@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.<br><br>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&#xa0;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.<br><br>RESULTS: CRISPR/CasRx system can significantly reduce HTT mRNA levels across various models, including HEK 293&#xa0;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.<br><br>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.}, }
@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.}, }
@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 &amp; 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.}, }
@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.}, }
@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 &amp; 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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.<br><br>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&#xa0;&#xd7;&#xa0;10[-3] and 3.820&#xa0;&#xd7;&#xa0;10[-3] U/mL, respectively, which were lower than those of most reported fluorescence methods.<br><br>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.}, }
@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 &amp; 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.}, }
@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 &amp; dosage ; *Neoplasms/therapy/immunology/genetics ; *mRNA Vaccines ; *Immunotherapy/methods ; RNA, Messenger/administration &amp; 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.}, }
@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 &amp; Human Services | National Institutes of Health (NIH)/ ; RM1HG009490//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; P30CA072720//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; T32HG003284//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; T32GM007388//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; F31HD113443//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01-GM076275//U.S. Department of Health &amp; 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.}, }
@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.}, }
@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 &amp; 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.}, }
@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.}, }
@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.}, }
@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 &amp; 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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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 &amp; 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.<br><br>OBJECTIVE: This review aims to highlight advancements in molecular diagnostics that address these limitations.<br><br>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.<br><br>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.<br><br>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.}, }
@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 &amp; 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.}, }
@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.}, }
@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 &amp; 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.<br><br>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.<br><br>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.}, }
@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.}, }
@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.<br><br>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.<br><br>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.}, }
@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.<br><br>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.<br><br>RESULTS: The T. gondiirop41 gene knockout strain (RH &#x394;ku80&#x394;rop41) 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.<br><br>CONCLUSIONS: The T. gondii RH &#x394;ku80&#x394;rop41 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.}, }
@article {pmid39788098, year = {2025}, author = {Lin, J and Bhoobalan-Chitty, Y and Peng, X}, title = {Cad1 turns ATP into phage poison.}, journal = {Cell host &amp; 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.}, }
@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.}, }
@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 &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 1R01NS123556-01A1//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 5R01GM141296//U.S. Department of Health &amp; 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.}, }
@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 &#xdc;niversitesi/ ; }, 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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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.}, }
@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//Coordena&#xe7;&#xe3;o de Aperfeicoamento de Pessoal de N&#xed;vel Superior/ ; VPGDI 024 INT 22//Funda&#xe7;&#xe3;o Oswaldo Cruz/ ; 001//Funda&#xe7;&#xe3;o Carlos Chagas Filho de Amparo &#xe0; 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.}, }
@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 &amp; 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.}, }
@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 &amp; 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.}, }
@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.}, }
@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.}, }
@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.}, }
@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 &amp; 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.}, }
@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.}, }
@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 &amp; 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 &amp; development/metabolism ; *Pupa/genetics/growth &amp; 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.}, }
@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.}, }
@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 &amp; 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.}, }
@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 = {ACCI&#xd3;-Eurecat TRA&#xc7;A 2023-FEREMIC//Agencia per a la Competitivit de l'Empresa/ ; SGR01275-IOMICS//Ag&#xe8;ncia de Gesti&#xf3; d'Ajuts Universitaris i de Recerca/ ; Vicente L&#xf3;pez to A.L-B.//Fundaci&#xf3; 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.}, }
@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.<br><br>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.<br><br>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.}, }
@article {pmid39762363, year = {2025}, author = {Kang, GH and Ko, Y and Lee, JM}, title = {Enhancing virus-mediated genome editing for cultivated tomato through low temperature.}, journal = {Plant cell reports}, volume = {44}, number = {1}, pages = {22}, pmid = {39762363}, issn = {1432-203X}, support = {RS-2024-00322217//New Breeding Technologies Development Program, Rural Development Administration/ ; 2021R1A2C2093789//National Research Foundation of Korea/ ; }, mesh = {*Solanum lycopersicum/genetics/virology ; *Gene Editing/methods ; *Cold Temperature ; *Plant Viruses/genetics ; Potexvirus/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; Genetic Vectors/genetics ; Genome, Plant/genetics ; Secoviridae/genetics ; Plant Breeding/methods ; Oxidoreductases/genetics ; Plant Leaves/genetics/virology ; }, abstract = {Viral vector-mediated gene editing is enhanced for cultivated tomato under low temperature conditions, enabling higher mutation rates, heritable, and virus-free gene editing for efficient breeding. The CRISPR/Cas system, a versatile gene-editing tool, has revolutionized plant breeding by enabling precise genetic modifications. The development of robust and efficient genome-editing tools for crops is crucial for their application in plant breeding. In this study, we highly improved virus-induced genome-editing (VIGE) system for cultivated tomato. Vectors of tobacco rattle virus (TRV) and potato virus X (PVX) were used to deliver sgRNA targeting phytoene desaturase (SlPDS), along with mobile RNA sequences of tFT or tRNA[Ileu], into Cas9-overexpressing cultivated tomato (S. lycopersicum cv. Moneymaker). Our results demonstrate that low temperature significantly enhanced viral vector-mediated gene editing efficiency in both cotyledons and systemic upper leaves. However, no mutant progeny was obtained from TRV- and PVX301-infected MM-Cas9 plants. To address this challenge, we employed tissue culture techniques and found that low-temperature incubations at the initiation stage of tissue culture lead to enhanced editing efficiency in both vectors, resulting in a higher mutation rate (> 70%) of SlPDS in regenerated plants. Heritable gene-edited and virus-free progenies were successfully identified. This study presents a straightforward approach to enhance VIGE efficiency and the expeditious production of gene-edited lines in tomato breeding.}, }
@article {pmid39762235, year = {2025}, author = {Hollingsworth, EW and Liu, TA and Alcantara, JA and Chen, CX and Jacinto, SH and Kvon, EZ}, title = {Rapid and quantitative functional interrogation of human enhancer variant activity in live mice.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {409}, pmid = {39762235}, issn = {2041-1723}, support = {T32 GM008620/GM/NIGMS NIH HHS/United States ; S10 OD021718/OD/NIH HHS/United States ; T32 NS082174/NS/NINDS NIH HHS/United States ; F30 HD110233/HD/NICHD NIH HHS/United States ; S10 OD010794/OD/NIH HHS/United States ; P30 CA062203/CA/NCI NIH HHS/United States ; S10 RR025496/RR/NCRR NIH HHS/United States ; R01 HD115268/HD/NICHD NIH HHS/United States ; }, mesh = {Animals ; *Enhancer Elements, Genetic/genetics ; Mice ; Humans ; *Otx Transcription Factors/genetics/metabolism ; MicroRNAs/genetics/metabolism ; Single-Cell Analysis ; Autism Spectrum Disorder/genetics/metabolism ; Craniofacial Abnormalities/genetics ; Genes, Reporter ; Female ; CRISPR-Cas Systems ; Genetic Variation ; Brain/metabolism ; Male ; Alleles ; }, abstract = {Functional analysis of non-coding variants associated with congenital disorders remains challenging due to the lack of efficient in vivo models. Here we introduce dual-enSERT, a robust Cas9-based two-color fluorescent reporter system which enables rapid, quantitative comparison of enhancer allele activities in live mice in less than two weeks. We use this technology to examine and measure the gain- and loss-of-function effects of enhancer variants previously linked to limb polydactyly, autism spectrum disorder, and craniofacial malformation. By combining dual-enSERT with single-cell transcriptomics, we characterise gene expression in cells where the enhancer is normally and ectopically active, revealing candidate pathways that may lead to enhancer misregulation. Finally, we demonstrate the widespread utility of dual-enSERT by testing the effects of fifteen previously uncharacterised rare and common non-coding variants linked to neurodevelopmental disorders. In doing so we identify variants that reproducibly alter the in vivo activity of OTX2 and MIR9-2 brain enhancers, implicating them in autism. Dual-enSERT thus allows researchers to go from identifying candidate enhancer variants to analysis of comparative enhancer activity in live embryos in under two weeks.}, }
