal Nanoparticles/chemistry
*Gold/chemistry
*Limit of Detection
Aptamers, Nucleotide/chemistry
CRISPR-Associated Proteins/metabolism/genetics
Biosensing Techniques/methods
Bacterial Proteins/genetics
Endodeoxyribonucleases/chemistry/metabolism

@article {pmid39806080,
year = {2025},
author = {Li, Y and Zeng, Z and Lv, X and Jiang, H and Li, A and Liu, Y and Deng, Y and Li, X},
title = {A POCT assay based on commercial HCG strip for miRNA21 detection by integrating with RCA-HCR cascade amplification and CRISPR/Cas12a.},
journal = {Mikrochimica acta},
volume = {192},
number = {2},
pages = {73},
pmid = {39806080},
issn = {1436-5073},
mesh = {*MicroRNAs/blood/genetics/analysis ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Point-of-Care Testing ; *Limit of Detection ; Chorionic Gonadotropin/blood ; Nucleic Acid Hybridization ; Reagent Strips ; },
abstract = {A point-of-care testing (POCT) assay based on commercial HCG strip was proposed for miRNA21 detection by integrating RCA-HCR cascaded isothermal amplification with CRISPR/Cas12a. Three modules were integrated in the proposed platform: target amplification module composed of rolling circle amplification (RCA) cascaded with hybridization chain reaction (HCR), signal transduction module composed of CRISPR/Cas12a combined with HCG-agarose gel beads probes, and signal readout module composed of commercial HCG strips. The proposed RCA-HCR-CRISPR/Cas12a-HCG strip assay for miRNA21 detection had high sensitivity, and the limit of detection was as low as 37 fM. The proposed assay showed excellent specificity for miRNA21, as other miRNAs did not caused interference for detection. The recoveries of miRNA21 were ranged from 89.0 to 118.0%. The intra-batch and inter-batch coefficient of variation (CV) were 10.1-13.4% and 11.9-14.5%, respectively, which indicated a high accuracy and precision, and the serum matrix did not cause any interference. With the advantages of low-cost, high sensitivity, visualization, and easy popularization, the proposed assay is expected to provide a powerful tool for early diagnosis of tumor disease miRNA, especially in resource-limited areas.},
}

*MicroRNAs/blood/genetics/analysis
*Nucleic Acid Amplification Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
*Point-of-Care Testing
*Limit of Detection
Chorionic Gonadotropin/blood
Nucleic Acid Hybridization
Reagent Strips

@article {pmid39804665,
year = {2025},
author = {Liu, X and Huang, L and Li, M and Fu, Y and Zhang, W and Zhang, S and Liang, X and Shen, Q},
title = {An Efficient and Cost-Effective Novel Strategy for Identifying CRISPR-Cas-Mediated Mutants in Plant Offspring.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1089/crispr.2024.0057},
pmid = {39804665},
issn = {2573-1602},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 system has revolutionized targeted mutagenesis, but screening for mutations in large sample pools can be time-consuming and costly. We present an efficient and cost-effective polymerase chain reaction (PCR)-based strategy for identifying edited mutants in the T1 generation. Unlike previous methods, our approach addresses the challenges of large progeny populations by using T0 generation sequencing results for genotype prediction. The T1 generation plants were then divided into two scenarios: ≥4 bp indels and 1-2 bp indels. Specific primers are designed for these categories, employing dual-primers critical annealing temperature PCR for ≥4 bp indels and the derived cleaved amplified polymorphic sequences (dCAPS) method for 1-2 bp indels. This method is straightforward, cost-effective, and allows rapid and precise identification of T1 editing outcomes, distinguishing between wild-type, heterozygous, and homozygous plants. This strategy accelerates gene functional analysis in plants and beyond.},
}

@article {pmid39804233,
year = {2025},
author = {Wang, H and Zhao, R and Zhang, B and Xiao, Y and Yu, C and Wang, Y and Yu, C and Tang, Y and Li, Y and Lu, B and Li, B},
title = {Accurate Molecular Sensing based on a Modular and Customizable CRISPR/Cas-Assisted Nanopore Operational Nexus (CANON).},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e202423473},
doi = {10.1002/anie.202423473},
pmid = {39804233},
issn = {1521-3773},
abstract = {Solid-state nanopore is a promising single molecular detection technique, but is largely limited by relatively low resolution to small-size targets and laborious design of signaling probes. Here we establish a universal, CRISPR/Cas-Assisted Nanopore Operational Nexus (CANON), which can accurately transduce different targeting sources/species into different DNA structural probes via a "Signal-ON" mode. Target recognition activates the cleavage activity of a Cas12a/crRNA system and then completely digest the blocker of an initiator. The unblocked initiator then triggers downstream DNA assembly reaction and generate a large-size structure easy for nanopore detection. Such integration of Cas12a/crRNA with DNA assembly establishes an accurate correspondence among the input targets, output DNA structures, and the ultimate nanopore signals. We demonstrated dsDNA, long RNA (i.e., Flu virus gene), short microRNA (i.e., let-7d) and non-nucleic acids (i.e., Pb2+) as input paradigms. Various structural assembly reactions, such as hybridization chain reaction (HCR), G-HCR and duplex polymerization strategy (DPS), are adapted as outputs for nanopore signaling. Simultaneous assay is also verified via transferring FluA and FluB genes into HCR and G-HCR, respectively. CANON is thus a modular sensing platform holding multiple advantages such as high accuracy, high resolution and high universality, which can be easily customized into various application scenes.},
}

@article {pmid39803585,
year = {2024},
author = {Wei, SC and Cantor, AJ and Walleshauser, J and Mepani, R and Melton, K and Bans, A and Khekare, P and Gupta, S and Wang, J and Soares, C and Kiwan, R and Lee, J and McCawley, S and Jani, V and Leong, WI and Shahi, PK and Chan, J and Boivin, P and Otoupal, P and Pattnaik, BR and Gamm, DM and Saha, K and Gowen, BG and Haak-Frendscho, M and Janatpour, MJ and Silverman, AP},
title = {Evaluation of subretinally delivered Cas9 ribonucleoproteins in murine and porcine animal models highlights key considerations for therapeutic translation of genetic medicines.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {39803585},
issn = {2692-8205},
abstract = {Genetic medicines, including CRISPR/Cas technologies, extend tremendous promise for addressing unmet medical need in inherited retinal disorders and other indications; however, there remain challenges for the development of therapeutics. Herein, we evaluate genome editing by engineered Cas9 ribonucleoproteins (eRNP) in vivo via subretinal administration using mouse and pig animal models. Subretinal administration of adenine base editor and double strand break-inducing Cas9 nuclease eRNPs mediate genome editing in both species. Editing occurs in retinal pigmented epithelium (RPE) and photoreceptor cells, with favorable tolerability in both species. Using transgenic reporter strains, we determine that editing primarily occurs close to the site of administration, within the bleb region associated with subretinal injection. Our results show that subretinal administration of eRNPs in mice mediates base editing of up to 12% of the total neural retina, with an average rate of 7% observed at the highest dose tested. In contrast, a substantially lower editing efficiency was observed in minipigs; even with direct quantification of only the treated region, a maximum base editing rate of 1.5%, with an average rate of <1%, was observed. Our data highlight the importance of species consideration in translational studies for genetic medicines targeting the eye and provide an example of a lack of translation between small and larger animal models in the context of subretinal administration of Cas9 eRNPs.},
}

@article {pmid39803448,
year = {2025},
author = {Hossain, KA and Nierzwicki, L and Orozco, M and Czub, J and Palermo, G},
title = {Flexibility in PAM Recognition Expands DNA Targeting in xCas9.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2024.08.26.609653},
pmid = {39803448},
issn = {2692-8205},
abstract = {xCas9 is an evolved variant of the CRISPR-Cas9 genome editing system, engineered to improve specificity and reduce undesired off-target effects. How xCas9 expands the DNA targeting capability of Cas9 by recognizing a series of alternative Protospacer Adjacent Motif (PAM) sequences while ignoring others is unknown. Here, we elucidate the molecular mechanism underlying xCas9's expanded PAM recognition and provide critical insights for expanding DNA targeting. We demonstrate that while wild-type Cas9 enforces stringent guanine selection through the rigidity of its interacting arginine dyad, xCas9 introduces flexibility in R1335, enabling selective recognition of specific PAM sequences. This increased flexibility confers a pronounced entropic preference, which also improves recognition of the canonical TGG PAM. Furthermore, xCas9 enhances DNA binding to alternative PAM sequences during the early evolution cycles, while favouring binding to the canonical PAM in the final evolution cycle. This dual functionality highlights how xCas9 broadens PAM recognition and underscores the importance of fine-tuning the flexibility of the PAM-interacting cleft as a key strategy for expanding the DNA targeting potential of CRISPR-Cas systems. These findings deepen our understanding of DNA recognition in xCas9 and may apply to other CRISPR-Cas systems with similar PAM recognition requirements.},
}

@article {pmid39801754,
year = {2025},
author = {Guha, S and Jagadeesan, Y and Pandey, MM and Mittal, A and Chitkara, D},
title = {Targeting the epigenome with advanced delivery strategies for epigenetic modulators.},
journal = {Bioengineering & translational medicine},
volume = {10},
number = {1},
pages = {e10710},
pmid = {39801754},
issn = {2380-6761},
abstract = {Epigenetics mechanisms play a significant role in human diseases by altering DNA methylation status, chromatin structure, and/or modifying histone proteins. By modulating the epigenetic status, the expression of genes can be regulated without any change in the DNA sequence itself. Epigenetic drugs exhibit promising therapeutic efficacy against several epigenetically originated diseases including several cancers, neurodegenerative diseases, metabolic disorders, cardiovascular disorders, and so forth. Currently, a considerable amount of research is focused on discovering new drug molecules to combat the existing research gap in epigenetic drug therapy. A novel and efficient delivery system can be established as a promising approach to overcome the drawbacks associated with the current epigenetic modulators. Therefore, formulating the existing epigenetic drugs with distinct encapsulation strategies in nanocarriers, including solid lipid nanoparticles, nanogels, bio-engineered nanocarriers, liposomes, surface modified nanoparticles, and polymer-drug conjugates have been examined for therapeutic efficacy. Nonetheless, several epigenetic modulators are untouched for their therapeutic potential through different delivery strategies. This review provides a comprehensive up to date discussion on the research findings of various epigenetics mechanism, epigenetic modulators, and delivery strategies utilized to improve their therapeutic outcome. Furthermore, this review also highlights the recently emerged CRISPR tool for epigenome editing.},
}

@article {pmid39801562,
year = {2025},
author = {Nguyen, T and Meleski, LWG and Belavatta, MP and Gurumoorthi, S and Zhang, C and Heins, AL and Zeng, AP},
title = {A Consecutive Genome Engineering Method Reveals a New Phenotype and Regulation of Glucose and Glycerol Utilization in Clostridium Pasteurianum.},
journal = {Engineering in life sciences},
volume = {25},
number = {1},
pages = {e202400026},
pmid = {39801562},
issn = {1618-0240},
abstract = {Clostridium pasteurianum is a microorganism for production of 1,3-propanediol (1,3-PDO) and butanol, but suffers from lacking genetic tools for metabolic engineering to improve product titers. Furthermore, previous studies of C. pasteurianum have mainly focused on single genomic modification. The aim of this work is the development and application of a method for modification of multiple gene targets in the genome of C. pasteurianum. To this end, a new approach for consecutive genome engineering is presented for the first time using a method based on endogenous CRISPR-Cas machineries. A total of three genome modifications were consecutively introduced in the same mutant and the effect of combined changes on the genome was observed by 39% decreased specific glycerol consumption rate and 29% increased 1,3-PDO yield in mixed substrate fermentations at laboratory scale in comparison to the wildtype strain. Additionally, examination of the phenotype of the generated mutant strain led to discovery of 2,3-butanediol (2,3-BDO) production of up to 0.48 g L[-1], and this metabolite was not reported to be produced by C. pasteurianum before. The developed procedure expands the genetic toolkit for C. pasteurianum and provides researchers an additional method which contributes to improved genetic accessibility of this strain.},
}

@article {pmid39787667,
year = {2025},
author = {Hall, R and Sikora, T and Suter, A and Kuah, JY and Christodoulou, J and Van Bergen, NJ},
title = {Generation and heterozygous repair of human iPSC lines from two individuals with the neurodevelopmental disorder, TRAPPC4 deficiency.},
journal = {Stem cell research},
volume = {82},
number = {},
pages = {103640},
doi = {10.1016/j.scr.2024.103640},
pmid = {39787667},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Neurodevelopmental Disorders/genetics ; *Heterozygote ; Male ; CRISPR-Cas Systems ; Female ; Cell Line ; Cell Differentiation ; },
abstract = {A rare neurodevelopmental disorder has been linked to a well-conserved splice site variant in the TRAPPC4 gene (c.454 + 3A > G), which causes mis-splicing of TRAPPC4 transcripts and reduced levels of TRAPPC4 protein. Patients present with severe progressive neurological symptoms including seizures, microcephaly, intellectual disability and facial dysmorphism. We have generated stem cells from fibroblasts of two individuals with the same homozygous TRAPPC4 c.454 + 3A > G pathogenic variant and used CRISPR/Cas9 editing to generate heterozygous gene-corrected isogenic controls. Clones were tested for pluripotency, differentiation potential, genotyped and karyotyped. These iPSC-based models will be used to understand disease mechanisms of TRAPPC4 disorder.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism
*Neurodevelopmental Disorders/genetics
*Heterozygote
Male
CRISPR-Cas Systems
Female
Cell Line
Cell Differentiation

@article {pmid39742627,
year = {2025},
author = {Wei, S and Zhen, Y and Sun, C and Ma, Y and Li, Q and Wen, L},
title = {Generation of a USP9Y knockout human embryonic stem cell line with CRISPR-Cas9 technology.},
journal = {Stem cell research},
volume = {82},
number = {},
pages = {103646},
doi = {10.1016/j.scr.2024.103646},
pmid = {39742627},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Knockout Techniques/methods ; Male ; Ubiquitin Thiolesterase/genetics/metabolism ; Cell Line ; Cell Differentiation ; },
abstract = {Human embryonic stem cell (hESC) lines are vital tools for studying gene function, disease modeling, and therapy. We generated a USP9Y knockout hESC line using CRISPR-Cas9 in the male-derived H1 line. Targeted deletion of the USP9Y gene was confirmed via PCR and sequencing. The modified line retained pluripotency markers, exhibited a normal karyotype, and differentiated into all three germ layers. This model provides a valuable platform for studying USP9Y's role in human development and male infertility, offering insights into related disorders and therapeutic potential.},
}

Humans
*CRISPR-Cas Systems
*Human Embryonic Stem Cells/metabolism/cytology
*Gene Knockout Techniques/methods
Male
Ubiquitin Thiolesterase/genetics/metabolism
Cell Line
Cell Differentiation

@article {pmid39708407,
year = {2025},
author = {Liu, X and Zhang, F and Chen, D and Yin, J and Bi, Z and Chen, L and Yan, J and Dong, Q and Peng, W and Xu, T and Guo, Y and Lin, H and Liu, H},
title = {Generation of INS-jGCaMP7f knock-in Ca[2+] reporter human embryonic stem cell line, GZLe001-C, using CRISPR/Cas9-based gene targeting.},
journal = {Stem cell research},
volume = {82},
number = {},
pages = {103633},
doi = {10.1016/j.scr.2024.103633},
pmid = {39708407},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems ; *Human Embryonic Stem Cells/metabolism/cytology ; *Calcium/metabolism ; Gene Targeting/methods ; Cell Line ; Gene Knock-In Techniques/methods ; Insulin/metabolism ; Genes, Reporter ; },
abstract = {As a member of the single-fluorophore genetically encoded calcium indicators (GECIs), jGCaMP7f is widely applied to investigate intracellular Ca[2+] concentrations. Here, we established an INS-jGCaMP7f knock-in H1 human embryonic stem cell (hESC) line by integrating jGCaMP7f gene into insulin locus via CRISPR/Cas9 system. The reporter cell line not only effectively labelled the insulin-producing cells induced from hESC, but also reflected the cytosolic change of Ca[2+] level in response to different stimuli. This reporter cell line is a valuable research tool for studying functional maturation of hESC-derived insulin-producing cells, conducting drug screenings, and exploring the mechanisms of diabetes.},
}

Humans
*CRISPR-Cas Systems
*Human Embryonic Stem Cells/metabolism/cytology
*Calcium/metabolism
Gene Targeting/methods
Cell Line
Gene Knock-In Techniques/methods
Insulin/metabolism
Genes, Reporter

@article {pmid39667064,
year = {2025},
author = {Lu, S and Chen, M and Liu, X and Li, J and Liu, H and Li, S},
title = {Generation of a BEST1 Pr-EGFP reporter human embryonic stem cell line via CRISPR/Cas9 editing.},
journal = {Stem cell research},
volume = {82},
number = {},
pages = {103625},
doi = {10.1016/j.scr.2024.103625},
pmid = {39667064},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *Green Fluorescent Proteins/metabolism/genetics ; Cell Line ; Cell Differentiation ; Bestrophins/metabolism/genetics ; Retinal Pigment Epithelium/cytology/metabolism ; Genes, Reporter ; },
abstract = {The retinal pigment epithelium (RPE) cell, located between the neural retina and choriocapillaris, is vital for retinal maintenance and photoreceptor function. Human embryonic stem cells (hESCs) provide a limitless source of RPE cells for transplantation. Using CRISPR/Cas9, we inserted a fusion of the BEST1 promoter (an RPE-specific marker) and the EGFP gene into the AAVS1 locus to track differentiation in hESC-induced RPE (hESC-iRPE). The resulting gene-edited line, WAe009-A-2 M, maintained a normal karyotype, expressed pluripotency markers, and demonstrated differentiation potential, making it invaluable for RPE development and therapeutic research.},
}

Humans
*CRISPR-Cas Systems
*Human Embryonic Stem Cells/metabolism/cytology
*Gene Editing/methods
*Green Fluorescent Proteins/metabolism/genetics
Cell Line
Cell Differentiation
Bestrophins/metabolism/genetics
Retinal Pigment Epithelium/cytology/metabolism
Genes, Reporter

@article {pmid39647515,
year = {2025},
author = {Luo, X and Germer, J and Burghardt, T and Grau, M and Lin, Y and Höhn, M and Lächelt, U and Wagner, E},
title = {Dual pH-responsive CRISPR/Cas9 ribonucleoprotein xenopeptide complexes for genome editing.},
journal = {European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences},
volume = {205},
number = {},
pages = {106983},
doi = {10.1016/j.ejps.2024.106983},
pmid = {39647515},
issn = {1879-0720},
mesh = {*Gene Editing/methods ; Humans ; *Ribonucleoproteins/genetics/metabolism ; *CRISPR-Cas Systems ; HeLa Cells ; Hydrogen-Ion Concentration ; RNA, Guide, CRISPR-Cas Systems ; Peptides/chemistry ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated (Cas) protein has been proved as a powerful tool for the treatment of genetic diseases. The Cas9 protein, when combined with single-guide RNA (sgRNA), forms a Cas9/sgRNA ribonucleoprotein (RNP) capable of targeting and editing the genome. However, the limited availability of effective carriers has restricted the broader application of CRISPR/Cas9 RNP. In this study, we evaluated dual pH-responsive amphiphilic xenopeptides (XPs) for delivering CRISPR/Cas9 RNP. These artificial lipo-XPs contain apolar cationizable lipoamino fatty acid (LAF) and polar cationizable oligoaminoethylene acid units such as succinoyl-tetraethylenepentamine (Stp) in various ratios and U-shaped topologies. The carriers were screened for functional Cas9/sgRNA RNP delivery in four different reporter cell lines, including a Duchenne muscular dystrophy (DMD) exon skipping reporter cell model. Significantly enhanced cellular uptake into HeLa cells, effective endosomal disruption in HeLa gal8-mRuby3 cells, and potent genome editing by several Cas9/sgRNA RNP complexes was observed in four different cell lines in the 5 nM sgRNA range. Comparing Cas9/sgRNA RNP complexes with Cas9 mRNA/sgRNA polyplexes in the DMD reporter cell model demonstrated similar splice site editing and high exon skipping of the two different molecular Cas9 modalities. Based on these studies, analogues of two potent U1 LAF2-Stp and LAF4-Stp2 structures were deployed, tuning the amphiphilicity of the polar Stp group by replacement with the six oligoamino acids dmGtp, chGtp, dGtp, Htp, Stt, or GEIPA. The most potent LAF2-Stp analogues (containing dGtp, chGtp or GEIPA) demonstrated further enhanced gene editing efficiency with EC50 values of 1 nM in the DMD exon skipping reporter cell line. Notably, the EC50 of LAF2-dGtp reached 0.51 nM even upon serum incubation. Another carrier (LAF4-GEIPA2) complexing Cas9/sgRNA RNP and donor DNA, facilitated up to 43 % of homology-directed repair (HDR) in HeLa eGFPd2 cells visualized by the switch from green fluorescent protein (eGFP) to blue fluorescent protein (BFP). This study presents a delivery system tunable for Cas9 RNP complexes or Cas9 RNP/donor DNA polyplexes, offering an effective and easily applicable strategy for gene editing.},
}

*Gene Editing/methods
Humans
*Ribonucleoproteins/genetics/metabolism
*CRISPR-Cas Systems
HeLa Cells
Hydrogen-Ion Concentration
RNA, Guide, CRISPR-Cas Systems
Peptides/chemistry
CRISPR-Associated Protein 9/genetics/metabolism

@article {pmid39644600,
year = {2025},
author = {Xu, Y and Wu, H and Jiang, J and Ye, L and Hao, K and Han, K and Hu, S and Lei, W and Guo, Z},
title = {Generation and characterization of the LINC01405 knockout human embryonic stem cell line.},
journal = {Stem cell research},
volume = {82},
number = {},
pages = {103619},
doi = {10.1016/j.scr.2024.103619},
pmid = {39644600},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *RNA, Long Noncoding/genetics/metabolism ; *Gene Knockout Techniques ; Cell Differentiation ; Cell Line ; CRISPR-Cas Systems ; },
abstract = {Long Intergenic Non-Protein Coding RNA 1405 (LINC01405), with known elevated expression in muscle, has been linked to a number of musculo-skeletal conditions. By utilizing the CRISPR/Cas9 gene editing system, we generated a LINC01405 knockout human embryonic stem cell (hESC) line. This line remains human stem cell-like morphology and pluripotency, exhibits a normal karyotype, and can differentiate into cells from all three germ layers. This cell line will be an invaluable model for the research on LINC01405's role in normal development of cardiac and skeletal muscle, and their diseases.},
}

Humans
*Human Embryonic Stem Cells/metabolism/cytology
*RNA, Long Noncoding/genetics/metabolism
*Gene Knockout Techniques
Cell Differentiation
Cell Line
CRISPR-Cas Systems

@article {pmid39380247,
year = {2025},
author = {Tsuji-Hosokawa, A and Tsuchiya, I and Shimizu, K and Terao, M and Yasuhara, M and Miyamoto, N and Kikuchi, S and Ogawa, Y and Nakamura, K and Matsubara, Y and Takada, S},
title = {Genetically humanized phenylketonuria mouse model as a testing tool for human genome editing in fertilized eggs.},
journal = {Journal of inherited metabolic disease},
volume = {48},
number = {1},
pages = {e12803},
doi = {10.1002/jimd.12803},
pmid = {39380247},
issn = {1573-2665},
support = {19ek0109290h0003//Japan Agency for Medical Research and Development/ ; 2023C-12//National Research Institute for Child Health and Development/ ; 20K18208//Japan Society for the Promotion of Science/ ; },
mesh = {*Phenylketonurias/genetics ; Animals ; *Gene Editing/methods ; *Disease Models, Animal ; Humans ; Mice ; Phenylalanine Hydroxylase/genetics ; Zygote ; Female ; Phenotype ; Male ; CRISPR-Cas Systems ; Genome, Human ; Phenylalanine/genetics ; },
abstract = {Targeted genome editing has made significant advancements; however, safety and ethical issues have not been fully elucidated, resulting in strict control of the technique. We tested genome editing tools on gametes from a genetically humanized mouse model using a phenylketonuria (PKU) mouse model to gain insights into genome editing in human embryos. The human PKU mouse model Pah[hR111X] mice was generated. The junctional region between exon 3 and intron 3 of Pah was replaced with a 120 bp corresponding human PAH sequence, including the pathogenic common variant c.331C > T in Pah[hR111X] mice. Pah[hR111X] mice successfully recapitulated the PKU phenotype and showed cognitive dysfunction and depressive-like behavior, which are observed in human patients with PKU. Genome editing was applied to fertilized eggs of Pah[hR111X] mice utilizing sgRNA that targets the human sequence. Mice with the corrected allele exhibited normal serum phenylalanine levels. Through genome editing, we validated the utility of sgRNA. The genetically humanized mouse model suggested that germ-line genome editing of the pathogenic variant may be feasible for monogenic disorders by revealing the recovery of the phenotype; however, there are remaining issues with the tool, including its efficiency and accuracy. This genome editing protocol using a genetically humanized mouse model will provide insights for improving current issues and contribute to the establishment of heritable human genome editing protocols.},
}

*Phenylketonurias/genetics
Animals
*Gene Editing/methods
*Disease Models, Animal
Humans
Mice
Phenylalanine Hydroxylase/genetics
Zygote
Female
Phenotype
Male
CRISPR-Cas Systems
Genome, Human
Phenylalanine/genetics

@article {pmid39109936,
year = {2025},
author = {Rose, JC and Belk, JA and Wong, IT and Luebeck, J and Horn, HT and Daniel, B and Jones, MG and Yost, KE and Hung, KL and Kolahi, KS and Curtis, EJ and Kuo, CJ and Bafna, V and Mischel, PS and Chang, HY},
title = {Disparate Pathways for Extrachromosomal DNA Biogenesis and Genomic DNA Repair.},
journal = {Cancer discovery},
volume = {15},
number = {1},
pages = {69-82},
doi = {10.1158/2159-8290.CD-23-1117},
pmid = {39109936},
issn = {2159-8290},
support = {K99-CA279512//National Cancer Institute (NCI)/ ; //Howard Hughes Medical Institute (HHMI)/ ; //A.P. Giannini Foundation/ ; CGCSDF-2021\100007//National Cancer Institute (NCI)/ ; CGCSDF-2021\100007//Cancer Research UK (CRUK)/ ; //Howard Hughes Medical Institute (HHMI)/ ; },
mesh = {Humans ; *DNA Repair ; DNA Breaks, Double-Stranded ; DNA, Circular/genetics ; DNA End-Joining Repair ; CRISPR-Cas Systems ; },
abstract = {Our study harnesses a CRISPR-based method to examine ecDNA biogenesis, uncovering efficient circularization between double-strand breaks. ecDNAs and their corresponding chromosomal scars can form via nonhomologous end joining or microhomology-mediated end joining, but the ecDNA and scar formation processes are distinct. Based on our findings, we establish a mechanistic model of excisional ecDNA formation.},
}

Humans
*DNA Repair
DNA Breaks, Double-Stranded
DNA, Circular/genetics
DNA End-Joining Repair
CRISPR-Cas Systems

@article {pmid39800582,
year = {2025},
author = {Zhang, Y and Dong, X and Jiang, C and Yu, Y and Zhang, H and Fu, J and Su, G and Liu, Y},
title = {Corrigendum to "A competitive aptamer binding-based CRISPR-cas biosensor for sensitive detection of tetracycline residues in biological samples" [Talanta, Volume 286 (2025), 127491].},
journal = {Talanta},
volume = {},
number = {},
pages = {127558},
doi = {10.1016/j.talanta.2025.127558},
pmid = {39800582},
issn = {1873-3573},
}

@article {pmid39800499,
year = {2025},
author = {Yu, F and Zhang, Q and Ma, T and Zhang, S and Wang, F and Yue, D and Liu, S and Liao, Y and Liu, LE and Wu, Y and Zang, W},
title = {Bifunctional probe propelling multipath strand displacement amplification tandem CRISPR/Cas12a for ultrasensitive and robust assay of DNA methyltransferase activity.},
journal = {Analytica chimica acta},
volume = {1337},
number = {},
pages = {343540},
doi = {10.1016/j.aca.2024.343540},
pmid = {39800499},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; DNA Methylation ; Limit of Detection ; DNA Modification Methylases/metabolism ; Enzyme Assays/methods ; DNA Probes/chemistry ; },
abstract = {BACKGROUND: DNA methylation catalyzed by various DNA methyltransferases (DNA MTases) is one of the important epigenetic regulations in both eukaryotes and prokaryotes. Therefore, the detection of DNA MTase activity is a vital target and direction in the study of methylation-related diseases.

RESULTS: In this study, an ultrasensitive and robust strategy was developed for DNA MTase activity sensing based on bifunctional probe propelling multipath strand displacement amplification and CRISPR/Cas12a techniques. First, a bifunctional hairpin probe (bHpDNA) was designed instead of a conventional single-function probe. In the presence of DNA MTase, the bHpDNA was methylated and cleaved by a restriction endonuclease into two independent primers, both of which bind with the templates to trigger strand displacement amplification and produce the active DNA of CRISPR/Cas12a. Second, annealing-assisted binding instead of free diffusion adhesion was used to improve hybridization efficiency between the primers and templates. Finally, the CRISPR/Cas12a system was used to achieve fluorescence signal output to analyze DNA MTase activity. If targets were absent, there was no signal because no primers were released from the bHpDNA. To verify the reliability of the method, two key DNA MTases, Dam and M. SssI, were analyzed, and their limits of detection were 2.458 × 10[-3] and 3.820 × 10[-3] U/mL, respectively, which were lower than those of most reported fluorescence methods.

SIGNIFICANCE: This method was successfully used in the evaluation of DNA MTase inhibitors and the detection of DNA MTase activity in complex biological systems with good recoveries and relative standard deviation at low spiked concentrations (0.1-1 U/mL), which all indicate that this method is an ultrasensitive and robust strategy in DNA MTase activity assay and has great potential in biomedical and clinical detection.},
}

*CRISPR-Cas Systems/genetics
*Nucleic Acid Amplification Techniques/methods
DNA Methylation
Limit of Detection
DNA Modification Methylases/metabolism
Enzyme Assays/methods
DNA Probes/chemistry

@article {pmid39799111,
year = {2025},
author = {Su-Tobon, Q and Fan, J and Goldstein, M and Feeney, K and Ren, H and Autissier, P and Wang, P and Huang, Y and Mohanty, U and Niu, J},
title = {CRISPR-Hybrid: A CRISPR-Mediated Intracellular Directed Evolution Platform for RNA Aptamers.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {595},
pmid = {39799111},
issn = {2041-1723},
support = {1DP2HG011027-01//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; Graduate Research Fellowship//National Science Foundation (NSF)/ ; },
mesh = {*Aptamers, Nucleotide/genetics/metabolism ; Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Directed Molecular Evolution/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; HEK293 Cells ; RNA-Binding Proteins/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Recent advances in gene editing and precise regulation of gene expression based on CRISPR technologies have provided powerful tools for the understanding and manipulation of gene functions. Fusing RNA aptamers to the sgRNA of CRISPR can recruit cognate RNA-binding protein (RBP) effectors to target genomic sites, and the expression of sgRNA containing different RNA aptamers permit simultaneous multiplexed and multifunctional gene regulations. Here, we report an intracellular directed evolution platform for RNA aptamers against intracellularly expressed RBPs. We optimize a bacterial CRISPR-hybrid system coupled with FACS, and identified high affinity RNA aptamers orthogonal to existing aptamer-RBP pairs. Application of orthogonal aptamer-RBP pairs in multiplexed CRISPR allows effective simultaneous transcriptional activation and repression of endogenous genes in mammalian cells.},
}

*Aptamers, Nucleotide/genetics/metabolism
Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*Directed Molecular Evolution/methods
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
HEK293 Cells
RNA-Binding Proteins/metabolism/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid39798882,
year = {2025},
author = {Kainov, DE and Ravlo, E and Ianevski, A},
title = {Seeking innovative concepts in development of antiviral drug combinations.},
journal = {Antiviral research},
volume = {},
number = {},
pages = {106079},
doi = {10.1016/j.antiviral.2025.106079},
pmid = {39798882},
issn = {1872-9096},
abstract = {Antiviral drugs are crucial for managing viral infections, but current treatment options remain limited, particularly for emerging viruses. These drugs can be classified based on their chemical composition, including neutralizing antibodies (nAbs), recombinant human receptors (rhRs), antiviral CRISPR/Cas systems, interferons, antiviral peptides (APs), antiviral nucleic acid polymers, and small molecules. Some of these agents target viral factors, host factors, or both. A major challenge for virus-targeted treatments is their narrow-spectrum effectiveness and the potential for drug resistance, while host-directed and virus-host-targeted therapies often suffer from significant side effects. The synergistic combination of multiple antiviral drugs holds promise for improving treatment outcomes by targeting different stages of the viral life cycle, reducing resistance, and minimizing side effects. However, developing such drug combinations presents its own set of challenges. Leveraging the accumulated knowledge, several drug combinations could be optimized, and new combinations developed, to more effectively treat both emerging and re-emerging viral infections.},
}

@article {pmid39798545,
year = {2025},
author = {Yaremenko, AV and Khan, MM and Zhen, X and Tang, Y and Tao, W},
title = {Clinical advances of mRNA vaccines for cancer immunotherapy.},
journal = {Med (New York, N.Y.)},
volume = {6},
number = {1},
pages = {100562},
doi = {10.1016/j.medj.2024.11.015},
pmid = {39798545},
issn = {2666-6340},
mesh = {Humans ; *Cancer Vaccines/immunology/therapeutic use/administration & dosage ; *Neoplasms/therapy/immunology/genetics ; *mRNA Vaccines ; *Immunotherapy/methods ; RNA, Messenger/administration & dosage/genetics ; CRISPR-Cas Systems ; Antigens, Neoplasm/immunology/genetics ; Clinical Trials as Topic ; },
abstract = {The development of mRNA vaccines represents a significant advancement in cancer treatment, with more than 120 clinical trials to date demonstrating their potential across various malignancies, including lung, breast, prostate, melanoma, and more challenging cancers such as pancreatic and brain tumors. These vaccines work by encoding tumor-specific antigens and immune-stimulating molecules, effectively activating the immune system to target and eliminate cancer cells. Despite these promising advancements, significant challenges remain, particularly in achieving efficient delivery and precise regulation of the immune response. This review provides a comprehensive overview of recent clinical progress in mRNA cancer vaccines, discusses the innovative strategies being employed to overcome existing hurdles, and explores future directions, including the integration of CRISPR-Cas9 technology and advancements in mRNA design. Our aim is to provide insights into the ongoing research and clinical trials, highlighting the transformative potential of mRNA vaccines in advancing oncology and improving patient outcomes.},
}

Humans
*Cancer Vaccines/immunology/therapeutic use/administration & dosage
*Neoplasms/therapy/immunology/genetics
*mRNA Vaccines
*Immunotherapy/methods
RNA, Messenger/administration & dosage/genetics
CRISPR-Cas Systems
Antigens, Neoplasm/immunology/genetics
Clinical Trials as Topic

@article {pmid39798420,
year = {2025},
author = {Gao, Z and Liu, J and Zhang, Y and Xu, R and Yang, Y and Wu, L and Lei, J and Ming, T and Ren, F and Liu, L and Chen, Q},
title = {TdT combined with Cas14a for the electrochemical biosensing of NPC-derived exosomes.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {163},
number = {},
pages = {108900},
doi = {10.1016/j.bioelechem.2025.108900},
pmid = {39798420},
issn = {1878-562X},
abstract = {In this work, the electrochemical biosensor based on the subtle combination of terminal deoxynucleotidyl transferase (TdT), CRISPR/Cas14a, and magnetic nanoparticles (MNPs) was developed for the detection of nasopharyngeal carcinoma (NPC)-derived exosomes. Due to the synergistic effect of the following factors: the powerful elongation capacity of TdT for single-stranded DNA (ssDNA) with 3-hydroxy terminus, the outstanding trans-cleavage ability of CRISPR/Cas14a specifcally activated by the crRNA binding to target DNA, and the excellent separation ability of MNPs, the developed electrochemical biosensor exhibited high sensitivity for the detection of NPC-derived exosome, with a linear range from 6.0 × 10[2] ∼ 1.0 × 10[5] particles/mL and a limit of detection as lown as 80 particles/mL. In addition, this electrochemical biosensor successfully distinguished exosomes from NPC patients and healthy individuals. This electrochemical biosensor opens up a new pathway for the early diagnosis of NPC. Abbreviations: NPC, Nasopharyngeal carcinoma; CRISPR/Cas, Clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins system; PAMs, Protospacer adjacent motifs; RCA, Rolling circle amplification; CHA, Catalytic hairpin assembly; LAMP, Loop-mediated isothermal amplification; TdT, Terminal deoxynucleotidyl transferase; SgRNA, Single guide RNA.},
}