@article {pmid39761113, year = {2025}, author = {Kardailsky, A and Durán-Vinet, B and Nester, G and Ayad, ME and Raes, EJ and Jeunen, GJ and Miller, AK and McVey, P and Corrigan, S and Fraser, M and Goncalves, P and Burnell, S and Bennett, A and Rauschert, S and Bayer, PE}, title = {Monitoring the Land and Sea: Enhancing Efficiency Through CRISPR-Cas Driven Depletion and Enrichment of Environmental DNA.}, journal = {The CRISPR journal}, volume = {}, number = {}, pages = {}, doi = {10.1089/crispr.2024.0050}, pmid = {39761113}, issn = {2573-1602}, abstract = {Characterizing biodiversity using environmental DNA (eDNA) represents a paradigm shift in our capacity for biomonitoring complex environments, both aquatic and terrestrial. However, eDNA biomonitoring is limited by biases toward certain species and the low taxonomic resolution of current metabarcoding approaches. Shotgun metagenomics of eDNA enables the collection of whole ecosystem data by sequencing all molecules present, allowing characterization and identification. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated proteins (Cas)-based methods have the potential to improve the efficiency of eDNA metagenomic sequencing of low-abundant target organisms and simplify data analysis by enrichment of target species or nontarget DNA depletion before sequencing. Implementation of CRISPR-Cas in eDNA has been limited due to a lack of interest and support in the past. This perspective synthesizes current approaches of CRISPR-Cas to study underrepresented taxa and advocate for further application and optimization of depletion and enrichment methods of eDNA using CRISPR-Cas, holding promise for eDNA biomonitoring.}, }
@article {pmid39760503, year = {2025}, author = {Zhu, Z and Lu, S and Wang, H and Wang, F and Xu, W and Zhu, Y and Xue, J and Yang, L}, title = {Innovations in Transgene Integration Analysis: A Comprehensive Review of Enrichment and Sequencing Strategies in Biotechnology.}, journal = {ACS applied materials &amp; interfaces}, volume = {}, number = {}, pages = {}, doi = {10.1021/acsami.4c14208}, pmid = {39760503}, issn = {1944-8252}, abstract = {Understanding the integration of transgene DNA (T-DNA) in transgenic crops, animals, and clinical applications is paramount for ensuring the stability and expression of inserted genes, which directly influence desired traits and therapeutic outcomes. Analyzing T-DNA integration patterns is essential for identifying potential unintended effects and evaluating the safety and environmental implications of genetically modified organisms (GMOs). This knowledge is crucial for regulatory compliance and fostering public trust in biotechnology by demonstrating transparency in genetic modifications. This review highlights recent advancements in T-DNA integration analysis, specifically focusing on targeted DNA enrichment and sequencing strategies. We examine key technologies, such as polymerase chain reaction (PCR)-based methods, hybridization capture, RNA/DNA-guided endonuclease-mediated enrichment, and high-throughput resequencing, emphasizing their contributions to enhancing precision and efficiency in transgene integration analysis. We discuss the principles, applications, and recent developments in these techniques, underscoring their critical role in advancing biotechnological products. Additionally, we address the existing challenges and future directions in the field, offering a comprehensive overview of how innovative DNA-targeted enrichment and sequencing strategies are reshaping biotechnology and genomics.}, }
@article {pmid39760089, year = {2024}, author = {Li, K and Luo, T and Zhang, Y and Li, C and Chen, H and Xia, C and Gao, C}, title = {Rapid detection of Mycoplasma hyopneumoniae by recombinase-aided amplification combined with the CRISPR/Cas12a system.}, journal = {Frontiers in cellular and infection microbiology}, volume = {14}, number = {}, pages = {1469558}, pmid = {39760089}, issn = {2235-2988}, mesh = {*Mycoplasma hyopneumoniae/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; Animals ; Swine ; *Nucleic Acid Amplification Techniques/methods ; *Pneumonia of Swine, Mycoplasmal/diagnosis/microbiology ; *Recombinases/metabolism/genetics ; Sensitivity and Specificity ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Molecular Diagnostic Techniques/methods ; Limit of Detection ; }, abstract = {Mycoplasma hyopneumoniae (M. hyopneumoniae) is one of the primary agents involved in porcine respiratory disease complex, and circulates in the swine industry worldwide. The prevention and control of M. hyopneumoniae is complicated. Thus, a recombinase-aided amplification (RAA) assay coupled with the clustered regularly-interspaced short palindromic repeats (CRISPR)/Cas12a system was established for the detection of M. hyopneumoniae. The most suitable primer pairs and CRISPR RNA (crRNA) were screened and selected for the RAA-CRISPR/Cas12a detection system. We have achieved a detection limit of 1 copy/µL and 5 copies/µL per reaction for the RAA-CRISPR/Cas12a-fluorescence assay and RAA-CRISPR/Cas12a-lateral flow assay (LFA), respectively. Furthermore, the RAA-CRISPR/Cas12a system displayed no cross-reactivity with other respiratory pathogens. The performance of the RAA-CRISPR/Cas12a system was compared with PCR as recommended by the Chinese national standard (GB/T 35909-2018) and qPCR as recommended by the Chinese entry-exit inspection and quarantine industry standard (SN/T4104-2015) for clinical samples, and good consistency with these methods was observed. Above all, the methods shed a light on the convenient, portable, visual, highly sensitive and specific detection of M. hyopneumoniae, demonstrating a great application potential for on-site monitoring of M. hyopneumoniae in the field.}, }
@article {pmid39759869, year = {2024}, author = {Gong, Z and Wang, W and Zhao, Y and Wang, Y and Sun, R and Zhang, H and Wang, F and Lu, Y and Zhang, J}, title = {Analysis of the pathogenicity and pathological characteristics of NOTCH3 gene-sparing cysteine mutations in vitro and in vivo models.}, journal = {Frontiers in molecular neuroscience}, volume = {17}, number = {}, pages = {1391040}, pmid = {39759869}, issn = {1662-5099}, abstract = {BACKGROUND: Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is one of the most common inherited cerebral small vessel diseases caused by the NOTCH3 gene mutation. This mutation leads to the accumulation of NOTCH3 extracellular domain protein (NOTCH3[ECD]) into the cerebral arterioles, causing recurrent stroke, white matter lesions, and cognitive impairment. With the development of gene sequencing technology, cysteine-sparing mutations can also cause CADASIL disease, however, the pathogenicity and pathogenic mechanisms of cysteine-sparing mutations remain controversial.<br><br>OBJECTIVE: To analyze the pathogenicity and pathological features of cysteine-sparing mutations in both in vitro and in vivo mouse models.<br><br>METHODS: A cysteine-sparing mutant of NOTCH3[ECD] R75Q was constructed by lentiviral transfection in vitro, and the NOTCH3 R75Q knock-in mouse model was constructed by CRISPR/Cas-mediated genome engineering in vivo. A cycloheximide pulse-chase experiment was used to analyze the degradation of NOTCH3 extracellular domain proteins, and the deposition characteristics of NOTCH3[ECD] were quantitatively analyzed by immunohistochemical staining. The characteristics of the smooth muscle cells and granular osmiophilic materials were observed using electron microscopy.<br><br>RESULTS: We elucidated that the NOTCH3 R75Q mutation is pathogenic. NOTCH3[ECD] R75Q was found to be resistant to protein degradation and more likely to cause abnormal aggregation of NOTCH3[ECD], resulting in reduced cell activity in vitro. The NOTCH3 R75Q mouse model showed pathological characteristics of CADASIL, with age-dependent NOTCH3[ECD], granular osmiophilic material, and degenerated smooth muscle cells detected in the brain.<br><br>CONCLUSION: To our knowledge, this is the first study to analyze the pathogenicity of NOTCH3 R75Q cysteine-sparing mutations in both in vitro and in vivo models. We demonstrate that NOTCH3[ECD] induced by NOTCH3 R75Q mutation has toxic effects on cells and reveal the deposition characteristics of NOTCH3[ECD] in the brain. This provides a feasible model and lays the foundation for further studies on the pathogenesis and therapeutic strategies of NOTCH3 cysteine-sparing mutations.}, }
@article {pmid39758988, year = {2024}, author = {Wu, X and Xiang, R and Yang, D and He, X and Zhu, L and Sun, F and Li, H and Pi, N and Li, Y}, title = {HRP-integrated CRISPR-Cas12a biosensor for rapid point-of-care detection of Langya henipavirus.}, journal = {iScience}, volume = {27}, number = {12}, pages = {111466}, pmid = {39758988}, issn = {2589-0042}, abstract = {Global pandemic has emphasized the needs for advanced pathogen diagnosis in dealing with newly emerged infectious threats, including the Langya henipavirus (LayV). LayV, as an emerging zoonotic pathogen, has potential to cause pandemic, but lacks of rapid diagnostic tools, particularly at point-of-care level. Here, we leveraged the merits of CRISPR-Cas12a biosensing and established a highly sensitive LayV detection method. By integrating CRISPR-Cas12a with RPA, 10 copies/μL ultra-sensitive LayV RNA detection has been achieved at room temperature within 30 min. More importantly, this study developed a special horseradish peroxidase (HRP)-single-stranded DNA (ssDNA) reporter enabling CRISPR-Cas12a to detect LayV RNA without pre-amplification, achieving a sensitivity of 1,200 copies/μL detectable by the naked eye. These explorations can serve as accelerator for CRISPR-Cas biosensing toward rapid response for newly emerged biological threats, and also provide a method to realize simple, precise, and amplicon-free point-of-care pathogen screening for resource limited or underdevelopment regions.}, }
@article {pmid39756423, year = {2025}, author = {Haeusser, LA and Becker, H and Kuhlburger, L and Zago, M and Walter, B and Tsiami, F and Erdmann, S and Trampert, J and Surender, S and Stahl, A and Templin, M and Wegner, E and Schmidt, T and Schmees, C and Casadei, N and Sevenich, L and Claassen, M and Nahnsen, S and Beck, S and Merk, DJ and Tabatabai, G}, title = {Genome-wide CRISPR-Cas 9 screens identify BCL family members as modulators of response to regorafenib in experimental glioma.}, journal = {Neuro-oncology}, volume = {}, number = {}, pages = {}, doi = {10.1093/neuonc/noae278}, pmid = {39756423}, issn = {1523-5866}, abstract = {BACKGROUND: Registered systemic treatment options for glioblastoma patients are limited. The phase II REGOMA trial suggested an improvement of median overall survival in progressive glioblastoma by the multi-tyrosine kinase inhibitor regorafenib. This has not been confirmed by GBM AGILE. So far, regorafenib has been administered as monotherapy or as an addition to standard of care in newly diagnosed glioblastoma. Rational combination therapies involving regorafenib might be a reasonable strategy. Here, we aimed at identifying functionally-instructed combination therapies involving regorafenib.<br><br>METHODS: We applied a genome-wide CRISPR-Cas9-based functional genomics target discovery approach using activation and knockout screens followed by genetic, pharmacological, functional validations. Regorafenib-induced molecular alterations were assessed by RNAsequencing and DigiWest. We investigated selected functionally-instructed combination therapies in three orthotopic glioma mouse models in vivo (syngeneic SMA560/VM/Dk model and two xenograft models) and performed immunohistochemistry of post-treatment brains.<br><br>RESULTS: We identified potential modifiers of regorafenib response including BCL2, BCL2L1, ITGB3, FOXC1, SERAC1, ARAF, and PLCE1. The combination of regorafenib with Bcl-2/Bcl-xL inhibition was superior to both monotherapies alone in vitro, ex vivo and in vivo. We identified regorafenib-induced regulations of the Bcl-2 downstream target chemokine receptor 1 (CCR1) as one potential underlying molecular mediator. Furthermore, regorafenib led to changes in the myeloid compartment of the glioma-associated microenvironment.<br><br>CONCLUSION: This preclinical study uses a functional genomics-based target discovery approach with subsequent validations involving regorafenib. It serves as a biological rationale for clinical translation. Particularly, an investigation of the combination of regorafenib plus navitoclax within a clinical trial is warranted.}, }