@article {pmid39562753,
year = {2025},
author = {Cirincione, A and Simpson, D and Yan, W and McNulty, R and Ravisankar, P and Solley, SC and Yan, J and Lim, F and Farley, EK and Singh, M and Adamson, B},
title = {A benchmarked, high-efficiency prime editing platform for multiplexed dropout screening.},
journal = {Nature methods},
volume = {22},
number = {1},
pages = {92-101},
pmid = {39562753},
issn = {1548-7105},
support = {R35GM138167//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; RM1HG009490//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; P30CA072720//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; T32HG003284//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; T32GM007388//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; F31HD113443//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01-GM076275//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; DGE-2039656//National Science Foundation (NSF)/ ; 2239957//National Science Foundation (NSF)/ ; },
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Benchmarking ; HEK293 Cells ; },
abstract = {Prime editing installs precise edits into the genome with minimal unwanted byproducts, but low and variable editing efficiencies have complicated application of the approach to high-throughput functional genomics. Here we assembled a prime editing platform capable of high-efficiency substitution editing suitable for functional interrogation of small genetic variants. We benchmarked this platform for pooled, loss-of-function screening using a library of ~240,000 engineered prime editing guide RNAs (epegRNAs) targeting ~17,000 codons with 1-3 bp substitutions. Comparing the abundance of these epegRNAs across screen samples identified negative selection phenotypes for 7,996 nonsense mutations targeted to 1,149 essential genes and for synonymous mutations that disrupted splice site motifs at 3' exon boundaries. Rigorous evaluation of codon-matched controls demonstrated that these phenotypes were highly specific to the intended edit. Altogether, we established a prime editing approach for multiplexed, functional characterization of genetic variants with simple readouts.},
}

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

@article {pmid39797940,
year = {2025},
author = {Fu, Y and Zhang, P and Chen, F and Xie, Z and Xiao, S and Huang, Z and Lau, CH and Zhu, H and Luo, J},
title = {CRISPR detection of cardiac tumor-associated microRNAs.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {114},
pmid = {39797940},
issn = {1573-4978},
support = {20212ACB206031//Jiangxi Provincial Natural Science Foundation/ ; 82260053//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *MicroRNAs/genetics ; *Heart Neoplasms/genetics/diagnosis ; *CRISPR-Cas Systems/genetics ; Myxoma/genetics/diagnosis ; Hemangiosarcoma/genetics/diagnosis ; Biomarkers, Tumor/genetics ; },
abstract = {As multiple imaging modalities cannot reliably diagnose cardiac tumors, the molecular approach offers alternative ways to detect rare ones. One such molecular approach is CRISPR-based diagnostics (CRISPR-Dx). CRISPR-Dx enables visual readout, portable diagnostics, and rapid and multiplex detection of nucleic acids such as microRNA (miRNA). Dysregulation of miRNA expressions has been associated with cardiac tumors such as atrial myxoma and angiosarcoma. Diverse CRISPR-Dx systems have been developed to detect miRNA in recent years. These CRISPR-Dx systems are generally classified into four classes, depending on the Cas proteins used (Cas9, Cas12, Cas13, or Cas12f). CRISPR/Cas systems are integrated with various isothermal amplifications to detect low-abundance miRNAs. Amplification-free CRISPR-Dx systems have also been recently developed to detect miRNA directly. Herein, we critically discuss the advances, pitfalls, and future perspectives for these CRISPR-Dx systems in detecting miRNA, focusing on the diagnosis and prognosis of cardiac tumors.},
}

Humans
*MicroRNAs/genetics
*Heart Neoplasms/genetics/diagnosis
*CRISPR-Cas Systems/genetics
Myxoma/genetics/diagnosis
Hemangiosarcoma/genetics/diagnosis
Biomarkers, Tumor/genetics

@article {pmid39797776,
year = {2025},
author = {Li, Q and Zhang, Z and Wu, X and Zhao, Y and Liu, Y},
title = {Cascade-Responsive Nanoparticles for Efficient CRISPR/Cas9-Based Glioblastoma Gene Therapy.},
journal = {ACS applied materials & interfaces},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsami.4c15671},
pmid = {39797776},
issn = {1944-8252},
abstract = {CRISPR/Cas9 (CRISPR, clustered regularly interspaced short palindromic repeats) gene editing technology represents great promise for treating glioblastoma (GBM) due to its potential to permanently eliminate tumor pathogenic genes. Unfortunately, delivering CRISPR to the GBM in a safe and effective manner is challenging. Herein, a glycosylated and cascade-responsive nanoparticle (GCNP) that can effectively cross the blood-brain barrier (BBB) and activate CRISPR/Cas9-based gene editing only in the GBM is designed. The GCNP possesses a cationic polyplex core and a glycosylated polymer layer that is capable of cascading response to low pH and high GSH concentration, so that the release of CRISPR/Cas9 only takes place after crossing the BBB and entering the GBM where the acidic tumor microenvironment and high concentration of glutathione (GSH) are present. By targeting the programmed death-ligand 1 (PD-L1) in GBM, GCNP effectively inhibited the tumor growth and greatly prolonged the survival time of GBM-bearing mice when combined with temozolomide (TMZ).},
}

@article {pmid39796169,
year = {2025},
author = {Rallabandi, HR and Singh, MK and Looger, LL and Nath, SK},
title = {Defining Mechanistic Links Between the Non-Coding Variant rs17673553 in CLEC16A and Lupus Susceptibility.},
journal = {International journal of molecular sciences},
volume = {26},
number = {1},
pages = {},
doi = {10.3390/ijms26010314},
pmid = {39796169},
issn = {1422-0067},
support = {5R01AI172255-02S1/NH/NIH HHS/United States ; },
mesh = {*Lectins, C-Type/genetics/metabolism ; Humans ; *Lupus Erythematosus, Systemic/genetics ; *Genetic Predisposition to Disease ; *Monosaccharide Transport Proteins/genetics ; *Polymorphism, Single Nucleotide ; Alleles ; Autophagy/genetics ; Genome-Wide Association Study ; Gene Editing ; STAT3 Transcription Factor/genetics/metabolism ; CRISPR-Cas Systems ; B-Lymphocytes/metabolism ; Chromosomes, Human, Pair 16/genetics ; },
abstract = {Systemic lupus erythematosus (SLE) is a complex autoimmune disorder characterized by widespread inflammation and autoantibody production. Its development and progression involve genetic, epigenetic, and environmental factors. Although genome-wide association studies (GWAS) have repeatedly identified a susceptibility signal at 16p13, its fine-scale source and its functional and mechanistic role in SLE remain unclear. We used bioinformatics to prioritize likely functional variants and validated the top candidate through various experimental techniques, including clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing in B cells. To assess the functional impact of the proposed causal variant in C-type lectin domain family 16, member A (CLEC16A), we compared autophagy levels between wild-type (WT) and knock-out (KO) cells. Systematic bioinformatics analysis identified the highly conserved non-coding intronic variant rs17673553, with the risk allele apparently affecting enhancer function and regulating several target genes, including CLEC16A itself. Luciferase reporter assays followed by chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) validated this enhancer activity, demonstrating that the risk allele increases the binding of enhancer histone marks (H3K27ac and H3K4me1), the CTCF-binding factor, and key immune transcription factors (GATA3 and STAT3). Knock-down of GATA3 and STAT3 via siRNA led to a significant decrease in CLEC16A expression. These regulatory effects on the target gene were further confirmed using CRISPR-based genome editing and CRISPR-dCas9-based epigenetic activation/silencing. Functionally, WT cells exhibited higher levels of starvation-induced autophagy compared to KO cells, highlighting the role of CLEC16A and the rs17673553 locus in autophagy regulation. These findings suggest that the rs17673553 locus-particularly the risk allele-drives significant allele-specific chromatin modifications and binding of multiple transcription factors, thereby mechanistically regulating the expression of target autophagy-associated genes, including CLEC16A itself. This mechanism could potentially explain the association between rs17673553 and SLE, and could underlie the signal at 16p13.},
}

*Lectins, C-Type/genetics/metabolism
Humans
*Lupus Erythematosus, Systemic/genetics
*Genetic Predisposition to Disease
*Monosaccharide Transport Proteins/genetics
*Polymorphism, Single Nucleotide
Alleles
Autophagy/genetics
Genome-Wide Association Study
Gene Editing
STAT3 Transcription Factor/genetics/metabolism
CRISPR-Cas Systems
B-Lymphocytes/metabolism
Chromosomes, Human, Pair 16/genetics

@article {pmid39795970,
year = {2024},
author = {Cerna-Chavez, R and Ortega-Gasco, A and Baig, HMA and Ehrenreich, N and Metais, T and Scandura, MJ and Bujakowska, K and Pierce, EA and Garita-Hernandez, M},
title = {Optimized Prime Editing of Human Induced Pluripotent Stem Cells to Efficiently Generate Isogenic Models of Mendelian Diseases.},
journal = {International journal of molecular sciences},
volume = {26},
number = {1},
pages = {},
doi = {10.3390/ijms26010114},
pmid = {39795970},
issn = {1422-0067},
support = {EY012910/EY/NEI NIH HHS/United States ; EY020902/EY/NEI NIH HHS/United States ; EY014104/NH/NIH HHS/United States ; Competitive Renewal Research Grant in Pediatric Ophthalmology//Knights Templar Eye Foundation/ ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Retinal Diseases/genetics/therapy ; Mutation ; },
abstract = {Prime editing (PE) is a CRISPR-based tool for genome engineering that can be applied to generate human induced pluripotent stem cell (hiPSC)-based disease models. PE technology safely introduces point mutations, small insertions, and deletions (indels) into the genome. It uses a Cas9-nickase (nCas9) fused to a reverse transcriptase (RT) as an editor and a PE guide RNA (pegRNA), which introduces the desired edit with great precision without creating double-strand breaks (DSBs). PE leads to minimal off-targets or indels when introducing single-strand breaks (SSB) in the DNA. Low efficiency can be an obstacle to its use in hiPSCs, especially when the genetic context precludes the screening of multiple pegRNAs, and other strategies must be employed to achieve the desired edit. We developed a PE platform to efficiently generate isogenic models of Mendelian disorders. We introduced the c.25G>A (p.V9M) mutation in the NMNAT1 gene with over 25% efficiency by optimizing the PE workflow. Using our optimized system, we generated other isogenic models of inherited retinal diseases (IRDs), including the c.1481C>T (p.T494M) mutation in PRPF3 and the c.6926A>C (p.H2309P) mutation in PRPF8. We modified several determinants of the hiPSC PE procedure, such as plasmid concentrations, PE component ratios, and delivery method settings, showing that our improved workflow increased the hiPSC editing efficiency.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Gene Editing/methods
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
Retinal Diseases/genetics/therapy
Mutation

@article {pmid39795964,
year = {2024},
author = {Tao, R and Zhang, J and Meng, L and Gao, J and Miao, C and Liu, W and Jin, W and Wan, Y},
title = {A Rapid Field-Visualization Detection Platform for Genetically Modified Soybean 'Zhonghuang 6106' Based on RPA-CRISPR.},
journal = {International journal of molecular sciences},
volume = {26},
number = {1},
pages = {},
doi = {10.3390/ijms26010108},
pmid = {39795964},
issn = {1422-0067},
support = {2022ZD0402003//Biological Breeding-Major Projects/ ; },
mesh = {*Glycine max/genetics ; *Plants, Genetically Modified/genetics ; *CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods ; Gene Editing/methods ; },
abstract = {Genetically modified (GM) herbicide-tolerant soybean 'Zhonghuang 6106', which introduces a glyphosate-resistant gene, ensures soybean yield while allowing farmers to reduce the use of other herbicides, thereby reducing weed management costs. To protect consumer rights and facilitate government supervision, we have established a simple and rapid on-site nucleic acid detection method for GM soybean 'Zhonghuang 6106'. This method leverages the isothermal amplification characteristics of RPA technology and the high specificity of CRISPR-Cas12a to achieve high sensitivity and accuracy in detecting GM soybean components. By optimizing experimental conditions, the platform can quickly produce visual detection results, significantly reducing detection time and improving efficiency. The system can detect down to 10 copies/μL of 'Zhonghuang 6106' DNA templates, and the entire detection process takes about 1 h. The technology also has strong editing capabilities; by redesigning the primers and crRNA in the method, it can become a specific detection method for other GM samples, providing strong technical support for the regulation and safety evaluation of GM crops.},
}

*Glycine max/genetics
*Plants, Genetically Modified/genetics
*CRISPR-Cas Systems
Nucleic Acid Amplification Techniques/methods
Gene Editing/methods

@article {pmid39794549,
year = {2025},
author = {Ahmed, R and Alghamdi, WN and Alharbi, FR and Alatawi, HD and Alenezi, KM and Alanazi, TF and Elsherbiny, NM},
title = {CRISPR/Cas9 System as a Promising Therapy in Thalassemia and Sickle Cell Disease: A Systematic Review of Clinical Trials.},
journal = {Molecular biotechnology},
volume = {},
number = {},
pages = {},
pmid = {39794549},
issn = {1559-0305},
abstract = {Clustered, regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system is a new gene editing tool that represents a revolution in gene therapy. This study aimed to review the clinical trials conducted to evaluate the efficacy and safety of the CRISPR/Cas9 system in treating thalassemia and sickle cell disease (SCD). We searched relevant literature using "CRISPR Cas", "thalassemia", "sickle cell" and "clinical trial" as subject terms in PubMed, Cochrane, Web of Science, and Google Scholar up to December 3rd, 2023. Following the PIO format (Patients, Intervention, Outcome), PRISMA guidelines were followed in the study selection, data extraction, and quality assessment processes. Out of 110 publications, 6 studies met our eligibility criteria with a total of 115 patients involved. CRISPR/Cas9 system was used to disrupt BCL11A gene enhancer in 4 studies and to disrupt γ-globin gene promoters in 2 studies. Patients demonstrated significant activation of fetal hemoglobin, elevated total hemoglobin, transfusion independence in thalassemia, and repression of vaso-occlusive episodes in SCD. Using CRISPR/Cas9 system to directly disrupt genes provides a safe and potential one-time functional cure for thalassemia and SCD, suggesting CRISPR/Cas9 as a potential therapeutic tool for the treatment of inherited hematological disorders.},
}

@article {pmid39747689,
year = {2025},
author = {Vaysset, H and Meers, C and Cury, J and Bernheim, A and Sternberg, SH},
title = {Evolutionary origins of archaeal and eukaryotic RNA-guided RNA modification in bacterial IS110 transposons.},
journal = {Nature microbiology},
volume = {10},
number = {1},
pages = {20-27},
pmid = {39747689},
issn = {2058-5276},
support = {2239685//National Science Foundation (NSF)/ ; GM143924//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; PECAN 101040529//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; },
mesh = {*DNA Transposable Elements/genetics ; *Evolution, Molecular ; *Transposases/genetics/metabolism ; *Phylogeny ; *Bacteria/genetics ; Archaea/genetics ; Eukaryota/genetics ; Archaeal Proteins/genetics/metabolism ; RNA, Small Nucleolar/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; RNA, Archaeal/genetics ; },
abstract = {Transposase genes are ubiquitous in all domains of life and provide a rich reservoir for the evolution of novel protein functions. Here we report deep evolutionary links between bacterial IS110-family transposases, which catalyse RNA-guided DNA recombination using bridge RNAs, and archaeal/eukaryotic Nop5-family proteins, which promote RNA-guided RNA 2'-O-methylation using C/D-box snoRNAs. On the basis of conservation of protein sequence, domain architecture, three-dimensional structure and non-coding RNA features, alongside phylogenetic analyses, we propose that programmable RNA modification emerged through the exaptation of components derived from IS110-like transposons. These findings underscore how recurrent domestication events of transposable elements have driven the evolution of RNA-guided mechanisms.},
}

*DNA Transposable Elements/genetics
*Evolution, Molecular
*Transposases/genetics/metabolism
*Phylogeny
*Bacteria/genetics
Archaea/genetics
Eukaryota/genetics
Archaeal Proteins/genetics/metabolism
RNA, Small Nucleolar/genetics/metabolism
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Bacterial Proteins/genetics/metabolism
RNA, Archaeal/genetics

@article {pmid39676659,
year = {2025},
author = {Lee, JM and Zeng, J and Liu, P and Nguyen, MA and Suchenski Loustaunau, D and Bauer, DE and Kurt Yilmaz, N and Wolfe, SA and Schiffer, CA},
title = {Direct delivery of Cas-embedded cytosine base editors as ribonucleoprotein complexes for efficient and accurate editing of clinically relevant targets.},
journal = {Nucleic acids research},
volume = {53},
number = {1},
pages = {},
doi = {10.1093/nar/gkae1217},
pmid = {39676659},
issn = {1362-4962},
support = {R01 AI150478/AI/NIAID NIH HHS/United States ; DDF #2022092//Doris Duke Foundation/ ; R01AI150478/NH/NIH HHS/United States ; //University of Massachusetts Chan Medical School/ ; R01AI150478/NH/NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; *Cytosine/chemistry/metabolism ; Humans ; *Ribonucleoproteins/metabolism/genetics ; *CRISPR-Cas Systems ; Animals ; Mice ; HEK293 Cells ; CRISPR-Associated Protein 9/genetics/metabolism ; Carrier Proteins/genetics/metabolism ; },
abstract = {Recently, cytosine base editors (CBEs) have emerged as a promising therapeutic tool for specific editing of single nucleotide variants and disrupting specific genes associated with disease. Despite this promise, the currently available CBEs have the significant liabilities of off-target and bystander editing activities, partly due to the mechanism by which they are delivered, causing limitations in their potential applications. In this study, we engineered optimized, soluble and stable Cas-embedded CBEs (CE_CBEs) that integrate several recent advances, which were efficiently formulated for direct delivery into cells as ribonucleoprotein (RNP) complexes. Our resulting CE_CBE RNP complexes efficiently target cytosines in TC dinucleotides with minimal off-target or bystander mutations. Delivery of additional uracil glycosylase inhibitor protein in trans further increased C-to-T editing efficiency and target purity in a dose-dependent manner, minimizing indel formation. A single electroporation was sufficient to effectively edit the therapeutically relevant locus BCL11A for sickle cell disease in hematopoietic stem and progenitor cells in a dose-dependent manner without cellular toxicity. Significantly, these CE_CBE RNPs permitted highly efficient editing and engraftment of transplanted cells in mice. Thus, our designed CBE proteins provide promising reagents for RNP-based editing at disease-related sites.},
}

*Gene Editing/methods
*Cytosine/chemistry/metabolism
Humans
*Ribonucleoproteins/metabolism/genetics
*CRISPR-Cas Systems
Animals
Mice
HEK293 Cells
CRISPR-Associated Protein 9/genetics/metabolism
Carrier Proteins/genetics/metabolism