@article {pmid39755699, year = {2025}, author = {Halegua, T and Risson, V and Carras, J and Rouyer, M and Coudert, L and Jacquier, A and Schaeffer, L and Ohlmann, T and Mangeot, PE}, title = {Delivery of Prime editing in human stem cells using pseudoviral NanoScribes particles.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {397}, pmid = {39755699}, issn = {2041-1723}, mesh = {Humans ; *Gene Editing/methods ; HEK293 Cells ; CRISPR-Cas Systems ; Induced Pluripotent Stem Cells/metabolism/cytology ; Ribonucleoproteins/metabolism/genetics ; Hematopoietic Stem Cells/metabolism/cytology/virology ; Myoblasts/metabolism/cytology ; Virion/metabolism/genetics ; Stem Cells/metabolism/cytology ; }, abstract = {Prime Editing can rewrite genes in living cells by allowing point mutations, deletions, or insertion of small DNA sequences with high precision. However, its safe and efficient delivery into human stem cells remains a technical challenge. In this report, we engineer Nanoscribes, virus-like particles that encapsidate ribonucleoprotein complexes of the Prime Editing system and allow their delivery into recipient cells. We identify key features that unlock the potential of Nanoscribes, including the use of multiple fusogens, the improvement of pegRNAs structures, their encoding by a Pol II system and the optimization of Prime-Editors. Nanoscribes edit HEK293T with an efficiency of 68% at the HEK3 locus with increased fidelity over DNA-transfection and support pegRNA-multiplexing. Importantly, Nanoscribes permit editing of myoblasts, hiPSCs and hiPSCs-derived hematopoietic stem cells with an editing efficiency up to 25%. Nanoscribes is an asset for development of next generation genome editing approaches using VLPs.}, }
@article {pmid39754035, year = {2025}, author = {Xiang, T and Feng, H and Xing, XH and Zhang, C}, title = {GLiDe: a web-based genome-scale CRISPRi sgRNA design tool for prokaryotes.}, journal = {BMC bioinformatics}, volume = {26}, number = {1}, pages = {1}, pmid = {39754035}, issn = {1471-2105}, support = {21938004//National Natural Science Foundation of China/ ; U2032210//National Natural Science Foundation of China/ ; 2023YFC3402300//National Key Research and Development Program of China/ ; }, mesh = {*Internet ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/genetics ; Software ; Prokaryotic Cells/metabolism ; Genomics/methods ; Genome, Bacterial ; }, abstract = {BACKGROUND: CRISPRi screening has become a powerful approach for functional genomic research. However, the off-target effects resulting from the mismatch tolerance between sgRNAs and their intended targets is a primary concern in CRISPRi applications.<br><br>RESULTS: We introduce Guide Library Designer (GLiDe), a web-based tool specifically created for the genome-scale design of sgRNA libraries tailored for CRISPRi screening in prokaryotic organisms. GLiDe incorporates a robust quality control framework, rooted in prior experimental knowledge, ensuring the accurate identification of off-target hits. It boasts an extensive built-in database, encompassing 1,397 common prokaryotic species as a comprehensive design resource. It also provides the capability to design sgRNAs for newly discovered organisms by accepting uploaded design resource. We further demonstrated that GLiDe exhibits enhanced precision in identifying off-target binding sites for the CRISPRi system.<br><br>CONCLUSIONS: We present a web server that allows the construction of genome-scale CRISPRi sgRNA libraries for prokaryotes. It mitigates off-target effects through a robust quality control framework, leveraging prior experimental knowledge within an end-to-end, user-friendly pipeline.}, }
@article {pmid39753078, year = {2024}, 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 = {A competitive aptamer binding-based CRISPR-cas biosensor for sensitive detection of tetracycline residues in biological samples.}, journal = {Talanta}, volume = {286}, number = {}, pages = {127491}, doi = {10.1016/j.talanta.2024.127491}, pmid = {39753078}, issn = {1873-3573}, abstract = {Tetracycline (TC) is widely used in veterinary medicine and animal feed; however, TC residues in food pose a risk to human health. Thus, the sensitive and selective detection of TC is needed to ensure food safety. Herein, we developed a CRISPR-Cas12a biosensor with competitive aptamer binding to detect TC residues. The aptasensor, formed by hybridizing activator DNA with TC-specific aptamers on streptavidin-modified magnetic beads, releases activator DNA in a TC concentration-dependent manner. This activated the Cas12a-crRNA complex, which cleaved single-strand DNA reporters to generate a detectable fluorescence signal. The TC signal was amplified through a two-step incubation reaction, with a detection limit as low as 9.45 × 10[-5] μg L[-1]. The assay showed high selectivity and good recovery rates in various biological samples (e.g., honey, milk, fish), demonstrating the applicability of the biosensors in pollutant detection.}, }
@article {pmid39752253, year = {2025}, author = {Mutte, SK and Barendse, P and Ugarte, PB and Swarts, DC}, title = {Distribution of bacterial DNA repair proteins and their co-occurrence with immune systems.}, journal = {Cell reports}, volume = {44}, number = {1}, pages = {115110}, doi = {10.1016/j.celrep.2024.115110}, pmid = {39752253}, issn = {2211-1247}, abstract = {Bacteria encode various DNA repair pathways to maintain genome integrity. However, the high degree of homology between DNA repair proteins or their domains hampers accurate identification. Here, we describe a stringent search strategy to identify DNA repair proteins and provide a systematic analysis of taxonomic distribution and co-occurrence of DNA repair proteins involved in RecA-dependent homologous recombination. Our results reveal the widespread presence of RecA, SSB, and RecOR proteins and phyla-specific distribution for the DNA repair complexes RecBCD, AddAB, and AdnAB. Furthermore, we report co-occurrences of DNA repair proteins with immune systems, including specific CRISPR-Cas subtypes, prokaryotic Argonautes (pAgos), dGTPases, GAPS2, and Wadjet. Our results imply that while certain DNA repair proteins and immune systems might function in conjunction, no immune system strictly relies on a specific DNA repair protein. As such, these findings offer an updated perspective on the distribution of DNA repair systems and their connection to immune systems in bacteria.}, }
@article {pmid39752243, year = {2025}, author = {Riaz, A and Uzair, M and Raza, A and Inam, S and Iqbal, R and Jameel, S and Bibi, B and Khan, MR}, title = {Enhancing the productivity and resilience of rice (Oryza sativa) under environmental stress conditions using clustered regularly interspaced short palindromic repeats (CRISPR) technology.}, journal = {Functional plant biology : FPB}, volume = {52}, number = {}, pages = {}, doi = {10.1071/FP24101}, pmid = {39752243}, issn = {1445-4416}, mesh = {*Oryza/genetics ; *Stress, Physiological/genetics ; *Gene Editing ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Plants, Genetically Modified/genetics ; }, abstract = {Rice (Oryza sativa) is a crucial staple crop worldwide, providing nutrition to more than half of the global population. Nonetheless, the sustainability of grain production is increasingly jeopardized by both biotic and abiotic stressors exacerbated by climate change, which increases the crop's rvulnerability to pests and diseases. Genome-editing by clustered regularly interspaced short palindromic repeats and CRISPR-associated Protein 9 (CRISPR-Cas9) presents a potential solution for enhancing rice productivity and resilience under climatic stress. This technology can alter a plant's genetic components without the introduction of foreign DNA or genes. It has become one of the most extensively used approaches for discovering new gene functions and creating novel varieties that exhibit a higher tolerance to both abiotic and biotic stresses, herbicide resistance, and improved yield production. This study examines numerous CRISPR-Cas9-based genome-editing techniques for gene knockout, gene knock-in, multiplexing for simultaneous disruption of multiple genes, base-editing, and prime-editing. This review elucidates the application of genome-editing technologies to enhance rice production by directly targeting yield-related genes or indirectly modulating numerous abiotic and biotic stress-responsive genes. We highlight the need to integrate genetic advancements with conventional and advanced agricultural methods to create rice varieties that are resilient to stresses, thereby safeguarding food security and promoting agricultural sustainability amid climatic concerns.}, }
@article {pmid39749289, year = {2024}, author = {Bhoobalan-Chitty, Y and Stouf, M and De Paepe, M}, title = {Genetic manipulation of bacteriophage T4 utilizing the CRISPR-Cas13b system.}, journal = {Frontiers in genome editing}, volume = {6}, number = {}, pages = {1495968}, pmid = {39749289}, issn = {2673-3439}, abstract = {CRISPR-Cas type II and type V systems are inefficient in modifying bacteriophage T4 genome, due to hypermodification of its DNA. Here, we present a genome editing technique for bacteriophage T4 using the type VI CRISPR-Cas system. Using BzCas13b targeting of T4 phage, we were able to individually delete both T4 glucosyl transferase genes, α-gt and β-gt. Furthermore, we employed this method to mutate two conserved residues within the T4 DNA polymerase and to introduce the yellow fluorescent protein (YFP) coding sequence into T4 phage genome, enabling us to visualize phage infections. This T4 genome editing protocol was optimized to generate recombinant phages within a 6-hour timescale. Finally, spacers homologous to a variety of T4 genes were used to study the generality of Cas13b targeting, revealing important variability in targeting efficiency. Overall, this method constitutes a rapid and effective means of generating specific T4 phage mutants, which could be extended to other T4-like phages.}, }
@article {pmid39748374, year = {2025}, author = {Wan, X and Kong, J and Hu, X and Liu, L and Yang, Y and Li, H and Liu, G and Niu, X and Chen, F and Zhang, D and Zhu, D and Zhang, Y}, title = {SiCLAT: simultaneous imaging of chromatin loops and active transcription in living cells.}, journal = {Genome biology}, volume = {26}, number = {1}, pages = {1}, pmid = {39748374}, issn = {1474-760X}, mesh = {Animals ; *Chromatin/metabolism ; Mice ; *Transcription, Genetic ; Enhancer Elements, Genetic ; CRISPR-Cas Systems ; Promoter Regions, Genetic ; Cell Differentiation ; }, abstract = {We present SiCLAT, which introduces a dCas9-dCas13d cassette into the mouse genome. This model enables the stable expression of both dCas9 and dCas13 proteins in diverse cell populations, facilitating concurrent labeling of DNA and RNA across various cell types. Using SiCLAT, we accurately labeled chromatin loop anchor interactions and associated gene transcription during myogenic differentiation. This imaging system offers a novel means of directly observing cis-element interactions and the corresponding gene transcription in living primary cells, thus providing real-time imaging for comprehensive mechanistic investigations of dynamic enhancer-promoter or enhancer-enhancer interactions in regulating transcription activation within living cells.}, }