@article {pmid39673524,
year = {2025},
author = {Prajapat, MK and Maria, AG and Vidigal, JA},
title = {CRISPR-based dissection of miRNA binding sites using isogenic cell lines is hampered by pervasive noise.},
journal = {Nucleic acids research},
volume = {53},
number = {1},
pages = {},
doi = {10.1093/nar/gkae1138},
pmid = {39673524},
issn = {1362-4962},
support = {1ZIABC011810-02/NH/NIH HHS/United States ; /RC/CCR NIH HHS/United States ; 1ZIABC011810-02/NH/NIH HHS/United States ; /RC/CCR NIH HHS/United States ; },
mesh = {*MicroRNAs/genetics/metabolism ; Binding Sites/genetics ; Humans ; *CRISPR-Cas Systems ; Gene Editing/methods ; Animals ; Cell Line ; Mutation ; HEK293 Cells ; },
abstract = {Non-coding regulatory sequences play essential roles in adjusting gene output to cellular needs and are thus critical to animal development and health. Numerous such sequences have been identified in mammalian genomes ranging from transcription factors binding motifs to recognition sites for RNA-binding proteins and non-coding RNAs. The advent of CRISPR has raised the possibility of assigning functionality to individual endogenous regulatory sites by facilitating the generation of isogenic cell lines that differ by a defined set of genetic modifications. Here we investigate the usefulness of this approach to assign function to individual miRNA binding sites. We find that the process of generating isogenic pairs of mammalian cell lines with CRISPR-mediated mutations introduces extensive molecular and phenotypic variability between biological replicates confounding attempts at assigning function to the binding site. Our work highlights an important consideration when employing CRISPR editing to characterize non-coding regulatory sequences in cell lines and calls for the development and adoption of alternative strategies to address this question in the future.},
}

*MicroRNAs/genetics/metabolism
Binding Sites/genetics
Humans
*CRISPR-Cas Systems
Gene Editing/methods
Animals
Cell Line
Mutation
HEK293 Cells

@article {pmid39657754,
year = {2025},
author = {Kiernan, KA and Kwon, J and Merrill, BJ and Simonović, M},
title = {Structural basis of Cas9 DNA interrogation with a 5' truncated sgRNA.},
journal = {Nucleic acids research},
volume = {53},
number = {1},
pages = {},
doi = {10.1093/nar/gkae1164},
pmid = {39657754},
issn = {1362-4962},
support = {R01 GM139894/GM/NIGMS NIH HHS/United States ; R35 GM138348/GM/NIGMS NIH HHS/United States ; F31 GM143822/GM/NIGMS NIH HHS/United States ; F31GM143822/GM/NIGMS NIH HHS/United States ; R01 GM097042/GM/NIGMS NIH HHS/United States ; 75N91019D00024/CA/NCI NIH HHS/United States ; F31GM143822/GM/NIGMS NIH HHS/United States ; 75N91019D00024/CA/NCI NIH HHS/United States ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; *CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; *CRISPR-Cas Systems ; *DNA/chemistry/metabolism/genetics ; Cryoelectron Microscopy ; Models, Molecular ; Nucleic Acid Conformation ; Gene Editing/methods ; },
abstract = {The efficiency and accuracy of CRISPR-Cas9 targeting varies considerably across genomic targets and remains a persistent issue for using this system in cells. Studies have shown that the use of 5' truncated single guide RNAs (sgRNAs) can reduce the rate of unwanted off-target recognition while still maintaining on-target specificity. However, it is not well-understood how reducing target complementarity enhances specificity or how truncation past 15 nucleotides (nts) prevents full Cas9 activation without compromising on-target binding. Here, we use biochemistry and cryogenic electron microscopy to investigate Cas9 structure and activity when bound to a 14-nt sgRNA. Our structures reveal that the shortened path of the displaced non-target strand (NTS) sterically occludes docking of the HNH L1 linker and prevents proper positioning of the nuclease domains. We show that cleavage inhibition can be alleviated by either artificially melting the protospacer adjacent motif (PAM)-distal duplex or providing a supercoiled substrate. Even though Cas9 forms a stable complex with its target, we find that plasmid cleavage is ∼1000-fold slower with a 14-nt sgRNA than with a full-length 20-nt sgRNA. Our results provide a structural basis for Cas9 target binding with 5' truncated sgRNAs and underline the importance of PAM-distal NTS availability in promoting Cas9 activation.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry
*CRISPR-Associated Protein 9/chemistry/metabolism/genetics
*CRISPR-Cas Systems
*DNA/chemistry/metabolism/genetics
Cryoelectron Microscopy
Models, Molecular
Nucleic Acid Conformation
Gene Editing/methods

@article {pmid39793042,
year = {2025},
author = {Ngo, W and Peukes, J and Baldwin, A and Xue, ZW and Hwang, S and Stickels, RR and Lin, Z and Satpathy, AT and Wells, JA and Schekman, R and Nogales, E and Doudna, JA},
title = {Mechanism-guided engineering of a minimal biological particle for genome editing.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {1},
pages = {e2413519121},
doi = {10.1073/pnas.2413519121},
pmid = {39793042},
issn = {1091-6490},
support = {1R21HL173710-01//HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; DE-AC52-07NA27344//DOE | NNSA | Lawrence Livermore National Laboratory (LLNL)/ ; 1R01CA248323-01//HHS | National Institutes of Health (NIH)/ ; PDF-578176-2023//Canadian Government | Natural Sciences and Engineering Research Council of Canada (NSERC)/ ; ALTF 1031-2021//European Molecular Biology Organization (EMBO)/ ; 1R01CA248323-01//HHS | National Institutes of Health (NIH)/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; Lentivirus/genetics ; Ribonucleoproteins/metabolism/genetics ; HEK293 Cells ; CRISPR-Associated Protein 9/metabolism/genetics ; Genetic Vectors ; },
abstract = {The widespread application of genome editing to treat and cure disease requires the delivery of genome editors into the nucleus of target cells. Enveloped delivery vehicles (EDVs) are engineered virally derived particles capable of packaging and delivering CRISPR-Cas9 ribonucleoproteins (RNPs). However, the presence of lentiviral genome encapsulation and replication proteins in EDVs has obscured the underlying delivery mechanism and precluded particle optimization. Here, we show that Cas9 RNP nuclear delivery is independent of the native lentiviral capsid structure. Instead, EDV-mediated genome editing activity corresponds directly to the number of nuclear localization sequences on the Cas9 enzyme. EDV structural analysis using cryo-electron tomography and small molecule inhibitors guided the removal of ~80% of viral residues, creating a minimal EDV (miniEDV) that retains full RNP delivery capability. MiniEDVs are 25% smaller yet package equivalent amounts of Cas9 RNPs relative to the original EDVs and demonstrated increased editing in cell lines and therapeutically relevant primary human T cells. These results show that virally derived particles can be streamlined to create efficacious genome editing delivery vehicles with simpler production and manufacturing.},
}

*Gene Editing/methods
Humans
*CRISPR-Cas Systems
Lentivirus/genetics
Ribonucleoproteins/metabolism/genetics
HEK293 Cells
CRISPR-Associated Protein 9/metabolism/genetics
Genetic Vectors

@article {pmid39792571,
year = {2025},
author = {Islam, MS and Habib, MA and Tonu, NS and Haque, MS and Rahman, MM},
title = {Beyond Serology: A Meta-Analysis of Advancements in Molecular Detection of Brucella spp. in Seronegative Animals and Biological Samples.},
journal = {Veterinary medicine and science},
volume = {11},
number = {1},
pages = {e70200},
doi = {10.1002/vms3.70200},
pmid = {39792571},
issn = {2053-1095},
mesh = {Animals ; *Brucella/isolation & purification/immunology ; *Brucellosis/veterinary/diagnosis/microbiology ; Molecular Diagnostic Techniques/veterinary/methods ; },
abstract = {BACKGROUND: Brucellosis is a zoonotic disease caused by Brucella spp., affecting various animals and humans, leading to significant economic and public health impacts. Traditional diagnostic methods, mainly serological, often fail to detect seronegative carriers, which continue to spread the infection.

OBJECTIVE: This review aims to highlight advancements in molecular diagnostics that address these limitations.

METHODS: A systematic search of PubMed, Web of Science and Scopus was conducted, focusing on studies using seronegative, PCR, qPCR and biosensor-based techniques. Data extraction and meta-analyses were performed, evaluating pooled detection rates and heterogeneity.

RESULTS: Through analysis of existing studies, we review key molecular techniques, including PCR, LAMP and biosensor-based assays, which offer high sensitivity and specificity by detecting bacterial DNA directly, thus overcoming the challenges of antibody-based tests. Meta-analysis of detection rates across different studies showed significant variability, with rates ranging from 0.96% to 100%, highlighting differences in sample types, animal species and regions. The pooled detection proportion from random-effects models was 35.08%, indicating that many seronegative animals still carry Brucella spp. A forest plot analysis further confirmed heterogeneity in detection, underlining the importance of using molecular diagnostics alongside serological tests to identify hidden carriers.

CONCLUSION: Innovations like nanoparticle-enhanced biosensors and CRISPR-Cas systems show promise for rapid, on-site diagnostics. The findings suggest that integrating molecular methods with traditional serology can improve surveillance and disease management. Future research should focus on developing portable, field-ready diagnostic devices and standardised protocols, along with exploring novel biomarkers to detect latent infections. A collaborative One Health approach, involving veterinary, public health and environmental sectors, is essential for comprehensive disease control and eradication efforts.},
}

Animals
*Brucella/isolation & purification/immunology
*Brucellosis/veterinary/diagnosis/microbiology
Molecular Diagnostic Techniques/veterinary/methods

@article {pmid39789283,
year = {2025},
author = {Hassan, YM and Mohamed, AS and Hassan, YM and El-Sayed, WM},
title = {Recent developments and future directions in point-of-care next-generation CRISPR-based rapid diagnosis.},
journal = {Clinical and experimental medicine},
volume = {25},
number = {1},
pages = {33},
pmid = {39789283},
issn = {1591-9528},
mesh = {Humans ; *CRISPR-Cas Systems ; *COVID-19/diagnosis/virology ; Point-of-Care Systems ; SARS-CoV-2/genetics/isolation & purification ; Point-of-Care Testing ; Molecular Diagnostic Techniques/methods ; },
abstract = {The demand for sensitive, rapid, and affordable diagnostic techniques has surged, particularly following the COVID-19 pandemic, driving the development of CRISPR-based diagnostic tools that utilize Cas effector proteins (such as Cas9, Cas12, and Cas13) as viable alternatives to traditional nucleic acid-based detection methods. These CRISPR systems, often integrated with biosensing and amplification technologies, provide precise, rapid, and portable diagnostics, making on-site testing without the need for extensive infrastructure feasible, especially in underserved or rural areas. In contrast, traditional diagnostic methods, while still essential, are often limited by the need for costly equipment and skilled operators, restricting their accessibility. As a result, developing accessible, user-friendly solutions for at-home, field, and laboratory diagnostics has become a key focus in CRISPR diagnostic innovations. This review examines the current state of CRISPR-based diagnostics and their potential applications across a wide range of diseases, including cancers (e.g., colorectal and breast cancer), genetic disorders (e.g., sickle cell disease), and infectious diseases (e.g., tuberculosis, malaria, Zika virus, and human papillomavirus). Additionally, the integration of machine learning (ML) and artificial intelligence (AI) to enhance the accuracy, scalability, and efficiency of CRISPR diagnostics is discussed, alongside the challenges of incorporating CRISPR technologies into point-of-care settings. The review also explores the potential for these cutting-edge tools to revolutionize disease diagnosis and personalized treatment in the future, while identifying the challenges and future directions necessary to address existing gaps in CRISPR-based diagnostic research.},
}

Humans
*CRISPR-Cas Systems
*COVID-19/diagnosis/virology
Point-of-Care Systems
SARS-CoV-2/genetics/isolation & purification
Point-of-Care Testing
Molecular Diagnostic Techniques/methods

@article {pmid39789078,
year = {2025},
author = {Lee, D and Muir, P and Lundberg, S and Lundholm, A and Sandegren, L and Koskiniemi, S},
title = {A CRISPR-Cas9 system protecting E. coli against acquisition of antibiotic resistance genes.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {1545},
pmid = {39789078},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; *Escherichia coli/genetics/drug effects ; Drug Resistance, Bacterial/genetics ; Gene Transfer, Horizontal ; Plasmids/genetics ; Anti-Bacterial Agents/pharmacology ; Conjugation, Genetic ; },
abstract = {Antimicrobial resistance (AMR) is an increasing problem worldwide, and new treatment options for bacterial infections are direly needed. Engineered probiotics show strong potential in treating or preventing bacterial infections. However, one concern with the use of live bacteria is the risk of the bacteria acquiring genes encoding for AMR or virulence factors through horizontal gene transfer (HGT), and the transformation of the probiotic into a superbug. Therefore, we developed an engineered CRISPR-Cas9 system that protects bacteria from horizontal gene transfer. We synthesized a CRISPR locus targeting eight AMR genes and cloned this with the Cas9 and transacting tracrRNA on a medium copy plasmid. We next evaluated the efficiency of the system to block HGT through transformation, transduction, and conjugation. Our results show that expression of the CRISPR-Cas9 system successfully protects E. coli MG1655 from acquiring the targeted resistance genes by transformation or transduction with 2-3 logs of protection depending on the system for transfer and the target gene. Furthermore, we show that the system blocks conjugation of a set of clinical plasmids, and that the system is also able to protect the probiotic bacterium E. coli Nissle 1917 from acquiring AMR genes.},
}

*CRISPR-Cas Systems
*Escherichia coli/genetics/drug effects
Drug Resistance, Bacterial/genetics
Gene Transfer, Horizontal
Plasmids/genetics
Anti-Bacterial Agents/pharmacology
Conjugation, Genetic

@article {pmid39789054,
year = {2025},
author = {Li, W and Jiang, X and Wang, W and Hou, L and Cai, R and Li, Y and Gu, Q and Chen, Q and Ma, P and Tang, J and Guo, M and Chuai, G and Huang, X and Zhang, J and Liu, Q},
title = {Author Correction: Discovering CRISPR-Cas system with self-processing pre-crRNA capability by foundation models.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {535},
doi = {10.1038/s41467-025-55913-y},
pmid = {39789054},
issn = {2041-1723},
}

@article {pmid39788669,
year = {2025},
author = {Li, X and Wang, C and Chai, J and Liu, H and Jiang, X and Li, Y and Li, Y},
title = {Structure-switchable branched inhibitors regulate the activity of CRISPR-Cas12a for nucleic acid diagnostics.},
journal = {Analytica chimica acta},
volume = {1336},
number = {},
pages = {343515},
doi = {10.1016/j.aca.2024.343515},
pmid = {39788669},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; CRISPR-Associated Proteins/metabolism ; Endodeoxyribonucleases/chemistry/metabolism/antagonists & inhibitors ; Nucleic Acids/chemistry ; Bacterial Proteins ; },
abstract = {BACKGROUND: In current years, the CRISPR (clustered regularly interspaced short palindromic repeats) based strategies have emerged as the most promising molecular tool in the field of gene editing, intracellular imaging, transcriptional regulation and biosensing. However, the recent CRISPR-based diagnostic technologies still require the incorporation of other amplification strategies (such as polymerase chain reaction) to improve the cis/trans cleavage activity of Cas12a, which complicates the detection workflow and lack of a uniform compatible system to respond to the target in one pot.

RESULTS: To better fully-functioning CRISPR/Cas12a, we reported a novel technique for straightforward nucleic acid detection by incorporating enzyme-responsive steric hindrance-based branched inhibitors with CRISPR/AsCas12a methodology. The construction-transferable branched inhibitors coupled with a specific overhang flap induce spatial steric effects and result in the loss of the binding ability of Cas12a, which inhibits the activity of Cas12a. Target as the input signal would trigger the site-directed APE1 enzyme incision of the inhibitors, thus transforming the conformation of the inhibitors into split activators to illumine the CRISPR/AsCas12a catalyst system. At the same time, we found that APE1 could drive the enzymatic positive feedback circuit and exhibited considerably high amplification efficiency to enhance the detection ability of nucleic acids. Besides, our method provides universal platforms and can be realized in real-time and one-pot detection of HIV-1 DNA by replacing the inhibitors and crRNA with different target recognition sequences.

SIGNIFICANCE AND NOVELTY: Overall, due to the high programmability of the nucleic acid network, this work proposed a feasible way to use the steric hindrance-based inhibitors as a switchable element, decorating the CRISPR/Cas12a-based strategy equipment for molecular diagnostics. Besides, this strategy could offer a simple tool for detecting trace nucleic acid, which opens avenues for future clinical application.},
}

*CRISPR-Cas Systems/genetics
Humans
CRISPR-Associated Proteins/metabolism
Endodeoxyribonucleases/chemistry/metabolism/antagonists & inhibitors
Nucleic Acids/chemistry
Bacterial Proteins

@article {pmid39788657,
year = {2025},
author = {Siler, T and Stanley, L and Saleem, M and Badalyan, A},
title = {A non-covalently bound redox indicator for electrochemical CRISPR-Cas12a and DNase I biosensors.},
journal = {Analytica chimica acta},
volume = {1336},
number = {},
pages = {343480},
doi = {10.1016/j.aca.2024.343480},
pmid = {39788657},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *Oxidation-Reduction ; *Electrochemical Techniques ; *DNA, Single-Stranded/chemistry/metabolism ; *Deoxyribonuclease I/metabolism/chemistry ; *CRISPR-Cas Systems ; Methylene Blue/chemistry ; CRISPR-Associated Proteins/metabolism/chemistry ; },
abstract = {A rapid and accurate biosensor for detecting disease biomarkers at point-of-care is essential for early disease diagnosis and preventing pandemics. CRISPR-Cas12a is a promising recognition element for DNA biosensors due to its programmability, specificity, and deoxyribonuclease activity initiated in the presence of a biomarker. The current electrochemical CRISPR-Cas12a-based biosensors utilize the single-stranded DNA (ssDNA) self-assembled on an electrode surface and covalently modified with the redox indicator, usually methylene blue (MB). In the presence of a biomarker, the nuclease domain is activated and cleaves ssDNA, decreasing the redox indicator signal. The covalent attachment of the MB to the ssDNA implies complexity and a higher production cost. Alternatively, some redox indicators can noncovalently bind to the ssDNA. Although such indicators have been applied for electrochemical nucleic acid detection, their potential for electrochemical CRISPR-Cas-based biosensors has not been explored. In this work, a ruthenium complex, [Ru(NH3)6][3+], was investigated as a redox indicator non-covalently binding to the ssDNA. Voltammetric studies and the optimization resulted in a simple and robust electrochemical method that was tested for deoxyribonuclease I (DNase I) activity detection and applied in the CRISPR-Cas12a-based biosensor for viral DNA (HPV-16). The biosensors revealed good analytical properties and represent an alternative to reported biosensors for nuclease activity requiring a covalent attachment of the redox indicator. Moreover, the developed method offers prospects for advancement and can be transformed to operate with other Cas nucleases to detect RNA and other analytes.},
}

*Biosensing Techniques/methods
*Oxidation-Reduction
*Electrochemical Techniques
*DNA, Single-Stranded/chemistry/metabolism
*Deoxyribonuclease I/metabolism/chemistry
*CRISPR-Cas Systems
Methylene Blue/chemistry
CRISPR-Associated Proteins/metabolism/chemistry

@article {pmid39788645,
year = {2025},
author = {Chen, Y and Zhao, R and Hu, X and Wang, X},
title = {The current status and future prospects of CRISPR-based detection of monkeypox virus: A review.},
journal = {Analytica chimica acta},
volume = {1336},
number = {},
pages = {343295},
doi = {10.1016/j.aca.2024.343295},
pmid = {39788645},
issn = {1873-4324},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Monkeypox virus/genetics ; Mpox (monkeypox)/diagnosis ; },
abstract = {BACKGROUND: The current pandemic of 2022 global mpox (formerly known as monkeypox), caused by infection with monkeypox virus (MPXV), has now reached over 120 countries. This constitutes a critical public health issue requiring effective disease management and surveillance. Rapid and reliable diagnosis is conducive to the control of infection, early intervention, and timely treatment. Clinical laboratories use various conventional diagnostic methods for detecting MPXV, including PCR, which can be regarded as a gold-standard diagnostic method. However, the application of PCR is limited by its requirements for high-cost equipment, skilled professionals, and a laboratory setting.

RESULTS: Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic systems have provided promising prospects for the rapid, sensitive, and specific detection of infectious diseases, especially in point-of-care settings. Over the past 2 years, an increasing number of researchers have concentrated on the application of the CRISPR method to mpox diagnosis. In the majority of cases, a two-step method was chosen, with CRISPR/Cas12a and recombinase polymerase amplification (RPA) as pre-amplification methods, followed by a fluorescence readout. Different strategies have been applied to overcome the encountered limitations of CRISPR detection, but no consensus on an integrated solution has been achieved. Thus, the application of the CRISPR/Cas system in mpox detection requires further exploration and improvement.

SIGNIFICANCE: This review discusses contemporary studies on MPXV CRISPR detection systems and the strategies proposed to address the challenges faced by CRISPR diagnosis with the hope of helping the development of CRISPR detection methods and improving pathogen detection technologies.},
}

Humans
*CRISPR-Cas Systems/genetics
*Monkeypox virus/genetics
Mpox (monkeypox)/diagnosis

@article {pmid39788509,
year = {2024},
author = {Wu, K and Xie, J and Liu, X and Yang, D and Wang, Y and Zhao, W and Shang, X and Jiang, L},
title = {Construction and biological function of Toxoplasma gondii rop41 gene knockout strain.},
journal = {Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences},
volume = {49},
number = {8},
pages = {1200-1209},
doi = {10.11817/j.issn.1672-7347.2024.240179},
pmid = {39788509},
issn = {1672-7347},
support = {32170510//the National Natural Science Foundation of China/ ; 20240026020055//the Innovation Training Program of Central South University/ ; },
mesh = {*Toxoplasma/genetics ; *Protozoan Proteins/genetics/metabolism ; *Gene Knockout Techniques ; CRISPR-Cas Systems ; Plasmids/genetics ; Virulence Factors/genetics ; Animals ; },
abstract = {OBJECTIVES: Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii (T. gondii), which can lead to complications such as encephalitis and ocular toxoplasmosis. The disease becomes more severe when the host's immune system is compromised. Rhoptry proteins are major virulence factors that enable T. gondii to invade host cells. This study aims to construct a T. gondii rhoptry protein 41 (rop41/ROP41) gene knockout strain and preliminarily investigate the biological function of rop41.

METHODS: Using CRISPR/Cas9 technology, a specific single-guide RNA (sgRNA) for the target gene was designed and linked to a recombinant plasmid. Homologous fragments were fused with a pyrimethamine resistance gene for selection purposes. The recombinant plasmid and the homologous fragments were electroporated into T. gondii, and PCR identification was performed after drug selection and monoclonal screening. Plaque assays were used to comprehensively assess whether rop41 affected the growth and proliferation of T. gondii in host cells. Invasion and proliferation assays were conducted to evaluate the invasion ability of the knockout strain into host cells and its intracellular proliferation capacity. The STRING database was utilized to construct a protein-protein interaction (PPI) network, and functional enrichment analysis was performed to predict the signaling pathways in which ROP41 might be involved.

RESULTS: The T. gondiirop41 gene knockout strain (RH Δku80Δrop41) was successfully constructed and stably inherited. Plaque assays showed that compared with the parental strain, the number of plaques formed by the rop41 gene knockout strain did not significantly decrease, but the reduction in plaque size was statistically significant (P<0.05). After the rop41 gene was knocked out, the invasion ability of T. gondii was reduced, but there was no statistically significant difference in its proliferation ability (P>0.05). The PPI network revealed that ROP41 was associated with other protein kinases and autophagy-related proteins. Enrichment analysis indicated that proteins interacting with ROP41 may be involved in signal transduction, biosynthesis, metabolism, and autophagy-related pathways and could be components of various kinase complexes and phagocytic vesicles.

CONCLUSIONS: The T. gondii RH Δku80Δrop41 strain has been successfully constructed. ROP41 primarily affects the ability of T. gondii to invade host cells and may play a role in signal transduction and autophagy-related pathways between T. gondii and the host.},
}

*Toxoplasma/genetics
*Protozoan Proteins/genetics/metabolism
*Gene Knockout Techniques
CRISPR-Cas Systems
Plasmids/genetics
Virulence Factors/genetics
Animals

@article {pmid39788098,
year = {2025},
author = {Lin, J and Bhoobalan-Chitty, Y and Peng, X},
title = {Cad1 turns ATP into phage poison.},
journal = {Cell host & microbe},
volume = {33},
number = {1},
pages = {8-10},
doi = {10.1016/j.chom.2024.12.013},
pmid = {39788098},
issn = {1934-6069},
mesh = {*Adenosine Triphosphate/metabolism ; *Bacteriophages/genetics/physiology/metabolism ; *CRISPR-Cas Systems ; Escherichia coli/genetics/virology/metabolism ; },
abstract = {Type III CRISPR-Cas executes a multifaceted anti-phage response, activating effectors such as a nuclease or membrane depolarizer. In a recent Cell paper, Baca and Majumder et al.[1] report an accessory effector, Cad1, which deaminates ATP into ITP, causing ITP accumulation and host growth arrest, thereby inhibiting phage propagation.},
}

*Adenosine Triphosphate/metabolism
*Bacteriophages/genetics/physiology/metabolism
*CRISPR-Cas Systems
Escherichia coli/genetics/virology/metabolism

@article {pmid39784310,
year = {2025},
author = {Dai, J and Wu, B and Ai, F and Yang, Z and Lu, Y and Zinian, C and Zeng, K and Zhang, Z},
title = {Exploiting the Potential of Spherical PAM Antenna for Enhanced CRISPR-Cas12a: A Paradigm Shift toward a Universal Amplification-Free Nucleic Acid Test Platform.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.4c04871},
pmid = {39784310},
issn = {1520-6882},
abstract = {The CRISPR-Cas12a system has shown tremendous potential for developing efficient biosensors. Albeit important, current CRISPR-Cas system-based diagnostic technologies (CRISPR-DX) highly rely on an additional preamplification procedure to obtain high sensitivity, inevitably leading to issues such as complicated assay workflow, cross-contamination, etc. Herein, a spherical protospacer-adjacent motif (PAM)-antenna-enhanced CRISPR-Cas12a system is fabricated for universal amplification-free nucleic acid detection with a detection limit of subfemtomolar. Meanwhile, the clinical detection capability of this sensor was further verified using gold-standard real-time quantitative polymerase chain reaction through Mycobacterium tuberculosis measurement, which demonstrated its good reliability for practical applications. Importantly, its excellent sensitivity is mainly ascribed to high efficiency of target search induced by a localized PAM-enriched microenvironment and improved catalytic activity of Cas12a (up to 4 folds). Our strategy provides some new insights for rapid and sensitive detection of nucleic acids in an amplification-free fashion.},
}

@article {pmid39781718,
year = {2025},
author = {Saberi, F and Yousefi-Najafabadi, Z and Shams, F and Dehghan, Z and Ahmadi, S and Pilehchi, T and Noori, E and Esmaeelzadeh, Z and Bazgiri, M and Mohammadi, R and Khani, F and Sameni, M and Moradbeigi, P and Kardar, GA and Salehi, M and Teng, Y and Jajarmi, V},
title = {CRISPR/Cas System: A Powerful Strategy to Improve Monogenic Human Diseases as Therapeutic Delivery; Current Applications and Challenges.},
journal = {Current gene therapy},
volume = {},
number = {},
pages = {},
doi = {10.2174/0115665232345516241119070150},
pmid = {39781718},
issn = {1875-5631},
abstract = {The 5,000 to 8,000 monogenic diseases are inherited disorders leading to mutations in a single gene. These diseases usually appear in childhood and sometimes lead to morbidity or premature death. Although treatments for such diseases exist, gene therapy is considered an effective and targeted method and has been used in clinics for monogenic diseases since 1989. Monogenic diseases are good candidates for novel therapeutic technologies like gene editing approaches to repair gene mutations. Clustered regularly interspaced short palindromic repeats (CRISPR)-based systems, the pioneer and effective gene editing tool, are utilized for ex vivo and in vivo treatment of monogenic diseases. The current review provides an overview of recent therapeutic applications of CRISPR-based gene editing in monogenic diseases in in vivo and ex vivo models. Furthermore, this review consolidates strategies aimed at providing new treatment options with gene therapy, thereby serving as a valuable reference for advancing the treatment landscape for patients with monogenic disorders.},
}

@article {pmid39779704,
year = {2025},
author = {Kempthorne, L and Vaizoglu, D and Cammack, AJ and Carcolé, M and Roberts, MJ and Mikheenko, A and Fisher, A and Suklai, P and Muralidharan, B and Kroll, F and Moens, TG and Yshii, L and Verschoren, S and Hölbling, BV and Moreira, FC and Katona, E and Coneys, R and de Oliveira, P and Zhang, YJ and Jansen, K and Daughrity, LM and McGown, A and Ramesh, TM and Van Den Bosch, L and Lignani, G and Rahim, AA and Coyne, AN and Petrucelli, L and Rihel, J and Isaacs, AM},
title = {Dual-targeting CRISPR-CasRx reduces C9orf72 ALS/FTD sense and antisense repeat RNAs in vitro and in vivo.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {459},
pmid = {39779704},
issn = {2041-1723},
support = {648716 - C9ND//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 217150/Z/19/Z//Wellcome Trust (Wellcome)/ ; },
mesh = {*C9orf72 Protein/genetics/metabolism ; *Amyotrophic Lateral Sclerosis/genetics/metabolism/therapy ; Humans ; *Frontotemporal Dementia/genetics/metabolism ; Animals ; *CRISPR-Cas Systems ; *RNA, Antisense/genetics ; Mice ; HEK293 Cells ; *Induced Pluripotent Stem Cells/metabolism ; DNA Repeat Expansion/genetics ; Disease Models, Animal ; Neurons/metabolism ; Genetic Therapy/methods ; },
abstract = {The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is an intronic G4C2 repeat expansion in C9orf72. The repeats undergo bidirectional transcription to produce sense and antisense repeat RNA species, which are translated into dipeptide repeat proteins (DPRs). As toxicity has been associated with both sense and antisense repeat-derived RNA and DPRs, targeting both strands may provide the most effective therapeutic strategy. CRISPR-Cas13 systems mature their own guide arrays, allowing targeting of multiple RNA species from a single construct. We show CRISPR-Cas13d variant CasRx effectively reduces overexpressed C9orf72 sense and antisense repeat transcripts and DPRs in HEK cells. In C9orf72 patient-derived iPSC-neuron lines, CRISPR-CasRx reduces endogenous sense and antisense repeat RNAs and DPRs and protects against glutamate-induced excitotoxicity. AAV delivery of CRISPR-CasRx to two distinct C9orf72 repeat mouse models significantly reduced both sense and antisense repeat-containing transcripts. This highlights the potential of RNA-targeting CRISPR systems as therapeutics for C9orf72 ALS/FTD.},
}

*C9orf72 Protein/genetics/metabolism
*Amyotrophic Lateral Sclerosis/genetics/metabolism/therapy
Humans
*Frontotemporal Dementia/genetics/metabolism
Animals
*CRISPR-Cas Systems
*RNA, Antisense/genetics
Mice
HEK293 Cells
*Induced Pluripotent Stem Cells/metabolism
DNA Repeat Expansion/genetics
Disease Models, Animal
Neurons/metabolism
Genetic Therapy/methods

@article {pmid39779681,
year = {2025},
author = {McCallister, TX and Lim, CKW and Singh, M and Zhang, S and Ahsan, NS and Terpstra, WM and Xiong, AY and Zeballos C, MA and Powell, JE and Drnevich, J and Kang, Y and Gaj, T},
title = {A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {460},
pmid = {39779681},
issn = {2041-1723},
support = {1U01NS122102-01A1//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 1R01NS123556-01A1//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 5R01GM141296//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; MDA602798//Muscular Dystrophy Association (Muscular Dystrophy Association Inc.)/ ; 20-IIP-516//Amyotrophic Lateral Sclerosis Association (ALS Association)/ ; },
mesh = {*Amyotrophic Lateral Sclerosis/genetics/pathology/metabolism ; *C9orf72 Protein/genetics/metabolism ; *Frontotemporal Dementia/genetics/pathology/metabolism ; *CRISPR-Cas Systems ; Humans ; Animals ; DNA Repeat Expansion/genetics ; Disease Models, Animal ; Motor Neurons/metabolism/pathology ; Mice ; },
abstract = {An abnormal expansion of a GGGGCC (G4C2) hexanucleotide repeat in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two debilitating neurodegenerative disorders driven in part by gain-of-function mechanisms involving transcribed forms of the repeat expansion. By utilizing a Cas13 variant with reduced collateral effects, we develop here a high-fidelity RNA-targeting CRISPR-based system for C9ORF72-linked ALS/FTD. When delivered to the brain of a transgenic rodent model, this Cas13-based platform curbed the expression of the G4C2 repeat-containing RNA without affecting normal C9ORF72 levels, which in turn decreased the formation of RNA foci, reduced the production of a dipeptide repeat protein, and reversed transcriptional deficits. This high-fidelity system possessed improved transcriptome-wide specificity compared to its native form and mediated targeting in motor neuron-like cells derived from a patient with ALS. These results lay the foundation for the implementation of RNA-targeting CRISPR technologies for C9ORF72-linked ALS/FTD.},
}

*Amyotrophic Lateral Sclerosis/genetics/pathology/metabolism
*C9orf72 Protein/genetics/metabolism
*Frontotemporal Dementia/genetics/pathology/metabolism
*CRISPR-Cas Systems
Humans
Animals
DNA Repeat Expansion/genetics
Disease Models, Animal
Motor Neurons/metabolism/pathology
Mice

@article {pmid39779650,
year = {2025},
author = {Aydin, A and Yerlikaya, BA and Yerlikaya, S and Yilmaz, NN and Kavas, M},
title = {CRISPR-mediated mutation of cytokinin signaling genes (SlHP2 and SlHP3) in tomato: Morphological, physiological, and molecular characterization.},
journal = {The plant genome},
volume = {18},
number = {1},
pages = {e20542},
pmid = {39779650},
issn = {1940-3372},
support = {PYO.ZRT.1904.22.006//Ondokuz Mayis Üniversitesi/ ; },
mesh = {*Solanum lycopersicum/genetics/physiology ; *Cytokinins/metabolism ; *Signal Transduction ; Mutation ; CRISPR-Cas Systems ; Droughts ; Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Stress, Physiological/genetics ; Gene Editing ; },
abstract = {Synergistic and antagonistic relationships between cytokinins and other plant growth regulators are important in response to changing environmental conditions. Our study aimed to determine the functions of SlHP2 and SlHP3, two members of cytokinin signaling in tomato, in drought stress response using CRISPR/Cas9-mediated mutagenesis. Ten distinct genome-edited lines were generated via Agrobacterium tumefaciens-mediated gene transfer and confirmed through Sanger sequencing. Stress experiments were conducted with two of these lines (slhp2,3-10 and slhp2,3-11), which harbored homozygous mutations in both genes. The responses of two lines carrying homozygous mutations in both genes under polyethylene glycol (PEG)-induced stress were examined using morphological, physiological, biochemical, and molecular methods. The genome-edited lines demonstrated enhanced water retention, reduced stomatal density, and less oxidative damage compared to the wild-type plants under PEG-induced stress. Moreover, the slhp2,3 double mutant plants exhibited improved root growth, showcasing their superior drought tolerance over wild-type plants by accessing deeper water sources and maintaining hydration in water-limited environments. To investigate the involvement of cytokinin signaling regulators and genes associated with stomatal formation and differentiation, the expression of genes (Speechless [SPCH], FAMA, MUTE, TMM, HB25, HB31, RR6, RR7, and Solyc02g080860) was assessed. The results revealed that all regulators were downregulated, with SPCH, TMM, RR7, and RR6 showing significant reductions under PEG-induced stress. These results emphasize the promise of utilizing CRISPR/Cas9 to target cytokinin signaling pathways, enhancing drought tolerance in tomatoes through improvements in water retention and root growth, along with a reduction in stomatal density and malondialdehyde content.},
}

*Solanum lycopersicum/genetics/physiology
*Cytokinins/metabolism
*Signal Transduction
Mutation
CRISPR-Cas Systems
Droughts
Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
Stress, Physiological/genetics
Gene Editing