@article {pmid39747875, year = {2025}, author = {Raghavan, R and Friedrich, MJ and King, I and Chau-Duy-Tam Vo, S and Strebinger, D and Lash, B and Kilian, M and Platten, M and Macrae, RK and Song, Y and Nivon, L and Zhang, F}, title = {Rational engineering of minimally immunogenic nucleases for gene therapy.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {105}, pmid = {39747875}, issn = {2041-1723}, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; Animals ; CRISPR-Associated Protein 9/metabolism/genetics ; Protein Engineering/methods ; Proprotein Convertase 9/immunology/genetics/metabolism ; Mice ; HEK293 Cells ; T-Lymphocytes, Cytotoxic/immunology ; Epitopes/immunology ; Endonucleases/metabolism/genetics ; Female ; }, abstract = {Genome editing using CRISPR-Cas systems is a promising avenue for the treatment of genetic diseases. However, cellular and humoral immunogenicity of genome editing tools, which originate from bacteria, complicates their clinical use. Here we report reduced immunogenicity (Red)(i)-variants of two clinically relevant nucleases, SaCas9 and AsCas12a. Through MHC-associated peptide proteomics (MAPPs) analysis, we identify putative immunogenic epitopes on each nuclease. Using computational modeling, we rationally design these proteins to evade the immune response. SaCas9 and AsCas12a Redi variants are substantially less recognized by adaptive immune components, including reduced binding affinity to MHC molecules and attenuated generation of cytotoxic T cell responses, yet maintain wild-type levels of activity and specificity. In vivo editing of PCSK9 with SaCas9.Redi.1 is comparable in efficiency to wild-type SaCas9, but significantly reduces undesired immune responses. This demonstrates the utility of this approach in engineering proteins to evade immune detection.}, }
@article {pmid39747848, year = {2025}, author = {Escobar, H and Di Francescantonio, S and Smirnova, J and Graf, R and Müthel, S and Marg, A and Zhogov, A and Krishna, S and Metzler, E and Petkova, M and Daumke, O and Kühn, R and Spuler, S}, title = {Gene-editing in patient and humanized-mice primary muscle stem cells rescues dysferlin expression in dysferlin-deficient muscular dystrophy.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {120}, pmid = {39747848}, issn = {2041-1723}, mesh = {*Dysferlin/genetics/metabolism ; Animals ; Humans ; *Gene Editing/methods ; Mice ; *Muscle, Skeletal/metabolism ; Disease Models, Animal ; Frameshift Mutation ; CRISPR-Cas Systems ; Muscular Dystrophies/therapy/genetics/metabolism ; Exons/genetics ; Stem Cells/metabolism ; Female ; Membrane Proteins/genetics/metabolism ; Male ; Muscular Dystrophies, Limb-Girdle/therapy/genetics/metabolism ; Genetic Therapy/methods ; }, abstract = {Dystrophy-associated fer-1-like protein (dysferlin) conducts plasma membrane repair. Mutations in the DYSF gene cause a panoply of genetic muscular dystrophies. We targeted a frequent loss-of-function, DYSF exon 44, founder frameshift mutation with mRNA-mediated delivery of SpCas9 in combination with a mutation-specific sgRNA to primary muscle stem cells from two homozygous patients. We observed a consistent >60% exon 44 re-framing, rescuing a full-length and functional dysferlin protein. A new mouse model harboring a humanized Dysf exon 44 with the founder mutation, hEx44mut, recapitulates the patients' phenotype and an identical re-framing outcome in primary muscle stem cells. Finally, gene-edited murine primary muscle stem-cells are able to regenerate muscle and rescue dysferlin when transplanted back into hEx44mut hosts. These findings are the first to show that a CRISPR-mediated therapy can ameliorate dysferlin deficiency. We suggest that gene-edited primary muscle stem cells could exhibit utility, not only in treating dysferlin deficiency syndromes, but also perhaps other forms of muscular dystrophy.}, }
@article {pmid39747609, year = {2025}, author = {Motooka, Y and Tanaka, H and Maeda, Y and Katabuchi, M and Mashimo, T and Toyokuni, S}, title = {Heterozygous mutation in BRCA2 induces accelerated age-dependent decline in sperm quality with male subfertility in rats.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {447}, pmid = {39747609}, issn = {2045-2322}, support = {JP23K08883//Japan Society for the Promotion of Science/ ; JP19H0546//Japan Society for the Promotion of Science/ ; JP16H06276 [AdAMS (Aa210038)]//Japan Society for the Promotion of Science/ ; JPMJCR19H4//JST CREST/ ; }, mesh = {Male ; Animals ; *BRCA2 Protein/genetics ; Rats ; *Spermatozoa/metabolism ; *Infertility, Male/genetics ; Heterozygote ; Mutation ; Exons/genetics ; DNA Breaks, Double-Stranded ; Aging/genetics ; Humans ; Apoptosis/genetics ; Testis/pathology/metabolism ; CRISPR-Cas Systems ; }, abstract = {Tumor suppressor BRCA2 executes homologous recombination to repair DNA double-strand breaks in collaboration with RAD51, involving exon 11 and 27. Exon 11 constitutes a region where pathogenic variants (PVs) accumulate, and mutations in this region are known to contribute to carcinogenesis. However, the impact of the heterozygous PVs of BRCA2 exon 11 on the life quality beyond cancer risk, including male fertility, remains unclear. Here, we established a rat model with a frameshift on the seventh BRC repeat in Brca2 exon 11 (Brca2[+/p.T1942fs]), which is homologous to human BRCA2[+/p.T1974fs], using CRISPR/Cas9 system. Our analyses revealed that the heterozygous rats with the PV in the BRCA2 exon 11 showed increased DNA double-strand breaks and apoptosis in spermatogonia and spermatocytes, accelerated testicular germ cell loss, and deterioration in sperm quality according with aging, ultimately resulting in early male reproductive dysfunction. Of note, these alterations in testes and sperm, including DNA fragmentation in spermatozoa, were observed from completion of sexual maturation. The present findings suggest that it is crucial to consider not only cancer risk but also potential declines in reproductive capacity in men carrying BRCA2 exon 11 PVs. Further investigation is warranted to determine whether similar traits appear in humans.}, }
@article {pmid39747369, year = {2025}, author = {Patil, S and Siji, A and Mallur, D and Kruthika, BS and Gheewalla, N and Karve, S and Kavathekar, M and Tarai, B and Naik, M and Hegde, V and Rangineni, J and Gupta, V and Chandru, V and Pradeep, BE and Arora, R}, title = {PathCrisp: an innovative molecular diagnostic tool for early detection of NDM-resistant infections.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {490}, pmid = {39747369}, issn = {2045-2322}, support = {2021 HTH 018//Rockefeller Foundation Grant/ ; 2021 HTH 018//Rockefeller Foundation Grant/ ; }, mesh = {*beta-Lactamases/genetics ; Humans ; *Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Enterobacteriaceae Infections/diagnosis/microbiology ; Carbapenem-Resistant Enterobacteriaceae/genetics/isolation &amp; purification ; Sensitivity and Specificity ; Early Diagnosis ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Polymerase Chain Reaction/methods ; Microbial Sensitivity Tests ; CRISPR-Cas Systems ; }, abstract = {The rapid and early detection of infections and antibiotic resistance markers is a critical challenge in healthcare. Currently, most commercial diagnostic tools for analyzing antimicrobial resistance patterns of pathogens require elaborate culture-based testing. Our study aims to develop a rapid, accurate molecular detection system that can be used directly from culture, thereby introducing molecular testing in conjunction with culture tests to reduce turnaround time and guide therapy. PathCrisp assay, a combination of loop-mediated isothermal amplification and CRISPR-based detection, maintained at a single temperature, was designed and tested on clinical isolates. The specificity and sensitivity of the assay was analyzed, post which the assay was compared with the polymerase chain reaction (PCR) method to detect the New Delhi metallo-beta-lactamase (NDM) gene in carbapenem-resistant enterobacteriaceae clinical samples. Our PathCrisp assay demonstrated the ability to detect as few as 700 copies of the NDM gene from clinical isolates. Our assay demonstrated 100% concordance with the PCR-Sanger sequencing method, more commonly used. Additionally, the lack of the need for a kit-based DNA purification step, rather a crude extraction via heating, enables the direct use of culture samples. The PathCrisp assay is precise, specific and rapid, providing results in approximately 2 h, and operates at a constant temperature, reducing the need for complex equipment handling. In the near future, we hope that this assay can be further optimized and designed as a point-of-care test kit, facilitating its use in various healthcare settings and aiding clinicians in the choice of antibiotics for therapy.}, }
@article {pmid39747289, year = {2025}, author = {Frusteri Chiacchiera, A and Casanova, M and Bellato, M and Piazza, A and Migliavacca, R and Batt, G and Magni, P and Pasotti, L}, title = {Harnessing CRISPR interference to resensitize laboratory strains and clinical isolates to last resort antibiotics.}, journal = {Scientific reports}, volume = {15}, number = {1}, pages = {261}, pmid = {39747289}, issn = {2045-2322}, support = {1139857//Regione Lombardia/ ; 20-PAMR-0010 (Seq2DiAg)//Agence Nationale de la Recherche/ ; 59576 (2021)//Fondazione Cassa di Risparmio di Padova e Rovigo/ ; INCEPTION//Institut Pasteur/ ; }, mesh = {*Anti-Bacterial Agents/pharmacology/therapeutic use ; *Escherichia coli/genetics/drug effects ; *CRISPR-Cas Systems ; Humans ; Drug Resistance, Bacterial/genetics ; Microbial Sensitivity Tests ; Mutation ; }, abstract = {The global race against antimicrobial resistance requires novel antimicrobials that are not only effective in killing specific bacteria, but also minimize the emergence of new resistances. Recently, CRISPR/Cas-based antimicrobials were proposed to address killing specificity with encouraging results. However, the emergence of target sequence mutations triggered by Cas-cleavage was identified as an escape strategy, posing the risk of generating new antibiotic-resistance gene (ARG) variants. Here, we evaluated an antibiotic re-sensitization strategy based on CRISPR interference (CRISPRi), which inhibits gene expression without damaging target DNA. The resistance to four antibiotics, including last resort drugs, was significantly reduced by individual and multi-gene targeting of ARGs in low- to high-copy numbers in recombinant E. coli. Escaper analysis confirmed the absence of mutations in target sequence, corroborating the harmless role of CRISPRi in the selection of new resistances. E. coli clinical isolates carrying ARGs of severe clinical concern were then used to assess the robustness of CRISPRi under different growth conditions. Meropenem, colistin and cefotaxime susceptibility was successfully increased in terms of MIC (up to > 4-fold) and growth delay (up to 11 h) in a medium-dependent fashion. ARG repression also worked in a pathogenic strain grown in human urine, as a demonstration of CRISPRi-mediated re-sensitization in host-mimicking media. This study laid the foundations for further leveraging CRISPRi as antimicrobial agent or research tool to selectively repress ARGs and investigate resistance mechanisms.}, }