@article {pmid39779219,
year = {2025},
author = {Tang, JX and Cabrera-Orefice, A and Meisterknecht, J and Taylor, LS and Monteuuis, G and Stensland, ME and Szczepanek, A and Stals, K and Davison, J and He, L and Hopton, S and Nyman, TA and Jackson, CB and Pyle, A and Winter, M and Wittig, I and Taylor, RW},
title = {COA5 has an essential role in the early stage of mitochondrial complex IV assembly.},
journal = {Life science alliance},
volume = {8},
number = {3},
pages = {},
pmid = {39779219},
issn = {2575-1077},
mesh = {Humans ; Male ; *Mitochondria/metabolism/genetics ; *Electron Transport Complex IV/metabolism/genetics ; Female ; Mitochondrial Proteins/metabolism/genetics ; Mitochondrial Diseases/genetics/metabolism/pathology ; Pedigree ; CRISPR-Cas Systems ; Fibroblasts/metabolism ; Mutation, Missense ; },
abstract = {Pathogenic variants in cytochrome c oxidase assembly factor 5 (COA5), a proposed complex IV (CIV) assembly factor, have been shown to cause clinical mitochondrial disease with two siblings affected by neonatal hypertrophic cardiomyopathy manifesting a rare, homozygous COA5 missense variant (NM_001008215.3: c.157G>C, p.Ala53Pro). The most striking observation in the affected individuals was an isolated impairment in the early stage of mitochondrial CIV assembly. In this study, we report an unrelated family in whom we have identified the same COA5 variant with patient-derived fibroblasts and skeletal muscle biopsies replicating an isolated CIV deficiency. A CRISPR/Cas9-edited homozygous COA5 knockout U2OS cell line with a similar biochemical profile was generated to interrogate the functional role of the human COA5 protein. Mitochondrial complexome profiling pinpointed a role of COA5 in early CIV assembly, more specifically, its involvement in the stage between MTCO1 maturation and the incorporation of MTCO2. We therefore propose that the COA5 protein plays an essential role in the biogenesis of MTCO2 and its integration into the early CIV assembly intermediate for downstream assembly of the functional holocomplex.},
}

Humans
Male
*Mitochondria/metabolism/genetics
*Electron Transport Complex IV/metabolism/genetics
Female
Mitochondrial Proteins/metabolism/genetics
Mitochondrial Diseases/genetics/metabolism/pathology
Pedigree
CRISPR-Cas Systems
Fibroblasts/metabolism
Mutation, Missense

@article {pmid39777467,
year = {2025},
author = {Xu, X and Lv, X and Liu, Y and Li, J and Du, G and Chen, J and Ledesma-Amaro, R and Liu, L},
title = {CRISPR/Cas13X-assisted programmable and multiplexed translation regulation for controlled biosynthesis.},
journal = {Nucleic acids research},
volume = {53},
number = {1},
pages = {},
pmid = {39777467},
issn = {1362-4962},
support = {32200050//National Natural Science Foundation of China/ ; BK20221079//Natural Science Foundation of Jiangsu Province/ ; JUSRP52019A//Fundamental Research Funds for the Central Universities/ ; 2020YFA0908300//National Key Research and Development Program of China/ ; BK20233003//Jiangsu Basic Research Center for Synthetic Biology/ ; },
mesh = {*CRISPR-Cas Systems ; *Bacillus subtilis/genetics/metabolism ; *Protein Biosynthesis ; *RNA, Messenger/genetics/metabolism ; RNA Stability ; Gene Expression Regulation, Bacterial ; Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Proteins/metabolism/genetics ; },
abstract = {Developing efficient gene regulation tools is essential for optimizing microbial cell factories, but most existing tools only modulate gene expression at the transcriptional level. Regulation at the translational level provides a faster dynamic response, whereas developing a programmable, efficient and multiplexed translational regulation tool remains a challenge. Here, we have developed CRISPRi and CRISPRa systems based on hfCas13X that can regulate gene translation in Bacillus subtilis. First, we constructed a CRISPRi system to regulate gene translation based on catalytically deactivated hfCas13X (dhfCas13X). Second, we designed unique mRNA-crRNA pairs to construct DiCRISPRa (degradation-inhibited CRISPRa) and TsCRISPRa (translation-started CRISPRa) systems, which can activate downstream gene translation by enhancing mRNA stability or initiating mRNA translation. In addition, we found that fusing dhfCas13X with the RNA-binding chaperone BHfq significantly improved the activation efficiency of the DiCRISPRa and TsCRISPRa systems (43.2-fold). Finally, we demonstrated that the constructed CRISPR systems could be used to optimize the metabolic networks of two biotechnologically relevant compounds, riboflavin and 2'-fucosyllactose, increasing their titers by 3- and 1.2-fold, respectively. The CRISPRa and CRISPRi systems developed here provide new tools for the regulation of gene expression at the translation level and offer new ideas for the construction of CRISPRa systems.},
}

*CRISPR-Cas Systems
*Bacillus subtilis/genetics/metabolism
*Protein Biosynthesis
*RNA, Messenger/genetics/metabolism
RNA Stability
Gene Expression Regulation, Bacterial
Bacterial Proteins/genetics/metabolism
CRISPR-Associated Proteins/metabolism/genetics

@article {pmid39775780,
year = {2025},
author = {Tong, G and Nath, P and Hiruta, Y and Citterio, D},
title = {Amplification-free CRISPR/Cas based dual-enzymatic colorimetric nucleic acid biosensing device.},
journal = {Lab on a chip},
volume = {},
number = {},
pages = {},
doi = {10.1039/d4lc01039f},
pmid = {39775780},
issn = {1473-0189},
abstract = {Nucleic acid testing (NAT) is widely considered the gold standard in analytical fields, with applications spanning environmental monitoring, forensic science and clinical diagnostics, among others. However, its widespread use is often constrained by complicated assay procedures, the need for specialized equipment, and the complexity of reagent handling. In this study, we demonstrate a fully integrated 3D-printed biosensensing device employing a CRISPR/Cas12a-based dual-enzymatic mechanism for highly sensitive and user-friendly nucleic acid detection. A plastic probe stick was designed to host small-sized gold nanoparticles, enhancing enzyme labeling density. Alkaline phosphatase (ALP) was then conjugated via single-stranded DNA, requiring only a single enzyme substrate addition to generate a simple visual signal change. This approach eliminates the need for amplification or centrifugation steps, achieving a limit of detection (LOD) as low as 10 pM - among the highest sensitivities reported for amplification-free colorimetric nucleic acid detection. Furthermore, we developed a device that incorporates this probe stick, integrates all necessary reagents, and features a smartphone-compatible accessory for quantitative analysis. This allows end-users to perform visual or quantitative DNA analysis with simple operations, achieving a visual detection limit of approximately 100 pM, comparable to other CRISPR-based non-amplified nucleic acid detection methods. Additionally, the system successfully distinguished perfectly matched from mismatched nucleic acid sequences, demonstrating its specificity and versatility. Although certain design limitations affected the sensitivity of the integrated device compared to the probe stick alone, the simplicity and portability of this device make it a promising tool for rapid nucleic acid screening in clinical diagnostics, environmental monitoring, and food safety control. This study paves the way for the development of practical biosensors for point-of-care testing (POCT) applications.},
}

@article {pmid39775585,
year = {2024},
author = {Zhang, WW and Matlashewski, G},
title = {Evidence for gene essentiality in Leishmania using CRISPR.},
journal = {PloS one},
volume = {19},
number = {12},
pages = {e0316331},
pmid = {39775585},
issn = {1932-6203},
mesh = {*Genes, Essential ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Leishmania/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genes, Protozoan ; Promoter Regions, Genetic ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Ribosomal/genetics ; Plasmids/genetics ; DNA-Directed RNA Polymerases ; Viral Proteins ; },
abstract = {The ability to determine the essentiality of a gene in the protozoan parasite Leishmania is important to identify potential targets for intervention and understanding the parasite biology. CRISPR gene editing technology has significantly improved gene targeting efficiency in Leishmania. There are two commonly used CRISPR gene targeting methods in Leishmania; the stable expression of the gRNA and Cas9 using a plasmid containing a Leishmania ribosomal RNA gene promoter (rRNA-P stable protocol) and the T7 RNA polymerase based transient gRNA expression system in promastigotes stably expressing Cas9 (T7 transient protocol). There are distinct advantages with both systems. The T7 transient protocol is excellent for high throughput gene deletions and has been used to successfully delete hundreds of Leishmania genes to study mutant phenotypes and several research labs are now using this protocol to target all the genes in L. mexicana genome. The rRNA-P stable protocol stably expresses the plasmid derived gRNA and has been used to delete or disrupt single and multicopy Leishmania genes, perform single nucleotide changes and provide evidence for gene essentiality by directly observing null mutant promastigotes dying in culture. In this study, the rRNA-P stable protocol was used to target 22 Leishmania genes in which null mutants were not generated using the T7 transient protocol. Notably, the rRNA-P stable protocol was able to generate alive null mutants for 8 of the 22 genes. These results demonstrate the rRNA-P stable protocol could be used alone or in combination with the T7 transient protocol to investigate gene essentiality in Leishmania.},
}

*Genes, Essential
*CRISPR-Cas Systems
*Gene Editing/methods
*Leishmania/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Genes, Protozoan
Promoter Regions, Genetic
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
RNA, Ribosomal/genetics
Plasmids/genetics
DNA-Directed RNA Polymerases
Viral Proteins

@article {pmid39774105,
year = {2025},
author = {Ocampo, RF and Bravo, JPK and Dangerfield, TL and Nocedal, I and Jirde, SA and Alexander, LM and Thomas, NC and Das, A and Nielson, S and Johnson, KA and Brown, CT and Butterfield, CN and Goltsman, DSA and Taylor, DW},
title = {DNA targeting by compact Cas9d and its resurrected ancestor.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {457},
pmid = {39774105},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Humans ; *DNA/metabolism/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; Cryoelectron Microscopy ; HEK293 Cells ; Endonucleases/metabolism/genetics/chemistry ; Protein Domains ; },
abstract = {Type II CRISPR endonucleases are widely used programmable genome editing tools. Recently, CRISPR-Cas systems with highly compact nucleases have been discovered, including Cas9d (a type II-D nuclease). Here, we report the cryo-EM structures of a Cas9d nuclease (747 amino acids in length) in multiple functional states, revealing a stepwise process of DNA targeting involving a conformational switch in a REC2 domain insertion. Our structures provide insights into the intricately folded guide RNA which acts as a structural scaffold to anchor small, flexible protein domains for DNA recognition. The sgRNA can be truncated by up to ~25% yet still retain activity in vivo. Using ancestral sequence reconstruction, we generated compact nucleases capable of efficient genome editing in mammalian cells. Collectively, our results provide mechanistic insights into the evolution and DNA targeting of diverse type II CRISPR-Cas systems, providing a blueprint for future re-engineering of minimal RNA-guided DNA endonucleases.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Humans
*DNA/metabolism/genetics
*CRISPR-Associated Protein 9/metabolism/genetics/chemistry
Cryoelectron Microscopy
HEK293 Cells
Endonucleases/metabolism/genetics/chemistry
Protein Domains

@article {pmid39774003,
year = {2025},
author = {Claiborne, DT and Detwiler, Z and Docken, SS and Borland, TD and Cromer, D and Simkhovich, A and Ophinni, Y and Okawa, K and Bateson, T and Chen, T and Hudson, W and Trifonova, R and Davenport, MP and Ho, TW and Boutwell, CL and Allen, TM},
title = {High frequency CCR5 editing in human hematopoietic stem progenitor cells protects xenograft mice from HIV infection.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {446},
pmid = {39774003},
issn = {2041-1723},
support = {U19 HL156247/HL/NHLBI NIH HHS/United States ; U19 HL156247/HL/NHLBI NIH HHS/United States ; },
mesh = {*Receptors, CCR5/genetics/metabolism ; Humans ; Animals ; *Hematopoietic Stem Cells/metabolism/virology ; *HIV Infections/virology/therapy/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Hematopoietic Stem Cell Transplantation ; Mice ; Heterografts ; HIV-1/genetics ; Mice, SCID ; },
abstract = {The only cure of HIV has been achieved in a small number of people who received a hematopoietic stem cell transplant (HSCT) comprising allogeneic cells carrying a rare, naturally occurring, homozygous deletion in the CCR5 gene. The rarity of the mutation and the significant morbidity and mortality of such allogeneic transplants precludes widespread adoption of this HIV cure. Here, we show the application of CRISPR/Cas9 to achieve >90% CCR5 editing in human, mobilized hematopoietic stem progenitor cells (HSPC), resulting in a transplant that undergoes normal hematopoiesis, produces CCR5 null T cells, and renders xenograft mice refractory to HIV infection. Titration studies transplanting decreasing frequencies of CCR5 edited HSPCs demonstrate that <90% CCR5 editing confers decreasing protective benefit that becomes negligible between 54% and 26%. Our study demonstrates the feasibility of using CRISPR/Cas9/RNP to produce an HSPC transplant with high frequency CCR5 editing that is refractory to HIV replication. These results raise the potential of using CRISPR/Cas9 to produce a curative autologous HSCT and bring us closer to the development of a cure for HIV infection.},
}

*Receptors, CCR5/genetics/metabolism
Humans
Animals
*Hematopoietic Stem Cells/metabolism/virology
*HIV Infections/virology/therapy/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
*Hematopoietic Stem Cell Transplantation
Mice
Heterografts
HIV-1/genetics
Mice, SCID

@article {pmid39773393,
year = {2025},
author = {Bogut, A and Kołodziejek, A and Minnich, SA and Hovde, CJ},
title = {CRISPR/Cas Systems as Diagnostic and Potential Therapeutic Tools for Enterohemorrhagic Escherichia coli.},
journal = {Archivum immunologiae et therapiae experimentalis},
volume = {73},
number = {1},
pages = {},
pmid = {39773393},
issn = {1661-4917},
mesh = {Humans ; *Enterohemorrhagic Escherichia coli/genetics ; *CRISPR-Cas Systems ; Animals ; *Escherichia coli Infections/diagnosis/therapy/immunology ; *Gene Editing/methods ; Cattle ; Virulence/genetics ; Hemolytic-Uremic Syndrome/diagnosis/therapy/immunology/microbiology ; Probiotics/therapeutic use ; Bacteriophages/genetics ; Genetic Engineering ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Following its discovery as an adaptive immune system in prokaryotes, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been developed into a multifaceted genome editing tool. This review compiles findings aimed at implementation of this technology for selective elimination or attenuation of enterohemorrhagic Escherichia coli (EHEC). EHEC are important zoonotic foodborne pathogens that cause hemorrhagic colitis and can progress to the life-threatening hemolytic uremic syndrome (HUS). Advancements in the application of CRISPR methodology include laboratory detection and identification of EHEC, genotyping, screening for pathogenic potential, and engineering probiotics to reduce microbial shedding by cattle, the primary source of human infection. Genetically engineered phages or conjugative plasmids have been designed to target and inactivate genes whose products are critical for EHEC virulence.},
}

Humans
*Enterohemorrhagic Escherichia coli/genetics
*CRISPR-Cas Systems
Animals
*Escherichia coli Infections/diagnosis/therapy/immunology
*Gene Editing/methods
Cattle
Virulence/genetics
Hemolytic-Uremic Syndrome/diagnosis/therapy/immunology/microbiology
Probiotics/therapeutic use
Bacteriophages/genetics
Genetic Engineering
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid39773308,
year = {2025},
author = {Chen, H and Fang, HQ and Liu, JT and Chang, SY and Cheng, LB and Sun, MX and Feng, JR and Liu, ZM and Zhang, YH and Rosen, CJ and Liu, P},
title = {Atlas of Fshr expression from novel reporter mice.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {39773308},
issn = {2050-084X},
mesh = {Animals ; *Receptors, FSH/genetics/metabolism ; Mice ; *Genes, Reporter ; Male ; CRISPR-Cas Systems ; Female ; Mice, Transgenic ; },
abstract = {The FSH-FSHR pathway has been considered an essential regulator in reproductive development and fertility. But there has been emerging evidence of FSHR expression in extragonadal organs. This poses new questions and long-term debates regarding the physiological role of the FSH-FSHR, and underscores the need for reliable, in vivo analysis of FSHR expression in animal models. However, conventional methods have proven insufficient for examining FSHR expression due to several limitations. To address this challenge, we developed Fshr-ZsGreen reporter mice under the control of Fshr endogenous promoter using CRISPR-Cas9. With this novel genetic tool, we provide a reliable readout of Fshr expression at single-cell resolution level in vivo and in real time. Reporter animals were also subjected to additional analyses,to define the accurate expression profile of FSHR in gonadal and extragonadal organs/tissues. Our compelling results not only demonstrated Fshr expression in intragonadal tissues but also, strikingly, unveiled notably increased expression in Leydig cells, osteoblast lineage cells, endothelial cells in vascular structures, and epithelial cells in bronchi of the lung and renal tubes. The genetic decoding of the widespread pattern of Fshr expression highlights its physiological relevance beyond reproduction and fertility, and opens new avenues for therapeutic options for age-related disorders of the bones, lungs, kidneys, and hearts, among other tissues. Exploiting the power of the Fshr knockin reporter animals, this report provides the first comprehensive genetic record of the spatial distribution of FSHR expression, correcting a long-term misconception about Fshr expression and offering prospects for extensive exploration of FSH-FSHR biology.},
}

Animals
*Receptors, FSH/genetics/metabolism
Mice
*Genes, Reporter
Male
CRISPR-Cas Systems
Female
Mice, Transgenic

@article {pmid39770835,
year = {2024},
author = {Tuli, SR and Ali, MF and Jamal, TB and Khan, MAS and Fatima, N and Ahmed, I and Khatun, M and Sharmin, SA},
title = {Characterization and Molecular Insights of a Chromium-Reducing Bacterium Bacillus tropicus.},
journal = {Microorganisms},
volume = {12},
number = {12},
pages = {},
pmid = {39770835},
issn = {2076-2607},
abstract = {Environmental pollution from metal toxicity is a widespread concern. Certain bacteria hold promise for bioremediation via the conversion of toxic chromium compounds into less harmful forms, promoting environmental cleanup. In this study, we report the isolation and detailed characterization of a highly chromium-tolerant bacterium, Bacillus tropicus CRB14. The isolate is capable of growing on 5000 mg/L Cr (VI) in an LB (Luria Bertani) agar plate while on 900 mg/L Cr (VI) in LB broth. It shows an 86.57% reduction ability in 96 h of culture. It can also tolerate high levels of As, Cd, Co, Fe, Zn, and Pb. The isolate also shows plant growth-promoting potential as demonstrated by a significant activity of nitrogen fixation, phosphate solubilization, IAA (indole acetic acid), and siderophore production. Whole-genome sequencing revealed that the isolate lacks Cr resistance genes in their plasmids and are located on its chromosome. The presence of the chrA gene points towards Cr(VI) transport, while the absence of ycnD suggests alternative reduction pathways. The genome harbors features like genomic islands and CRISPR-Cas systems, potentially aiding adaptation and defense. Analysis suggests robust metabolic pathways, potentially involved in Cr detoxification. Notably, genes for siderophore and NRP-metallophore production were identified. Whole-genome sequencing data also provides the basis for molecular validation of various genes. Findings from this study highlight the potential application of Bacillus tropicus CRB14 for bioremediation while plant growth promotion can be utilized as an added benefit.},
}