@article {pmid39747101, year = {2025}, author = {Lin, JF and Liu, ZX and Chen, DL and Huang, RZ and Cao, F and Yu, K and Li, T and Mo, HY and Sheng, H and Liang, ZB and Liao, K and Han, Y and Li, SS and Zeng, ZL and Gao, S and Ju, HQ and Xu, RH}, title = {Nucleus-translocated GCLM promotes chemoresistance in colorectal cancer through a moonlighting function.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {263}, pmid = {39747101}, issn = {2041-1723}, support = {82303306//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82373376//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {Humans ; *Colorectal Neoplasms/drug therapy/genetics/metabolism/pathology ; *Drug Resistance, Neoplasm/genetics ; *NF-kappa B/metabolism ; *Cell Nucleus/metabolism ; Cell Line, Tumor ; *Glutamate-Cysteine Ligase/metabolism/genetics ; Phosphorylation ; Animals ; Antineoplastic Agents/therapeutic use/pharmacology ; Mice ; Gene Expression Regulation, Neoplastic ; p38 Mitogen-Activated Protein Kinases/metabolism ; Female ; CRISPR-Cas Systems ; Male ; Active Transport, Cell Nucleus ; Mice, Nude ; }, abstract = {Metabolic enzymes perform moonlighting functions during tumor progression, including the modulation of chemoresistance. However, the underlying mechanisms of these functions remain elusive. Here, utilizing a metabolic clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 knockout library screen, we observe that the loss of glutamate-cysteine ligase modifier subunit (GCLM), a rate-limiting enzyme in glutathione biosynthesis, noticeably increases the sensitivity of colorectal cancer (CRC) cells to platinum-based chemotherapy. Mechanistically, we unveil a noncanonical mechanism through which nuclear GCLM competitively interacts with NF-kappa-B (NF-κB)-repressing factor (NKRF), to promote NF-κB activity and facilitate chemoresistance. In response to platinum drug treatment, GCLM is phosphorylated by P38 MAPK at T17, resulting in its recognition by importin a5 and subsequent nuclear translocation. Furthermore, elevated expression of nuclear GCLM and phospho-GCLM correlate with an unfavorable prognosis and poor benefit from standard chemotherapy. Overall, our work highlights the essential nonmetabolic role and posttranslational regulatory mechanism of GCLM in enhancing NF-κB activity and subsequent chemoresistance.}, }
@article {pmid39747086, year = {2025}, author = {Albuquerque-Wendt, A and McCoy, C and Neish, R and Dobramysl, U and Alagöz, &#xc7; and Beneke, T and Cowley, SA and Crouch, K and Wheeler, RJ and Mottram, JC and Gluenz, E}, title = {TransLeish: Identification of membrane transporters essential for survival of intracellular Leishmania parasites in a systematic gene deletion screen.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {299}, pmid = {39747086}, issn = {2041-1723}, support = {221944/A/20/Z//Wellcome Trust (Wellcome)/ ; trans-LEISHion-EU FP7, No. 798736//EC | EC Seventh Framework Programm | FP7 People: Marie-Curie Actions (FP7-PEOPLE - Specific Programme "People" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013))/ ; 211075/Z/18/Z//Wellcome Trust (Wellcome)/ ; 104111/Z/14/Z//Wellcome Trust (Wellcome)/ ; /WT_/Wellcome Trust/United Kingdom ; 15/16_MSD_836338//RCUK | Medical Research Council (MRC)/ ; 101064428 - LeishMOM//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 Marie Sk&#x142;odowska-Curie Actions (H2020 Excellent Science - Marie Sk&#x142;odowska-Curie Actions)/ ; 200807/Z/16/Z//Wellcome Trust (Wellcome)/ ; UF160661//Royal Society/ ; ALTF 727-2021//European Molecular Biology Organization (EMBO)/ ; }, mesh = {Animals ; Mice ; *Gene Deletion ; *Macrophages/parasitology/metabolism ; *CRISPR-Cas Systems ; *Protozoan Proteins/genetics/metabolism ; *Membrane Transport Proteins/genetics/metabolism ; Leishmania mexicana/genetics/metabolism ; Vacuolar Proton-Translocating ATPases/genetics/metabolism ; Female ; Mice, Inbred BALB C ; }, abstract = {For the protozoan parasite Leishmania, completion of its life cycle requires sequential adaptation of cellular physiology and nutrient scavenging mechanisms to the different environments of a sand fly alimentary tract and the acidic mammalian host cell phagolysosome. Transmembrane transporters are the gatekeepers of intracellular environments, controlling the flux of solutes and ions across membranes. To discover which transporters are vital for survival as intracellular amastigote forms, we carried out a systematic loss-of-function screen of the L. mexicana transportome. A total of 312 protein components of small molecule carriers, ion channels and pumps were identified and targeted in a CRISPR-Cas9 gene deletion screen in the promastigote form, yielding 188 viable null mutants. Forty transporter deletions caused significant loss of fitness in macrophage and mouse infections. A striking example is the Vacuolar H[+] ATPase (V-ATPase), which, unexpectedly, was dispensable for promastigote growth in vitro but essential for survival of the disease-causing amastigotes.}, }
@article {pmid39747083, year = {2025}, author = {Fei, J and Zhao, D and Pang, C and Li, J and Li, S and Qiao, W and Tan, J and Bi, C and Zhang, X}, title = {Mismatch prime editing gRNA increased efficiency and reduced indels.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {139}, pmid = {39747083}, issn = {2041-1723}, support = {32171449//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32271483//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32225031//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2022177//Youth Innovation Promotion Association of the Chinese Academy of Sciences (Youth Innovation Promotion Association CAS)/ ; }, mesh = {*Gene Editing/methods ; Humans ; *INDEL Mutation ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; HEK293 Cells ; Base Pair Mismatch ; }, abstract = {Prime editing enables precise and efficient genome editing, but its efficacy is hindered by pegRNA's 3' extension, forming secondary structures due to high complementarity with the protospacer. The continuous presence of the prime editing system also leads to unintended indel formation, raising safety concerns for therapeutic applications. To address these challenges, we develop a mismatched pegRNA (mpegRNA) strategy that introduces mismatched bases into the pegRNA protospacer, reducing complementarity and secondary structure formation, and preventing sustained activity. Our findings show that mpegRNA enhances editing efficiency by up to 2.3 times and reduces indel levels by 76.5% without compromising performance. Combining mpegRNA with epegRNA further increases efficiency up to 14-fold, or 2.4-fold in PE4max/PE5max systems, underscoring its potential in research and therapy. AlphaFold 3 analysis suggests that the optimal mpegRNA structure contributes significantly to improved editing outcomes. Overall, mpegRNA advances prime editing technology, improving efficiency while reducing indels.}, }
@article {pmid39747012, year = {2025}, author = {Tolosana, I and Willis, K and Gribble, M and Phillimore, L and Burt, A and Nolan, T and Crisanti, A and Bernardini, F}, title = {A Y chromosome-linked genome editor for efficient population suppression in the malaria vector Anopheles gambiae.}, journal = {Nature communications}, volume = {16}, number = {1}, pages = {206}, pmid = {39747012}, issn = {2041-1723}, support = {INV006610 "Target Malaria Phase II"//Bill and Melinda Gates Foundation (Bill &amp; Melinda Gates Foundation)/ ; INV006610 "Target Malaria Phase II"//Bill and Melinda Gates Foundation (Bill &amp; Melinda Gates Foundation)/ ; }, mesh = {*Anopheles/genetics ; Animals ; Female ; *Y Chromosome/genetics ; Male ; *Mosquito Vectors/genetics ; *Malaria/prevention &amp; control/transmission ; *CRISPR-Cas Systems ; *Mosquito Control/methods ; *Gene Editing/methods ; Genome, Insect ; }, abstract = {Genetic control - the deliberate introduction of genetic traits to control a pest or vector population - offers a powerful tool to augment conventional mosquito control tools that have been successful in reducing malaria burden but that are compromised by a range of operational challenges. Self-sustaining genetic control strategies have shown great potential in laboratory settings, but hesitancy due to their invasive and persistent nature may delay their implementation. Here, instead, we describe a self-limiting strategy, designed to have geographically and temporally restricted effect, based on a Y chromosome-linked genome editor (YLE). The YLE comprises a CRISPR-Cas9 construct that is always inherited by males yet generates an autosomal dominant mutation that is transmitted to over 90% of the offspring and results in female-specific sterility. To our knowledge, our system represents a pioneering approach in the engineering of the Y chromosome to generate a genetic control strain for mosquitoes. Mathematical modelling shows that this YLE technology is up to seven times more efficient for population suppression than optimal versions of other self-limiting strategies, such as the widely used Sterile Insect Technique or the Release of Insects carrying a Dominant Lethal gene.}, }
@article {pmid39745650, year = {2025}, author = {Salvador-Martínez, I}, title = {Computational Methods for Lineage Reconstruction.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2886}, number = {}, pages = {355-373}, pmid = {39745650}, issn = {1940-6029}, mesh = {*Software ; *Cell Lineage/genetics ; *Algorithms ; *Phylogeny ; *Computational Biology/methods ; Animals ; CRISPR-Cas Systems ; Humans ; }, abstract = {The recent development of genetic lineage recorders, designed to register the genealogical history of cells using induced somatic mutations, has opened the possibility of reconstructing complete animal cell lineages. To reconstruct a cell lineage tree from a molecular recorder, it is crucial to use an appropriate reconstruction algorithm. Current approaches include algorithms specifically designed for cell lineage reconstruction and the repurposing of phylogenetic algorithms. These methods have, however, the same objective: to uncover the hierarchical relationships between cells and the sequence of cell divisions that have occurred during development. In this chapter, I will use the phylogenetic software FastTree to reconstruct a lineage tree, in a step-by-step manner, using data from a simulated CRISPR-Cas9 recorder. To ensure reproducibility, the code is presented as a Jupyter Notebook, available (together with the necessary input files) at https://github.com/irepansalvador/lineage_reconstruction_chapter .}, }
@article {pmid39745649, year = {2025}, author = {Espinosa-Medina, I}, title = {Design and Generation of TEMPO Reagents for Sequential Labeling and Manipulation of Vertebrate Cell Lineages.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2886}, number = {}, pages = {327-353}, pmid = {39745649}, issn = {1940-6029}, mesh = {Animals ; *Zebrafish ; Mice ; *Cell Lineage ; CRISPR-Cas Systems ; Staining and Labeling/methods ; Luminescent Proteins/genetics/metabolism ; }, abstract = {During development, cells undergo a sequence of specification events to form functional tissues and organs. To investigate complex tissue development, it is crucial to visualize how cell lineages emerge and to be able to manipulate regulatory factors with temporal control. We recently developed TEMPO (Temporal Encoding and Manipulation in a Predefined Order), a genetic tool to label with different colors and genetically manipulate consecutive cell generations in vertebrates. TEMPO relies on CRISPR to activate a cascade of fluorescent proteins which can be imaged in vivo. Here, we explain the steps to design, generate, and express TEMPO constructs in zebrafish and mice.}, }
@article {pmid39745647, year = {2025}, author = {Raj, B}, title = {Single-Cell Profiling of Lineages and Cell Types in the Vertebrate Brain.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2886}, number = {}, pages = {299-310}, pmid = {39745647}, issn = {1940-6029}, mesh = {Animals ; *Single-Cell Analysis/methods ; *Brain/cytology/metabolism ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; *Cell Lineage/genetics ; *Gene Editing/methods ; Transcriptome ; Gene Expression Profiling/methods ; }, abstract = {CRISPR-Cas tools have recently been adapted for cell lineage tracing during development. Combined with single-cell RNA sequencing, these methods enable scalable lineage tracing with single-cell resolution. Here, I describe, scGESTALTv2, which combines cumulative CRISPR-Cas9 editing of a lineage barcode array with transcriptional profiling via droplet-based single-cell RNA sequencing (scRNA-seq). The technique is applied in developing zebrafish brains to generate mutations in the barcode array during development. The recorded lineages along with cellular transcriptomes are then extracted via scRNA-seq to define cell relationships among thousands of profiled brain cells and dozens of cell types.}, }
@article {pmid39745646, year = {2025}, author = {Bowling, S and Camargo, FD}, title = {CARLIN: A Mouse Line for Simultaneous Readout of Lineage Histories and Gene Expression.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2886}, number = {}, pages = {281-298}, pmid = {39745646}, issn = {1940-6029}, mesh = {Animals ; Mice ; *Cell Lineage/genetics ; *DNA Barcoding, Taxonomic/methods ; *High-Throughput Nucleotide Sequencing/methods ; *Single-Cell Analysis/methods ; CRISPR-Cas Systems ; Gene Editing/methods ; Gene Expression/genetics ; }, abstract = {The CRISPR-activated repair lineage tracing (CARLIN) mouse line uses DNA barcoding to enable high-resolution tracing of cell lineages in vivo (Bowling et al, Cell 181, 1410-1422.e27, 2020). CARLIN mice contain expressed barcodes that allow simultaneous interrogation of lineage and gene expression information from single cells. Furthermore, barcode editing is fully inducible, resulting in cell lineage labeling that can be performed at any time point in development or adulthood. This chapter details the protocols followed for maintaining CARLIN mice, inducing barcoding, and amplifying the CARLIN barcode from DNA, RNA, and single-cell RNA-sequencing libraries for next-generation sequencing.}, }
@article {pmid39745643, year = {2025}, author = {Baron, CS and Alemany, A}, title = {Paired Single-Cell Transcriptome and DNA Barcode Detection in Zebrafish Using ScarTrace.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2886}, number = {}, pages = {221-241}, pmid = {39745643}, issn = {1940-6029}, mesh = {Animals ; *Zebrafish/genetics ; *Single-Cell Analysis/methods ; *DNA Barcoding, Taxonomic/methods ; *CRISPR-Cas Systems ; *Transcriptome/genetics ; Gene Expression Profiling/methods ; Embryo, Nonmammalian/metabolism ; }, abstract = {ScarTrace is a CRISPR/Cas9-based genetic lineage tracing method that allows for uniquely barcoding the DNA of single cells at a target GFP sequence during developing zebrafish embryos. Single cells from barcoded adult zebrafish can be isolated from various tissues (e.g., marrow, brain, eyes, fins), and their transcriptome and barcode sequences are captured by single-cell cDNA amplification and genomic DNA nested PCR, respectively. Computationally, cell type and barcode identification permit clone tracing and lineage tree reconstruction of tissues to unravel fate decisions during embryogenesis.}, }
@article {pmid39745637, year = {2025}, author = {Gentile, E and Maynard, A and He, Z and Treutlein, B}, title = {Lineage Recording in Human Brain Organoids with iTracer.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2886}, number = {}, pages = {85-101}, pmid = {39745637}, issn = {1940-6029}, mesh = {Humans ; *Organoids/cytology/metabolism ; *Brain/cytology ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Cell Lineage/genetics ; *Single-Cell Analysis/methods ; *CRISPR-Cas Systems ; Transcriptome ; Cell Differentiation ; }, abstract = {Induced pluripotent stem cell (iPSC)-derived organoids provide models to study human organ development. Single-cell transcriptomics enables highly resolved descriptions of cell states within these systems; however, approaches are needed to directly determine the lineage relationship between cells. Here we provide a detailed protocol (Fig. 1) for the application of iTracer (He Z, Maynard A, Jain A, et al., Nat Methods 19:90-99, 2022), a recently published lineage recorder that combines reporter barcodes with inducible CRISPR-Cas9 scarring and is compatible with single-cell and spatial transcriptomics. iTracer is used to explore clonality and lineage dynamics during brain organoid development. More broadly, iTracer can be adapted to any iPSC-derived culture system to dissect lineage dynamics during normal or perturbed development.}, }
@article {pmid39745602, year = {2025}, author = {Hajirnis, N}, title = {Re-arranging the Cis-regulatory Modules of Hox Complex in Drosophila via FLP-FRT and CRISPR/Cas9.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2889}, number = {}, pages = {11-24}, pmid = {39745602}, issn = {1940-6029}, mesh = {Animals ; *CRISPR-Cas Systems ; *Drosophila melanogaster/genetics/growth &amp; development ; Drosophila Proteins/genetics/metabolism ; Homeodomain Proteins/genetics/metabolism ; Genes, Homeobox/genetics ; Gene Editing/methods ; DNA Nucleotidyltransferases ; }, abstract = {FLP-FRT, a well-established technique for genome manipulation, and the revolutionary CRISPR/Cas9, known for its targeted indels, are combined in a novel approach. This unique method is applied to the Hox genes in the Drosophila melanogaster bithorax complex, which are closely located to the cis-regulatory modules that define their spatial-temporal regulation. The number and position of these genes are directly correlated to their expression pattern. This chapter unveils the exciting potential of this combinatorial use of FLP-FRT and CRISPR-Cas9 to rearrange the cis-regulatory modules of the Hox complex in Drosophila melanogaster.}, }
@article {pmid39745317, year = {2025}, author = {Hou, M and Yang, X and Gong, L and Shen, X}, title = {Surveillance of antimicrobial resistance using isothermal amplification: a review.}, journal = {Chemical communications (Cambridge, England)}, volume = {}, number = {}, pages = {}, doi = {10.1039/d4cc05488a}, pmid = {39745317}, issn = {1364-548X}, abstract = {The monitoring of antibiotic resistance genes (ARGs) is crucial for understanding the level of antimicrobial resistance and the associated health burden, which in turn is essential for the control and prevention of antimicrobial resistance (AMR). Isothermal amplification, an emerging molecular biology technology, has been widely used for drug resistance detection. Furthermore, its compatibility with a range of technologies enables high-specificity, high-throughput, and portable and integrated detection in drug resistance, particularly in resource-limited areas. However, to date, reviews involved in isothermal amplification all concentrate on its technological advancements and its application in nucleic acid point-of-care testing. Few reviews have been published that focus specifically on the application of isothermal amplification in the detection of drug resistance. This review summarizes the detection principles of different isothermal amplification techniques and discusses their strengths and weaknesses as well as the applicable scenarios for drug resistance detection. It also summarizes advances in the application, challenges and prospects of isothermal amplification technologies in conjunction with different methods such as base mismatch, CRISPR-Cas, lateral flow immunoassay, sensing and microfluidic technologies for improvement of specificity, throughput and integration for drug resistance detection. It is anticipated that this review will assist scientists in comprehending the evolution of isothermal amplification in the context of drug resistance detection and provide insights into the prospective applications of isothermal amplification for highly integrated and immediate on-site detection of drug resistance.}, }
@article {pmid39744681, year = {2025}, author = {Park, M and Ryu, H and Heo, S and Kim, B and Park, J and Lim, KH and Han, SB and Park, H}, title = {Targeted demethylation of cathepsin D via epigenome editing rescues pathology in Alzheimer's disease mouse model.}, journal = {Theranostics}, volume = {15}, number = {2}, pages = {428-438}, pmid = {39744681}, issn = {1838-7640}, mesh = {Animals ; *Alzheimer Disease/genetics/therapy/metabolism ; *Disease Models, Animal ; Mice ; *Amyloid beta-Peptides/metabolism ; *Cathepsin D/metabolism/genetics ; *Gene Editing/methods ; *Mice, Transgenic ; *Epigenome ; Neurons/metabolism ; Amyloid beta-Protein Precursor/genetics/metabolism ; Humans ; Brain/metabolism/pathology ; Genetic Therapy/methods ; CRISPR-Cas Systems/genetics ; Male ; }, abstract = {Background: Cathepsin D (Ctsd) has emerged as a promising therapeutic target for Alzheimer's disease (AD) due to its role in degrading intracellular amyloid beta (Aβ). Enhancing Ctsd activity could reduce Aβ42 accumulation and restore the Aβ42/40 ratio, offering a potential AD treatment strategy. Methods: This study explored Ctsd demethylation in AD mouse models using dCas9-Tet1-mediated epigenome editing. We identified dCas9-Tet1 as an effective tool for demethylating the endogenous Ctsd gene in primary neurons and in vivo brains. Results: Treatment with Ctsd-targeted dCas9-Tet1 in primary neurons overexpressing mutant APP (mutAPP) reduced Aβ peptide levels and the Aβ42/40 ratio. Additionally, in vivo demethylation of Ctsd via dCas9-Tet1 in 5xFAD mice significantly altered Aβ levels and alleviated cognitive and behavioral deficits. Conclusion: These findings offer valuable insights into developing epigenome editing-based gene therapy strategies for AD.}, }
@article {pmid39743757, year = {2024}, author = {Yu, CH and Lai, KL}, title = {Cure the Incurable: Update of Treatment in Inherited Neuromuscular Disorders.}, journal = {Acta neurologica Taiwanica}, volume = {33(4)}, number = {}, pages = {129-135}, pmid = {39743757}, issn = {1028-768X}, mesh = {Humans ; *Genetic Therapy/methods ; *Neuromuscular Diseases/therapy/genetics ; *CRISPR-Cas Systems ; }, abstract = {Originally thought to be incurable, huge therapeutic progress has been made in recent years in the field of inherited neuromuscular disorders. Approaches aiming to rescue the underlying pathophysiology, i.e. loss-of-function or gain-of-function mutations, have been developed via end-product replacement or gene delivery/modulation, with promising results. In the review, advanced treatment in some of the inherited neuromuscular disorders will be discussed. On the other hand, it has been found more than 1000 genes are responsible for the clinical diversities in this group of diseases, and finding a way which owns the therapeutic potential to various diseases is the optimal goal. The discovery of CRISPR-Cas9 system in the last decade offers such an opportunity and is under rigorous investigation. This important issue will also be discussed. Keywords: CRISPR-Cas9, gene therapy, gene delivery/modulation, inherited neuromuscular disorders, therapeutic advances.}, }