@article {pmid39770798,
year = {2024},
author = {Muriuki, R and Ndichu, M and Githigia, S and Svitek, N},
title = {CRISPR-Cas-Based Pen-Side Diagnostic Tests for Anaplasma marginale and Babesia bigemina.},
journal = {Microorganisms},
volume = {12},
number = {12},
pages = {},
pmid = {39770798},
issn = {2076-2607},
support = {7200AA20CA00022//USAID/ ; },
abstract = {Anaplasma marginale and Babesia bigemina are tick-borne pathogens, posing significant threats to the health and productivity of cattle in tropical and subtropical regions worldwide. Currently, detection of Babesia bigemina and Anaplasma marginale in infected animals relies primarily on microscopic examination of Giemsa-stained blood or organ smears, which has limited sensitivity. Molecular methods offer higher sensitivity but are costly and impractical in resource-limited settings. Following the development of a pen-side test for detecting Theileria parva infections in cattle, we have created two additional CRISPR-Cas12a assays targeting Anaplasma marginale and Babesia bigemina. The assays target the major surface protein 5 (MSP5) for A. marginale and rhoptry-associated protein 1a (RAP1a) for B. bigemina. These additional tests involve a 20 min recombinase polymerase amplification (RPA) reaction followed by a 60 min CRISPR-Cas12a detection with a lateral strip readout. Results demonstrate high specificity, with no cross-reactivity against other tick-borne parasites, and a limit of detection down to 10[2] DNA copies/µL of each target marker. The findings pave the way for sensitive and user-friendly pen-side tests to diagnose A. marginale and B. bigemina infections.},
}

@article {pmid39770347,
year = {2024},
author = {de Sousa, RMP and Garcia, LS and Lemos, FS and de Campos, VS and Machado Ferreira, E and de Almeida, NAA and Maron-Gutierrez, T and de Souza, EM and de Paula, VS},
title = {CRISPR/Cas9 Eye Drop HSV-1 Treatment Reduces Brain Viral Load: A Novel Application to Prevent Neuronal Damage.},
journal = {Pathogens (Basel, Switzerland)},
volume = {13},
number = {12},
pages = {},
pmid = {39770347},
issn = {2076-0817},
support = {GM-GD 2022//National Council for Scientific and Technological Development/ ; 001//Coordenação de Aperfeicoamento de Pessoal de Nível Superior/ ; VPGDI 024 INT 22//Fundação Oswaldo Cruz/ ; 001//Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro/ ; },
mesh = {Animals ; *Herpesvirus 1, Human/drug effects/genetics/physiology/pathogenicity ; *CRISPR-Cas Systems/genetics ; Mice ; *Viral Load/drug effects ; *Mice, Inbred BALB C ; *Antiviral Agents/pharmacology ; *Brain/virology/pathology/drug effects/metabolism ; *Herpes Simplex/drug therapy/virology ; Female ; Virus Replication/drug effects ; Disease Models, Animal ; Gene Editing/methods ; },
abstract = {Herpes simplex virus-1 (HSV-1) can invade the central nervous system (CNS). However, antiviral drugs used to treat HSV-1 have significant toxicity and resistance. An alternative approach involves the use of the CRISPR/Cas9 complex as a viral replication inhibitor. Editing the UL39 gene with CRISPR/Cas9 results in >95% inhibition of HSV-1 replication in vitro; however, few studies have investigated alternative therapies in in vivo models. This study aimed to investigate the efficacy of CRISPR/Cas9 targeting the UL39 region, which was administered via the ocular route, to reduce the HSV-1 viral count in the CNS of BALB/c mice. Mice were inoculated with HSV-1 and treated using CRISPR/Cas9. The kinetics of CNS infection were assessed, and the effects of CRISPR/Cas9 were compared with those of topical acyclovir treatments. The brain viral load was analyzed, and histopathology and immunofluorescence of the nervous tissue were performed. The group treated with CRISPR/Cas9 showed a reduced viral load on the seventh day post-infection, and no brain inflammation or chromatin compaction was observed in animals that received CRISPR/Cas9 therapy. These findings suggest that CRISPR/Cas9 anti-UL39 therapy can reduce the HSV-1 viral load in brain tissue. Therefore, investigating viral detection and evaluating antiviral treatments in the brain is essential.},
}

Animals
*Herpesvirus 1, Human/drug effects/genetics/physiology/pathogenicity
*CRISPR-Cas Systems/genetics
Mice
*Viral Load/drug effects
*Mice, Inbred BALB C
*Antiviral Agents/pharmacology
*Brain/virology/pathology/drug effects/metabolism
*Herpes Simplex/drug therapy/virology
Female
Virus Replication/drug effects
Disease Models, Animal
Gene Editing/methods

@article {pmid39770308,
year = {2024},
author = {Islam, MM and Jung, DE and Shin, WS and Oh, MH},
title = {Colistin Resistance Mechanism and Management Strategies of Colistin-Resistant Acinetobacter baumannii Infections.},
journal = {Pathogens (Basel, Switzerland)},
volume = {13},
number = {12},
pages = {},
doi = {10.3390/pathogens13121049},
pmid = {39770308},
issn = {2076-0817},
support = {Grant Nos. 2022R1F1A1071415 and NRF-RS-2023-00275307 and Grant No. 2019R1A6C1010033//National Research Foundation of Korea (NRF) funded by the Ministry of Education & Basic Science Research Capacity Enhancement Project through Korea Basic Science Institute (National research Facilities and Equipment Center) grant funded by the Ministry o/ ; },
mesh = {*Acinetobacter baumannii/drug effects/genetics ; *Colistin/pharmacology/therapeutic use ; Humans ; *Acinetobacter Infections/drug therapy ; *Anti-Bacterial Agents/therapeutic use/pharmacology ; Drug Resistance, Multiple, Bacterial/genetics/drug effects ; Drug Resistance, Bacterial/genetics/drug effects ; Animals ; },
abstract = {The emergence of antibiotic-resistant Acinetobacter baumannii (A. baumannii) is a pressing threat in clinical settings. Colistin is currently a widely used treatment for multidrug-resistant A. baumannii, serving as the last line of defense. However, reports of colistin-resistant strains of A. baumannii have emerged, underscoring the urgent need to develop alternative medications to combat these serious pathogens. To resist colistin, A. baumannii has developed several mechanisms. These include the loss of outer membrane lipopolysaccharides (LPSs) due to mutation of LPS biosynthetic genes, modification of lipid A (a constituent of LPSs) structure through the addition of phosphoethanolamine (PEtN) moieties to the lipid A component by overexpression of chromosomal pmrCAB operon genes and eptA gene, or acquisition of plasmid-encoded mcr genes through horizontal gene transfer. Other resistance mechanisms involve alterations of outer membrane permeability through porins, the expulsion of colistin by efflux pumps, and heteroresistance. In response to the rising threat of colistin-resistant A. baumannii, researchers have developed various treatment strategies, including antibiotic combination therapy, adjuvants to potentiate antibiotic activity, repurposing existing drugs, antimicrobial peptides, nanotechnology, photodynamic therapy, CRISPR/Cas, and phage therapy. While many of these strategies have shown promise in vitro and in vivo, further clinical trials are necessary to ensure their efficacy and widen their clinical applications. Ongoing research is essential for identifying the most effective therapeutic strategies to manage colistin-resistant A. baumannii. This review explores the genetic mechanisms underlying colistin resistance and assesses potential treatment options for this challenging pathogen.},
}

*Acinetobacter baumannii/drug effects/genetics
*Colistin/pharmacology/therapeutic use
Humans
*Acinetobacter Infections/drug therapy
*Anti-Bacterial Agents/therapeutic use/pharmacology
Drug Resistance, Multiple, Bacterial/genetics/drug effects
Drug Resistance, Bacterial/genetics/drug effects
Animals

@article {pmid39770154,
year = {2024},
author = {Shang, Z and Liu, S and Liu, D and Pei, X and Li, S and He, Y and Tong, Y and Liu, G},
title = {CRISPR/Cas12a with Universal crRNA for Indiscriminate Virus Detection.},
journal = {Molecules (Basel, Switzerland)},
volume = {29},
number = {24},
pages = {},
pmid = {39770154},
issn = {1420-3049},
support = {No. 22004005//National Natural Science Foundation of China/ ; 223777118D//Special Project on Biomedical Innovation/ ; },
mesh = {*CRISPR-Cas Systems ; *SARS-CoV-2/genetics/isolation & purification ; Humans ; *RNA, Viral/genetics/analysis ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/genetics ; COVID-19/diagnosis/virology ; Molecular Diagnostic Techniques/methods ; Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins/genetics ; Biosensing Techniques/methods ; },
abstract = {Viruses, known for causing widespread biological harm and even extinction, pose significant challenges to public health. Virus detection is crucial for accurate disease diagnosis and preventing the spread of infections. Recently, the outstanding analytical performance of CRISPR/Cas biosensors has shown great potential and they have been considered as augmenting methods for reverse-transcription polymerase chain reaction (RT-PCR), which was the gold standard for nucleic acid detection. We herein utilized Cas12a with universal CRISPR RNA (crRNA) for indiscriminate virus detection by attaching the target to a longer track strand for isothermal amplification. The amplified products contain a domain that is recognized by the Cas12a/crRNA complex, triggering the cleavage of surrounding reporters to produce signals, thereby escaping the target dependence of crRNA recognition. The proposed method allows the same crRNA to detect multiple viral nucleic acids with high sensitivity, including but not limited to SARS-CoV-2, human papillomaviruses (HPV), HCOV-NL63, HCOV-HKU1, and miRNA biomarkers. Taking SARS-CoV-2 and HPV16 pseudoviruses as examples, this method was proved as a versatile and sensitive platform for molecular diagnostic applications.},
}

*CRISPR-Cas Systems
*SARS-CoV-2/genetics/isolation & purification
Humans
*RNA, Viral/genetics/analysis
Nucleic Acid Amplification Techniques/methods
CRISPR-Associated Proteins/genetics
COVID-19/diagnosis/virology
Molecular Diagnostic Techniques/methods
Endodeoxyribonucleases/genetics/metabolism
Bacterial Proteins/genetics
Biosensing Techniques/methods

@article {pmid39769261,
year = {2024},
author = {Taki, T and Morimoto, K and Mizuno, S and Kuno, A},
title = {KOnezumi-AID: Automation Software for Efficient Multiplex Gene Knockout Using Target-AID.},
journal = {International journal of molecular sciences},
volume = {25},
number = {24},
pages = {},
pmid = {39769261},
issn = {1422-0067},
support = {JP24ama121047//Japan Agency for Medical Research and Development/ ; JPMJFR221H//Japan Science and Technology Agency/ ; 24K18045//Japan Society for the Promotion of Science/ ; },
mesh = {*Gene Knockout Techniques/methods ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; Mice ; Humans ; *Software ; },
abstract = {With the groundbreaking advancements in genome editing technologies, particularly CRISPR-Cas9, creating knockout mutants has become highly efficient. However, the CRISPR-Cas9 system introduces DNA double-strand breaks, increasing the risk of chromosomal rearrangements and posing a major obstacle to simultaneous multiple gene knockout. Base-editing systems, such as Target-AID, are safe alternatives for precise base modifications without requiring DNA double-strand breaks, serving as promising solutions for existing challenges. Nevertheless, the absence of adequate tools to support Target-AID-based gene knockout highlights the need for a comprehensive system to design guide RNAs (gRNAs) for the simultaneous knockout of multiple genes. Here, we aimed to develop KOnezumi-AID, a command-line tool for gRNA design for Target-AID-mediated genome editing. KOnezumi-AID facilitates gene knockout by inducing the premature termination codons or promoting exon skipping, thereby generating experiment-ready gRNA designs for mouse and human genomes. Additionally, KOnezumi-AID exhibits batch processing capacity, enabling rapid and precise gRNA design for large-scale genome editing, including CRISPR screening. In summary, KOnezumi-AID is an efficient and user-friendly tool for gRNA design, streamlining genome editing workflows and advancing gene knockout research.},
}

*Gene Knockout Techniques/methods
*CRISPR-Cas Systems
*RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Editing/methods
Animals
Mice
Humans
*Software

@article {pmid39769183,
year = {2024},
author = {Kappler, M and Thielemann, L and Glaß, M and Caggegi, L and Güttler, A and Pyko, J and Blauschmidt, S and Gutschner, T and Taubert, H and Otto, S and Eckert, AW and Tavassol, F and Bache, M and Vordermark, D and Kaune, T and Rot, S},
title = {Functional and Biological Characterization of the LGR5Δ5 Splice Variant in HEK293T Cells.},
journal = {International journal of molecular sciences},
volume = {25},
number = {24},
pages = {},
pmid = {39769183},
issn = {1422-0067},
support = {NA//Dr. Kleist Stiftung (Foundation)/ ; NA//Open Access Publication Fund of the Martin Luther University Halle-Wittenberg/ ; },
mesh = {Humans ; *Receptors, G-Protein-Coupled/genetics/metabolism ; HEK293 Cells ; *Thrombospondins/genetics/metabolism ; Wnt Signaling Pathway/genetics ; Protein Isoforms/genetics/metabolism ; CRISPR-Cas Systems ; Cell Movement/genetics ; },
abstract = {The regulator of the canonical Wnt pathway, leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), is expressed in the stem cell compartment of several tissues and overexpressed in different human carcinomas. The isoform of the stem cell marker LGR5, named LGR5Δ5 and first described by our group, is associated with prognosis and metastasis in oral squamous cell carcinoma (OSCC) and soft tissue sarcoma (STS). In a proof-of-principle analysis, the function of LGR5Δ5 was investigated in HEK293T cells, a model cell line of the Wnt pathway, compared to full-length LGR5 (FL) expression. The CRISPR/CAS knockout of LGR5 and LGR4 (thereby avoiding the side effects of LGR4) resulted in a loss of Wnt activity that cannot be restored by LGR5Δ5 but by LGR5FL rescue. The ability to migrate was not affected by LGR5Δ5, but was reduced by LGR5FL overexpression. The CRISPR/CAS of LGR4 and 5 induced radiosensitization, which was enhanced by the overexpression of LGR5FL or LGR5Δ5. RNA sequencing analysis revealed a significant increase in the ligand R-spondin 1 (RSPO1) level by LGR5Δ5. Furthermore, LGR5Δ5 appears to be involved in the regulation of genes related to the cytoskeleton, extracellular matrix stiffness, and angiogenesis, while LGR5FL is associated with the regulation of collagens and histone proteins.},
}

Humans
*Receptors, G-Protein-Coupled/genetics/metabolism
HEK293 Cells
*Thrombospondins/genetics/metabolism
Wnt Signaling Pathway/genetics
Protein Isoforms/genetics/metabolism
CRISPR-Cas Systems
Cell Movement/genetics

@article {pmid39769084,
year = {2024},
author = {Bairqdar, A and Karitskaya, PE and Stepanov, GA},
title = {Expanding Horizons of CRISPR/Cas Technology: Clinical Advancements, Therapeutic Applications, and Challenges in Gene Therapy.},
journal = {International journal of molecular sciences},
volume = {25},
number = {24},
pages = {},
pmid = {39769084},
issn = {1422-0067},
support = {075-15-2021-1085//Ministry of Science and Higher Education of the Russian Federation/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; *Gene Editing/methods ; Clinical Trials as Topic ; Animals ; },
abstract = {CRISPR-Cas technology has transformed the field of gene editing, opening new possibilities for treatment of various genetic disorders. Recent years have seen a surge in clinical trials using CRISPR-Cas-based therapies. This review examines the current landscape of CRISPR-Cas implementation in clinical trials, with data from key registries, including the Australian New Zealand Clinical Trials Registry, the Chinese Clinical Trial Register, and ClinicalTrials.gov. Emphasis is placed on the mechanism of action of tested therapies, the delivery method, and the most recent findings of each clinical trial.},
}

Humans
*CRISPR-Cas Systems
*Genetic Therapy/methods
*Gene Editing/methods
Clinical Trials as Topic
Animals

@article {pmid39769020,
year = {2024},
author = {Sun, X and Fu, Q and Song, Y and Deng, X and Li, Y and Wu, K and Li, S and Fu, J},
title = {Research Progress and Prospects of Molecular Breeding in Bermudagrass (Cynodon dactylon).},
journal = {International journal of molecular sciences},
volume = {25},
number = {24},
pages = {},
pmid = {39769020},
issn = {1422-0067},
support = {2024LZGC00303//Key R & D Plan of Shandong Province/ ; },
mesh = {*Cynodon/genetics ; *Plant Breeding/methods ; Genomics/methods ; Gene Editing/methods ; Stress, Physiological ; Genome, Plant ; },
abstract = {Bermudagrass (Cynodon dactylon L.) is a warm-season grass species of significant ecological and economic importance. It is widely utilized in turf management and forage production due to its resilience to drought, salt, and other environmental stresses. Recent advancements in molecular breeding, particularly through genomics technology and gene editing, have enabled the efficient identification of key genes associated with stress tolerance and turf quality. The use of techniques such as overexpression and CRISPR/Cas has enhanced resistance to drought, salt, cold, and heat, while the application of molecular markers has accelerated the development of superior varieties. The integration of multi-omics, such as genomics, transcriptomics, and proteomics, provides deeper insights into the molecular mechanisms of bermudagrass, thereby improving breeding efficiency and precision. Additionally, artificial intelligence is emerging as a powerful tool for analyzing genomic data, predicting optimal trait combinations, and accelerating breeding processes. These technologies, when combined with traditional breeding methods, hold great potential for optimizing bermudagrass varieties for both turf and forage use. Future research will focus on further integrating these tools to address the challenges of breeding posed by climate change to breeding climate-resilient turf and forage crops.},
}

*Cynodon/genetics
*Plant Breeding/methods
Genomics/methods
Gene Editing/methods
Stress, Physiological
Genome, Plant

@article {pmid39768143,
year = {2024},
author = {Zhang, XH and Tang, FL and Trouten, AM and Morad, M},
title = {Attempts to Create Transgenic Mice Carrying the Q3924E Mutation in RyR2 Ca[2+] Binding Site.},
journal = {Cells},
volume = {13},
number = {24},
pages = {},
pmid = {39768143},
issn = {2073-4409},
support = {R01HL153504/GF/NIH HHS/United States ; },
mesh = {Animals ; *Ryanodine Receptor Calcium Release Channel/genetics/metabolism ; *Mice, Transgenic ; Mice ; *Calcium/metabolism ; *Myocytes, Cardiac/metabolism ; Binding Sites ; Humans ; Mutation/genetics ; Female ; Calcium Signaling/genetics ; CRISPR-Cas Systems/genetics ; Caffeine/pharmacology ; },
abstract = {Over 200 point mutations in the ryanodine receptor (RyR2) of the cardiac sarcoplasmic reticulum (SR) are known to be associated with cardiac arrhythmia. We have already reported on the calcium signaling phenotype of a point mutation in RyR2 Ca[2+] binding site Q3925E expressed in human stem-cell-derived cardiomyocytes (hiPSC-CMs) that was found to be lethal in a 9-year-old girl. CRISPR/Cas9-gene-edited mutant cardiomyocytes carrying the RyR2-Q3925E mutation exhibited a loss of calcium-induced calcium release (CICR) and caffeine-triggered calcium release but continued to beat arrhythmically without generating significant SR Ca[2+] release, consistent with a remodeling of the calcium signaling pathway. An RNAseq heat map confirmed significant changes in calcium-associated genes, supporting the possibility of remodeling. To determine the in situ cardiac phenotype in an animal model of this mutation, we generated a knock-in mouse model of RyR2-Q3924E+/- using the CRISPR/Cas9 technique. We obtained three homozygous and one chimera mice, but they all died before reaching 3 weeks of age, preventing the establishment of germline mutation transmission in their offspring. A histo-pathological analysis of the heart showed significant cardiac hypertrophy, suggesting the Q3924E-RyR2 mutation was lethal to the mice.},
}