@article {pmid39743295, year = {2024}, author = {Jiang, Y and Pan, Q and Wang, Z and Lu, K and Xia, B and Chen, T}, title = {Efficient genome editing in medaka (Oryzias latipes) using a codon-optimized SaCas9 system.}, journal = {Journal of Zhejiang University. Science. B}, volume = {25}, number = {12}, pages = {1083-1096}, pmid = {39743295}, issn = {1862-1783}, support = {32273127, 31771648 and 31672653//the National Natural Science Foundation of China/ ; ZQ2020003//the Scientific Research Foundation of Jimei University/ ; }, mesh = {Animals ; *Oryzias/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Codon ; RNA, Guide, CRISPR-Cas Systems/genetics ; Monophenol Monooxygenase/genetics ; CRISPR-Associated Protein 9/genetics ; RNA, Transfer/genetics ; Staphylococcus aureus/genetics ; PAX6 Transcription Factor/genetics ; }, abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, belonging to the type II CRISPR/Cas system, is an effective gene-editing tool widely used in different organisms, but the size of Streptococcus pyogenes Cas9 (SpCas9) is quite large (4.3 kb), which is not convenient for vector delivery. In this study, we used a codon-optimized Staphylococcus aureus Cas9 (SaCas9) system to edit the tyrosinase (tyr), oculocutaneous albinism II (oca2), and paired box 6.1 (pax6.1) genes in the fish model medaka(Oryzias latipes), in which the size of SaCas9 (3.3 kb) is much smaller and the necessary protospacer-adjacent motif (PAM) sequence is 5'-NNGRRT-3'. We also used a transfer RNA (tRNA)‍-single-guide RNA (sgRNA) system to express the functional sgRNA by transcription eitherin vivo or in vitro, and the combination of SaCas9 and tRNA-sgRNA was used to edit the tyr gene in the medaka genome. The SaCas9/sgRNA and SaCas9/tRNA-sgRNA systems were shown to edit the medaka genome effectively, while the PAM sequence is an essential part for the efficiency of editing. Besides, tRNA can improve the flexibility of the system by enabling the sgRNA to be controlled by a common promoter such as cytomegalovirus. Moreover, the all-in-one cassette cytomegalovirus (CMV)‍-SaCas9-tRNA-sgRNA-tRNA is functional in medaka gene editing. Taken together, the codon-optimized SaCas9 system provides an alternative and smaller tool to edit the medaka genome and potentially other fish genomes.}, }
@article {pmid39743283, year = {2024}, author = {He, YM and Wu, ZP and He, J and Zhang, W and Zhu, FM}, title = {[Establishment and Application of Efficient Gene Editing Method for Classical HLA-I Molecules].}, journal = {Zhongguo shi yan xue ye xue za zhi}, volume = {32}, number = {6}, pages = {1896-1902}, doi = {10.19746/j.cnki.issn.1009-2137.2024.06.040}, pmid = {39743283}, issn = {1009-2137}, mesh = {Humans ; HEK293 Cells ; *Gene Editing ; *Transfection ; *Hematopoietic Stem Cells ; RNA, Guide, CRISPR-Cas Systems ; beta 2-Microglobulin/genetics ; Histocompatibility Antigens Class I/genetics ; Flow Cytometry ; }, abstract = {OBJECTIVE: To establish an efficient gene editing method of HLA-I gene to prepare HLA-I universal hematopoietic stem cells.<br><br>METHODS: The easyedit small guide RNA(sgRNA) was designed according to the sequences of &#x3b2;2 microglobulin gene and synthesized by GenScript company. RNP complexes were formed by NLS-Cas9-NLS nuclease and Easyedit sgRNA according to different molar ratios (1&#x2236;1~1&#x2236;4). Control group and four transfection groups were performed respectively. HEK-293 cells and CD34[+] hematopoietic stem cells were nucleotransfected with RNP complex by Lonza 4D Nucleofector system. The expression of HLA-I on the surface of HEK-293 cells was detected by flow cytometry after transfection for 72 hours, the cleavage effect was determined by T7E1 enzyme digestion reaction and the presence of nested peak in the DNA sequence was identified by direct sequencing.<br><br>RESULTS: The transfection groups had different levels of HLA-I negative expression cell populations by flow cytometry after transient transfection of HEK-293 cells and CD34[+] hematopoietic stem cells with different molar concentrations of RNP complex for 72 hours. There were nested peaks proximal to the sgRNA PAM sequence in the transfection groups by direct DNA sequencing, indicating that sgRNA had obvious editing effect. In the transfection of HEK-293 cells, the highest proportion of HLA-I negative expression cells was (87.69&#xb1;0.83)% when the molar ratio of NLS-Cas9-NLS nuclease to Easyedit sgRNA was 1&#x2236;4. The cutting efficiency of T7E1 was the highest up to (38&#xb1;2.0)% when the molar ratio was 1&#x2236;3. In the transfection of CD34[+] hematopoietic stem cells, the proportion of HLA-I negative expression cells was (91.56&#xb1;3.39)% when the molar ratio was 1&#x2236;2, and the cutting efficiency of T7E1 was (64&#xb1;8.45)% when the molar ratio was 1&#x2236;1.<br><br>CONCLUSION: This study provides an efficient gene editing method for classical HLA-I molecules, which can effectively silence the expression of class HLA-I molecules on the cell surface, and is suitable for stem cell system with difficult transfection.}, }
@article {pmid39741289, year = {2024}, author = {Yazdi, ZF and Roshannezhad, S and Sharif, S and Abbaszadegan, MR}, title = {Recent progress in prompt molecular detection of liquid biopsy using Cas enzymes: innovative approaches for cancer diagnosis and analysis.}, journal = {Journal of translational medicine}, volume = {22}, number = {1}, pages = {1173}, pmid = {39741289}, issn = {1479-5876}, mesh = {Humans ; Liquid Biopsy/methods ; *Neoplasms/diagnosis/genetics/pathology ; *Neoplastic Cells, Circulating/pathology/metabolism ; CRISPR-Cas Systems/genetics ; Cell-Free Nucleic Acids/blood ; Circulating Tumor DNA/blood ; }, abstract = {Creating fast, non-invasive, precise, and specific diagnostic tests is crucial for enhancing cancer treatment outcomes. Among diagnostic methods, those relying on nucleic acid detection are highly sensitive and specific. Recent developments in diagnostic technologies, particularly those leveraging Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), are revolutionizing cancer detection, providing accurate and timely results. In clinical oncology, liquid biopsy has become a noninvasive and early-detectable alternative to traditional biopsies over the last two decades. Analyzing the nucleic acid content of liquid biopsy samples, which include Circulating Tumor Cells (CTCs), Circulating Tumor DNA (ctDNA), Circulating Cell-Free RNA (cfRNA), and tumor extracellular vesicles, provides a noninvasive method for cancer detection and monitoring. In this review, we explore how the characteristics of various Cas (CRISPR-associated) enzymes have been utilized in diagnostic assays for cancer liquid biopsy and highlight their main applications of innovative approaches in monitoring, as well as early and rapid detection of cancers.}, }
@article {pmid39741007, year = {2025}, author = {Lima, BA and Pais, AC and Dupont, J and Dias, P and Custódio, N and Sousa, AB and Carmo-Fonseca, M and Carvalho, C}, title = {Genetic modulation of RNA splicing rescues BRCA2 function in mutant cells.}, journal = {Life science alliance}, volume = {8}, number = {3}, pages = {}, pmid = {39741007}, issn = {2575-1077}, mesh = {Humans ; *BRCA2 Protein/genetics/metabolism ; *RNA Splicing/genetics ; *CRISPR-Cas Systems/genetics ; *Mutation ; Gene Editing/methods ; DNA Repair/genetics ; Nonsense Mediated mRNA Decay/genetics ; RNA, Messenger/genetics/metabolism ; Cell Line, Tumor ; Female ; BRCA1 Protein/genetics/metabolism ; Rad51 Recombinase/genetics/metabolism ; Breast Neoplasms/genetics ; }, abstract = {Variants in the hereditary cancer-associated BRCA1 and BRCA2 genes can alter RNA splicing, producing transcripts that encode internally truncated yet potentially functional proteins. However, few studies have quantitatively analyzed variant-specific splicing isoforms. Here, we investigated cells heterozygous and homozygous for the BRCA2:c.681+5G>C variant. Using droplet digital RT-PCR, we identified two variant-specific mRNA isoforms. The predominant transcript is out-of-frame, contains a premature termination codon, and is degraded via the nonsense-mediated mRNA decay pathway. In addition, we detected a novel minor isoform encoding an internally truncated protein lacking non-essential domains. Homozygous mutant cells expressed low levels of BRCA2 protein and were defective in DNA repair. Using CRISPR-Cas9 gene editing, we induced the production of in-frame transcripts in mutant cells, which resulted in increased protein expression, enhanced RAD51 focus formation, and reduced chromosomal breaks after exposure to genotoxic agents. Our findings highlight the therapeutic potential of splicing modulation to restore BRCA2 function in mutant cells, offering a promising strategy to prevent cancer development.}, }
@article {pmid39740568, year = {2024}, author = {Zhan, X and Jiang, Y and Lei, J and Chen, H and Liu, T and Lan, F and Ying, B and Wu, Y}, title = {DNA Tetrahedron-enhanced single-particle counting integrated with cascaded CRISPR Program for ultrasensitive dual RNAs logic sensing.}, journal = {Journal of colloid and interface science}, volume = {683}, number = {Pt 2}, pages = {521-531}, doi = {10.1016/j.jcis.2024.12.182}, pmid = {39740568}, issn = {1095-7103}, abstract = {CRISPR-Cas-based technology, emerging as a leading platform for molecular assays, has been extensively researched and applied in bioanalysis. However, achieving simultaneous and highly sensitive detection of multiple nucleic acid targets remains a significant challenge for most current CRISPR-Cas systems. Herein, a CRISPR Cas12a based calibratable single particle counting-mediated biosensor was constructed for dual RNAs logic and ultra-sensitive detection in one tube based on DNA Tetrahedron (DTN)-interface supported fluorescent particle probes coupled with a novel synergistic cascaded strategy between CRISPR Cas13a system and strand displacement amplification (SDA). As expected, our platform enables dual RNA molecules intelligent detection using only one crRNA of Cas13a, achieving a sensitivity enhancement of three orders of magnitude assisted with multiple signal amplification and accurate fluorescence particle counting with DTN mediated nano-biointerface enhancement, compared to traditional bulk Cas13a assays. Moreover, the effectiveness and universality of our strategy are experimentally investigated and demonstrated through the detection of mRNAs (cervical cancer swab clinical samples and cultured cancer cells) and bacterial 16s rRNAs. This work not only proposes a highly promising avenue for designing CRISPR-based multiplex detection systems that excel in ultra-sensitivity, specificity, and clinical molecular diagnostics, but also provide new insights into the potential applications of nanotechnology in molecular diagnostics, functional surface engineering, and interface-mediated bioreactions.}, }
@article {pmid39740040, year = {2025}, author = {Cai, W and Yang, M and Zhao, Q and Yi, G and Lin, P and Chen, A and De, G}, title = {hURAT1 Transgenic Mouse Model for Evaluating Targeted Urate-Lowering Agents.}, journal = {International journal of rheumatic diseases}, volume = {28}, number = {1}, pages = {e70034}, doi = {10.1111/1756-185X.70034}, pmid = {39740040}, issn = {1756-185X}, support = {ZXKT23002//the Fundamental Research Funds for the Central Public Welfare Research Institutes/ ; 32273019//National Natural Science Foundation of China/ ; }, mesh = {Animals ; *Hyperuricemia/drug therapy/genetics/blood ; *Disease Models, Animal ; *Organic Anion Transporters/genetics/metabolism ; *Uric Acid/blood ; Humans ; *Organic Cation Transport Proteins/genetics/metabolism ; *Mice, Transgenic ; Uricosuric Agents/pharmacology/therapeutic use ; Gout/drug therapy/genetics/metabolism ; Mice, Inbred C57BL ; Male ; Gout Suppressants/pharmacology/therapeutic use ; Phenotype ; Gene Knock-In Techniques ; Hypoxanthine/metabolism ; Mice ; CRISPR-Cas Systems ; }, abstract = {BACKGROUND: Urate transporter 1 (URAT1) is a well-known therapeutic target for reducing urate levels in the treatment of hyperuricemia and gout. However, current pharmacological studies have failed to evaluate the efficacy of URAT1 inhibitors in non-primate animal models. We established a human URAT1 (hURAT1) transgenic knock-in (KI) mouse model to assess uricosuric agents' effectiveness and characterize URAT1-caused pathogenesis.<br><br>METHODS: We generated hURAT1 transgenic mice using CRISPR/Cas9 KI technique. mUrat1 knockout was achieved by replacing exon 1 coding sequence with a human SLC22A12 coding sequence (CDS)-pA cassette. Based on the above transgenic mice, a hyperuricemia model was further established by hypoxanthine administration.<br><br>RESULTS: The hURAT1-KI mice successfully expressed hURAT1 protein to the apical side of the kidney proximal tubule epithelium, where native human URAT1 is localized in human kidney. Upon hypoxanthine challenge, the blood uric acid (UA) level was elevated in hURAT1-KI mice (251&#x2009;&#x3bc;mol/L), showing an approximately 37% increase compared to wild-type (WT) mice (183.5&#x2009;&#x3bc;mol/L). The elevated blood UA level could be alleviated by hURAT1 inhibitor benzbromarone treatment in the hURAT1-KI mice (164.2&#x2009;&#x3bc;mol/L vs. 251&#x2009;&#x3bc;mol/L, p&#x2009;<&#x2009;0.05) whereas no response was observed in WT littermates (168.8&#x2009;&#x3bc;mol/L vs. 183.5&#x2009;&#x3bc;mol/L).<br><br>CONCLUSION: The hURAT1-KI hyperuricemia mouse model would be valuable for preclinical evaluation of gout treatment with urate-lowering drugs and for studying UA metabolic complexities in humans.}, }