Animals
*Ryanodine Receptor Calcium Release Channel/genetics/metabolism
*Mice, Transgenic
Mice
*Calcium/metabolism
*Myocytes, Cardiac/metabolism
Binding Sites
Humans
Mutation/genetics
Female
Calcium Signaling/genetics
CRISPR-Cas Systems/genetics
Caffeine/pharmacology

@article {pmid39766827,
year = {2024},
author = {Yang, R and Guo, H and Sun, J and Gui, T and Li, X and Qian, H and Chen, A},
title = {The ebony Gene in Silkworm Black Pupae Significantly Affects 30 K Proteins During the Pupal Stage.},
journal = {Genes},
volume = {15},
number = {12},
pages = {},
pmid = {39766827},
issn = {2073-4425},
support = {CARS-18-ZJ0101//China Agriculture Research System of MOF and MARA/ ; BE2020418//Key R & D plan of Jiangsu Province (Modern Agriculture)/ ; 2023-JC-YB-188//Basic Research Programs of the Shaanxi Provincial Science and Technology Department/ ; 2023-YBNY-134//Key Industrial Chain Projects of Shaanxi Provincial Government/ ; },
mesh = {Animals ; *Bombyx/genetics/growth & development/metabolism ; *Pupa/genetics/growth & development/metabolism ; *Insect Proteins/genetics/metabolism ; Gene Expression Regulation, Developmental ; Pigmentation/genetics ; CRISPR-Cas Systems ; },
abstract = {Background/Objectives: The body color and patterns of insects play important roles in foraging, evading predators, mating, thermoregulation, and environmental adaptation. During the rearing of the QiufengN silkworm strain, a mutant with black pupal cuticle (QiufengNBP) was discovered. Preliminary map-based cloning and sequence analysis indicated that the ebony gene might significantly influence the formation of the black pupa mutant and the expression of 30K proteins. This study aims to determine the function of the ebony gene and its effect on the expression of the 30K protein during the pupal stage; Methods and Results: We employed CRISPR/Cas9 gene-editing technology to knock out the ebony gene in the Nistari strain, resulting in individuals with black pupae, named Nistari Black Pupa (NisBP). This confirmed that the ebony gene plays a crucial role in black pupa formation. Two-dimensional electrophoresis (2-DE) analysis of the pupal cuticle of NisBP and its wild-type Nistari found that the ebony gene has a significant impact on the expression of 30K proteins, which are vital for embryonic development and serve as key storage proteins; Conclusions: This study is the first to demonstrate that the ebony gene affects the expression of 30K proteins, laying the foundation for further research on their functions and providing insights into the developmental mechanisms of silkworms.},
}

Animals
*Bombyx/genetics/growth & development/metabolism
*Pupa/genetics/growth & development/metabolism
*Insect Proteins/genetics/metabolism
Gene Expression Regulation, Developmental
Pigmentation/genetics
CRISPR-Cas Systems

@article {pmid39766530,
year = {2024},
author = {de la Fuente Tagarro, C and Martín-González, D and De Lucas, A and Bordel, S and Santos-Beneit, F},
title = {Current Knowledge on CRISPR Strategies Against Antimicrobial-Resistant Bacteria.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {13},
number = {12},
pages = {},
pmid = {39766530},
issn = {2079-6382},
abstract = {CRISPR/Cas systems have emerged as valuable tools to approach the problem of antimicrobial resistance by either sensitizing or lysing resistant bacteria or by aiding in antibiotic development, with successful applications across diverse organisms, including bacteria and fungi. CRISPR/Cas systems can target plasmids or the bacterial chromosome of AMR-bacteria, and it is especially necessary to have an efficient entry into the target cells, which can be achieved through nanoparticles or bacteriophages. Regarding antibiotic development and production, though the use of CRISPR/Cas in this field is still modest, there is an untapped reservoir of bacterial and fungal natural products, with over 95% yet to be characterized. In Streptomyces, a key antibiotic-producing bacterial genus, CRISPR/Cas has been successfully used to activate silent biosynthetic gene clusters, leading to the discovery of new antibiotics. CRISPR/Cas is also applicable to non-model bacteria and different species of fungi, making it a versatile tool for natural products discovery. Moreover, CRISPR/Cas-based studies offer insights into metabolic regulation and biosynthetic pathways in both bacteria and fungi, highlighting its utility in understanding genetic regulation and improving industrial strains. In this work, we review ongoing innovations on ways to treat antimicrobial resistances and on antibiotic discovery using CRISPR/Cas platforms, highlighting the role of bacteria and fungi in these processes.},
}

@article {pmid39766302,
year = {2024},
author = {Middlezong, W and Stinnett, V and Phan, M and Phan, B and Morsberger, L and Klausner, M and Ghabrial, J and DeMetrick, N and Zhu, J and James, T and Pallavajjala, A and Gocke, CD and Baer, MR and Zou, YS},
title = {Rapid Detection of PML::RARA Fusions in Acute Promyelocytic Leukemia: CRISPR/Cas9 Nanopore Sequencing with Adaptive Sampling.},
journal = {Biomolecules},
volume = {14},
number = {12},
pages = {},
pmid = {39766302},
issn = {2218-273X},
support = {ATIP grant//Johns Hopkins Institute for Clinical and Translational Research (ICTR)/ ; },
mesh = {Humans ; *Leukemia, Promyelocytic, Acute/genetics/diagnosis ; *Oncogene Proteins, Fusion/genetics ; *Nanopore Sequencing/methods ; *CRISPR-Cas Systems/genetics ; Retinoic Acid Receptor alpha/genetics ; Promyelocytic Leukemia Protein/genetics ; },
abstract = {Acute promyelocytic leukemia (APL) accounts for approximately 10-15% of newly diagnosed acute myeloid leukemia cases and presents with coagulopathy and bleeding. Prompt diagnosis and treatment are required to minimize early mortality in APL as initiation of all-trans retinoic acid therapy rapidly reverses coagulopathy. The PML::RARA fusion is a hallmark of APL and its rapid identification is essential for rapid initiation of specific treatment to prevent early deaths from coagulopathy and bleeding and optimize patient outcomes. Given limitations and long turnaround time of current gene fusion diagnostic strategies, we have developed a novel amplification-free nanopore sequencing-based approach with low cost, easy setup, and fast turnaround time. We termed the approach CRISPR/Cas9-enriched nanopore sequencing with adaptive sampling (CENAS). Using CENAS, we successfully sequenced breakpoints of typical and atypical PML::RARA fusions in APL patients. Compared with the standard-of-care genetic diagnostic tests, CENAS achieved good concordance in detecting PML::RARA fusions in this study. CENAS allowed for the identification of sequence information of fusion breakpoints involved in typical and atypical PML::RARA fusions and identified additional genes (ANKFN1 and JOSD1) and genomic regions (13q14.13) involving the atypical fusions. To the best of our knowledge, involvements of the ANKFN1 gene, the JOSD1 gene, and the 13q14.13 genomic region flanking with the SIAH3 and ZC3H13 genes have not been reported in the atypical PML::RARA fusions. CENAS has great potential to develop as a point-of-care test enabling immediate, low-cost bedside diagnosis of APL patients with a PML::RARA fusion. Given the early death rate in APL patients still reaches 15%, and ~10% of APL patients are resistant to initial therapy or prone to relapse, further sequencing studies of typical and atypical PML::RARA fusion might shed light on the pathophysiology of the disease and its responsiveness to treatment. Understanding the involvement of additional genes and positional effects related to the PML and RARA genes could shed light on their role in APL and may aid in the development of novel targeted therapies.},
}

Humans
*Leukemia, Promyelocytic, Acute/genetics/diagnosis
*Oncogene Proteins, Fusion/genetics
*Nanopore Sequencing/methods
*CRISPR-Cas Systems/genetics
Retinoic Acid Receptor alpha/genetics
Promyelocytic Leukemia Protein/genetics

@article {pmid39766265,
year = {2024},
author = {Hirose, J and Aizawa, E and Yamamoto, S and Xu, M and Iwai, S and Suzuki, K},
title = {Lipid Nanoparticles Enable Efficient In Vivo DNA Knock-In via HITI-Mediated Genome Editing.},
journal = {Biomolecules},
volume = {14},
number = {12},
pages = {},
pmid = {39766265},
issn = {2218-273X},
support = {21H04811//The Japan Society for the Promotion of Science KAKENHI/ ; 22K19405//The Japan Society for the Promotion of Science KAKENHI/ ; JP23ek0109521//Japan Agency for Medical Research and Development/ ; N.A.//Daiichi Sankyo Foundation of Life Science/ ; N.A.//Osaka University Honors Program for Graduate Schools in Science, Engineering and Informatics/ ; JPMXP1224 OS1059//Ministry of Education, Culture, Sports, Science and Technology/ ; },
mesh = {*Gene Editing/methods ; Animals ; *Nanoparticles/chemistry ; Mice ; *Gene Knock-In Techniques ; *Lipids/chemistry ; *DNA/genetics/administration & dosage ; Humans ; Liver/metabolism ; CRISPR-Cas Systems ; Green Fluorescent Proteins/genetics/metabolism ; Liposomes ; },
abstract = {In vivo genome editing holds great therapeutic potential for treating monogenic diseases by enabling precise gene correction or addition. However, improving the efficiency of delivery systems remains a key challenge. In this study, we investigated the use of lipid nanoparticles (LNPs) for in vivo knock-in of ectopic DNA. Our in vitro experiments demonstrated that the homology-independent targeted integration (HITI)-mediated genome-editing method achieved significantly higher knock-in efficiency at the Alb locus in hepatic cells compared to the traditional homology-directed repair (HDR)-mediated approach. By optimizing LNP composition and administration routes, we successfully achieved HITI-mediated GFP knock-in (2.1-2.7%) in the livers of mice through intravenous delivery of LNP-loaded genome editing components. Notably, repeated intravenous dosing led to a twofold increase in liver GFP knock-in efficiency (4.3-7.0%) compared to a single dose, highlighting the potential for cumulative genome editing effects. These findings provide a solid foundation for the use of LNPs in in vivo knock-in strategies, paving the way for future genome-editing therapies.},
}

*Gene Editing/methods
Animals
*Nanoparticles/chemistry
Mice
*Gene Knock-In Techniques
*Lipids/chemistry
*DNA/genetics/administration & dosage
Humans
Liver/metabolism
CRISPR-Cas Systems
Green Fluorescent Proteins/genetics/metabolism
Liposomes

@article {pmid39766243,
year = {2024},
author = {Lopez-Barbera, A and Abasolo, N and Torrell, H and Canela, N and Fernández-Arroyo, S},
title = {Integrative Transcriptomic and Target Metabolite Analysis as a New Tool for Designing Metabolic Engineering in Yeast.},
journal = {Biomolecules},
volume = {14},
number = {12},
pages = {},
pmid = {39766243},
issn = {2218-273X},
support = {ACCIÓ-Eurecat TRAÇA 2023-FEREMIC//Agencia per a la Competitivit de l'Empresa/ ; SGR01275-IOMICS//Agència de Gestió d'Ajuts Universitaris i de Recerca/ ; Vicente López to A.L-B.//Fundació Eurecat/ ; },
mesh = {*Metabolic Engineering/methods ; *Saccharomyces cerevisiae/metabolism/genetics ; *Transcriptome/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Gene Expression Profiling/methods ; Terpenes/metabolism ; Metabolomics/methods ; },
abstract = {Precision fermentation processes, especially when using edited microorganisms, demand accuracy in the bioengineering process to maximize the desired outcome and to avoid adverse effects. The selection of target sites to edit using CRISPR/Cas9 can be complex, resulting in non-controlled consequences. Therefore, the use of multi-omics strategies can help in the design, selection and efficiency of genetic editing. In this study, we present a multi-omics approach based on targeted metabolite analysis and transcriptomics for the designing of CRISPR/Cas9 in baker's yeast as a more efficient strategy to select editing regions. Multi-omics shows potential to reveal new metabolic bottlenecks and to elucidate new metabolic fluxes, which could be a key factor in minimizing the metabolic burden in edited microorganisms. In our model, we focus our attention on the isoprenoid synthesis due to their industrial interest. Targeted metabolite detection combined with a transcriptomic analysis revealed hydroxymethylglutaryl-CoA reductases (HMGs) as the best target gene to induce an increase in isoprenoid synthesis. Thus, an extra copy of HMG1 was introduced using, for the first time, the synthetic UADH1 promoter. The multi-omics analysis of the recombinant strain results in an accurate assessment of yeast behavior during the most important growth phases, highlighting the metabolic burden, Crabtree effect or the diauxic shift during culture.},
}

*Metabolic Engineering/methods
*Saccharomyces cerevisiae/metabolism/genetics
*Transcriptome/genetics
*CRISPR-Cas Systems/genetics
Gene Editing/methods
Gene Expression Profiling/methods
Terpenes/metabolism
Metabolomics/methods

@article {pmid39764496,
year = {2025},
author = {Tamaki, M and Chiku, T and Suzuki, S and Misaki, A and Funakubo, A and Matsushima, Y and Yokota, K and Igimi, S and Kajikawa, A},
title = {Application of the SpCas9 inhibitor BRD0539 for CRISPR/Cas9-based genetic tools in Lacticaseibacillus paracasei.},
journal = {Bioscience of microbiota, food and health},
volume = {44},
number = {1},
pages = {70-79},
pmid = {39764496},
issn = {2186-6953},
abstract = {Although the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has been extensively developed since its discovery for eukaryotic and prokaryotic genome editing and other genetic manipulations, there are still areas warranting improvement, especially regarding bacteria. In this study, BRD0539, a small-molecule inhibitor of Streptococcus pyogenes Cas9 (SpCas9), was used to suppress the activity of the nuclease during genetic modification of Lacticaseibacillus paracasei, as well as to regulate CRISPR interference (CRISPRi). First, we developed and validated a CRISPR-SpCas9 system targeting the sirA gene of L. paracasei. Then BRD0539 was used for CRISPR-dependent DNA cleavage in vivo. Our results suggested that the inhibitor worked partially in both Escherichia coli and L. paracasei. Next, we designed a CRISPRi system in a L. paracasei strain by inserting an inactive SpCas9 gene into the chromosome and introducing a plasmid encoding for a single guide RNA (sgRNA) targeting the sirA gene. Expression of sirA was successfully inhibited in the recombinant strains, and CRISPRi was abolished in an inhibitor-dependent manner. Our findings may help expand the CRISPR toolbox for research on lactic acid bacteria and other microbes.},
}

@article {pmid39762743,
year = {2025},
author = {Shelenkov, A and Slavokhotova, A and Mikhaylova, Y and Akimkin, V},
title = {Genomic typing, antimicrobial resistance gene, virulence factor and plasmid replicon database for the important pathogenic bacteria Klebsiella pneumoniae.},
journal = {BMC microbiology},
volume = {25},
number = {1},
pages = {3},
pmid = {39762743},
issn = {1471-2180},
support = {075-15-2019-1666//The Ministry of Science and Higher Education of the Russian Federation/ ; 075-15-2019-1666//The Ministry of Science and Higher Education of the Russian Federation/ ; 075-15-2019-1666//The Ministry of Science and Higher Education of the Russian Federation/ ; 075-15-2019-1666//The Ministry of Science and Higher Education of the Russian Federation/ ; },
mesh = {*Klebsiella pneumoniae/genetics/drug effects/pathogenicity ; *Plasmids/genetics ; *Virulence Factors/genetics ; *Genome, Bacterial/genetics ; Humans ; *Drug Resistance, Bacterial/genetics ; Klebsiella Infections/microbiology/epidemiology ; Replicon/genetics ; Multilocus Sequence Typing ; Anti-Bacterial Agents/pharmacology ; Databases, Genetic ; Genomics ; },
abstract = {BACKGROUND: The infections of bacterial origin represent a significant problem to the public healthcare worldwide both in clinical and community settings. Recent decade was marked by limiting treatment options for bacterial infections due to growing antimicrobial resistance (AMR) acquired and transferred by various bacterial species, especially the ones causing healthcare-associated infections, which has become a dangerous issue noticed by the World Health Organization. Numerous reports shown that the spread of AMR is often driven by several species-specific lineages usually called the 'global clones of high risk'. Thus, it is essential to track the isolates belonging to such clones and investigate the mechanisms of their pathogenicity and AMR acquisition. Currently, the whole genome-based analysis is more and more often used for these purposes, including the epidemiological surveillance and analysis of mobile elements involved in resistance transfer. However, in spite of the exponential growth of available bacterial genomes, their representation usually lack uniformity and availability of supporting metadata, which creates a bottleneck for such investigations.

DESCRIPTION: In this database, we provide the results of a thorough genomic analysis of 61,857 genomes of a highly dangerous bacterial pathogen Klebsiella pneumoniae. Important isolate typing information including multilocus sequence typing (MLST) types (STs), assignment of the isolates to known global clones, capsular (KL) and lipooligosaccharide (O) types, the presence of CRISPR-Cas systems, and cgMLST profiles are given, and the information regarding the presence of AMR, virulence genes and plasmid replicons within the genomes is provided.

CONCLUSION: This database is freely available under CC BY-NC-SA at https://doi.org/10.5281/zenodo.11069018 . The database will facilitate selection of the proper reference isolate sets for any types of genome-based investigations. It will be helpful for investigations in the field of K. pneumoniae genomic epidemiology, as well as antimicrobial resistance analysis and the development of prevention measures against this important pathogen.},
}

*Klebsiella pneumoniae/genetics/drug effects/pathogenicity
*Plasmids/genetics
*Virulence Factors/genetics
*Genome, Bacterial/genetics
Humans
*Drug Resistance, Bacterial/genetics
Klebsiella Infections/microbiology/epidemiology
Replicon/genetics
Multilocus Sequence Typing
Anti-Bacterial Agents/pharmacology
Databases, Genetic
Genomics