@article {pmid39739718, year = {2024}, author = {Delgado-Nungaray, JA and Figueroa-Yáñez, LJ and Reynaga-Delgado, E and Corona-España, AM and Gonzalez-Reynoso, O}, title = {Unveiling the endogenous CRISPR-Cas system in Pseudomonas aeruginosa PAO1.}, journal = {PloS one}, volume = {19}, number = {12}, pages = {e0312783}, pmid = {39739718}, issn = {1932-6203}, mesh = {*Pseudomonas aeruginosa/genetics/drug effects ; *CRISPR-Cas Systems ; Genome, Bacterial ; Bacterial Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Multidrug resistance in Pseudomonas aeruginosa, a high-priority pathogen per the World Health Organization, poses a global threat due to carbapenem resistance and limited antibiotic treatments. Using the bioinformatic tools CRISPRCasFinder, CRISPRCasTyper, CRISPRloci, and CRISPRImmunity, we analyzed the genome of P. aeruginosa PAO1 and revealed an orphan CRISPR system, suggesting it may be a remnant of a type IV system due to the presence of the DinG protein. This system comprises two CRISPR arrays and noteworthy DinG and Cas3 proteins, supporting recent evidence about the association between type IV and I CRISPR systems. Additionally, we demonstrated a co-evolutionary relationship between the orphan CRISPR system in P. aeruginosa PAO1 and the mobile genetic element and prophages identified. One self-targeting spacer was identified, often associated with bacterial evolution and autoimmunity, and no Acr proteins. This research opens avenues for studying how these CRISPR arrays regulate pathogenicity and for developing alternative strategies using its endogenous orphan CRISPR system against carbapenem-resistant P. aeruginosa strains.}, }
@article {pmid39738995, year = {2024}, author = {Argentel-Martínez, L and Peñuelas-Rubio, O and Herrera-Sepúlveda, A and González-Aguilera, J and Sudheer, S and Salim, LM and Lal, S and Pradeep, CK and Ortiz, A and Sansinenea, E and Hathurusinghe, SHK and Shin, JH and Babalola, OO and Azizoglu, U}, title = {Biotechnological advances in plant growth-promoting rhizobacteria for sustainable agriculture.}, journal = {World journal of microbiology &amp; biotechnology}, volume = {41}, number = {1}, pages = {21}, pmid = {39738995}, issn = {1573-0972}, mesh = {*Agriculture/methods ; *Bacteria/genetics/classification/metabolism ; *Biotechnology/methods ; CRISPR-Cas Systems ; *Crops, Agricultural/microbiology/growth &amp; development ; Gene Editing/methods ; *Genetic Engineering/methods ; Microbiota ; Plant Development ; Plant Roots/microbiology/growth &amp; development ; *Rhizosphere ; Soil Microbiology ; }, abstract = {The rhizosphere, the soil zone surrounding plant roots, serves as a reservoir for numerous beneficial microorganisms that enhance plant productivity and crop yield, with substantial potential for application as biofertilizers. These microbes play critical roles in ecological processes such as nutrient recycling, organic matter decomposition, and mineralization. Plant growth-promoting rhizobacteria (PGPR) represent a promising tool for sustainable agriculture, enabling green management of crop health and growth, being eco-friendly alternatives to replace chemical fertilizers and pesticides. In this sense, biotechnological advancements respecting genomics and gene editing have been crucial to develop microbiome engineering which is pivotal in developing microbial consortia to improve crop production. Genome mining, which involves comprehensive analysis of the entire genome sequence data of PGPR, is crucial for identifying genes encoding valuable bacterial enzymes and metabolites. The CRISPR-Cas system, a cutting-edge genome-editing technology, has shown significant promise in beneficial microbial species. Advances in genetic engineering, particularly CRISPR-Cas, have markedly enhanced grain output, plant biomass, resistance to pests, and the sensory and nutritional quality of crops. There has been a great advance about the use of PGPR in important crops; however, there is a need to go further studying synthetic microbial communities, microbiome engineering, and gene editing approaches in field trials. This review focuses on future research directions involving several factors and topics around the use of PGPR putting special emphasis on biotechnological advances.}, }
@article {pmid39738767, year = {2024}, author = {Lei, Z and Cai, H and Yan, Q}, title = {NF2 can mediate the expression of CAMK2A in a tissue specific manner.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {31992}, pmid = {39738767}, issn = {2045-2322}, mesh = {Humans ; *Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism/genetics ; *Gene Expression Regulation, Neoplastic ; Meningioma/genetics/metabolism/pathology ; Neurofibromin 2/metabolism/genetics ; Organ Specificity/genetics ; Meningeal Neoplasms/genetics/metabolism/pathology ; Calcium/metabolism ; }, abstract = {Meningioma is the most prevalent primary intracranial tumor, with approximately half of patients harboring NF2 alteration. The rationale behind the presence of NF2 alteration in meningiomas and its absence in non-nerve system tumors remains elusive. Therefore, meningiomas and several non-nerve system tumor types were analyzed using KEGG analysis and CRISPR/Cas 9 technology to determine the role of NF2 in regulating tissue specificity. Moreover, the different regulatory patterns of Ca[2+] and calcium/calmodulin-dependent protein kinase II alpha (CAMK2A) that play a decisive role in NF2 tissue-specific regulation were deciphered. Our results revealed that NF2 has a positive correlation in CAMK2A expression in both meningiomas and normal nervous system tissues but not in non-nervous system tumors and tissues, implying NF2 tissue-specificity is mediated by CAMK2A-related pathways. Thus, targeting CAMK2A may represent a promising strategy for drug screening and the development of therapeutics for NF2-associated meningiomas and other nervous system tumors.}, }
@article {pmid39738613, year = {2024}, author = {Koshi, D and Sugano, J and Yamasaki, F and Kawauchi, M and Nakazawa, T and Oh, M and Honda, Y}, title = {Trans-nuclei CRISPR/Cas9: safe approach for genome editing in the edible mushroom excluding foreign DNA sequences.}, journal = {Applied microbiology and biotechnology}, volume = {108}, number = {1}, pages = {548}, pmid = {39738613}, issn = {1432-0614}, support = {K-2019-002//Institute for Fermentation, Osaka/ ; 18KK0178//Japan Society for the Promotion of Science/ ; 22H00380//Japan Society for the Promotion of Science/ ; 22KK0090//Japan Society for the Promotion of Science/ ; 23KJ1317//Japan Society for the Promotion of Science/ ; PJ015554//National Institute of Horticultural and Herbal Science, Rural Development Administration/ ; PJ015554//National Institute of Horticultural and Herbal Science, Rural Development Administration/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Pleurotus/genetics ; Genome, Fungal/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Nucleus/genetics ; Plasmids/genetics ; CRISPR-Associated Protein 9/genetics ; }, abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-assisted genome editing has been applied to several major edible agaricomycetes, enabling efficient gene targeting. This method is promising for rapid and efficient breeding to isolate high-value cultivars and overcome cultivation challenges. However, the integration of foreign DNA fragments during this process raises concerns regarding genetically modified organisms (GMOs) and their regulatory restrictions. In this study, we developed a foreign-DNA-free genome editing method in Pleurotus ostreatus by transferring the Cas9/guide RNA (gRNA) complex between nuclei in the dikaryotic state. We isolated a donor monokaryotic P. ostreatus strain expressing Cas9 and gRNA targeting pyrG by introducing a recombinant plasmid, which exhibited uracil auxotrophy and 5-fluoroorotic acid (5-FOA) resistance. This strain was then crossed with a pyrG[+] recipient monokaryon, resulting in dikaryotic strains exhibiting 5-FOA resistance after mycelial growth. When these strains were de-dikaryonized into monokaryons through protoplasting, we obtained monokaryotic isolates harboring the recipient nucleus with small indels at the pyrG target site. Importantly, these isolates were confirmed to be free of foreign DNA through genomic PCR, Southern blotting, and whole-genome resequencing analyses. This is the first report of an efficient genome editing protocol in agaricomycetes that ensures no integration of exogenous DNA. This approach is expected to be applicable to other fungi with a dikaryotic life cycle, opening new possibilities for molecular breeding without the concerns associated with GMOs. KEY POINTS: • Successful genome editing via CRISPR/Cas9 trans-nuclei manner in P. ostreatus. • Recipient monokaryons from gene-edited dikaryons showed no exogenous DNA sequences. • Efficient genome editing protocol for safer molecular breeding in mushroom fungus.}, }
@article {pmid39738601, year = {2024}, author = {Esmaeili, M and Smith, DA and Mead, B}, title = {miRNA changes associated with differentiation of human embryonic stem cells into human retinal ganglion cells.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {31895}, pmid = {39738601}, issn = {2045-2322}, support = {5183/5184//Fight for Sight UK/ ; }, mesh = {*MicroRNAs/genetics/metabolism ; Humans ; *Retinal Ganglion Cells/metabolism/cytology ; *Cell Differentiation/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; Animals ; Rats ; Cell Line ; CRISPR-Cas Systems ; }, abstract = {miRNA, short non-coding RNA, are rapidly emerging as important regulators in cell homeostasis, as well as potential players in cellular degeneration. The latter has led to interest in them as both biomarkers and as potential therapeutics. Retinal ganglion cells (RGC), whose axons connect the eye to the brain, are central nervous system cells of great interest, yet their study is largely restricted to animals due to the difficulty in obtaining healthy human RGC. Using a CRISPR/Cas9-based reporter embryonic stem cell line, human RGC were generated and their miRNA profile characterized using NanoString miRNA assays. We identified a variety of retinal specific miRNA upregulated in ESC-derived RGC, with half of the most abundant miRNA also detectable in purified rat RGC. Several miRNA were however identified to be unique to RGC from human. The findings show which miRNA are abundant in RGC and the limited congruence with animal derived RGC. These data could be used to understand miRNA's role in RGC function, as well as potential biomarkers or therapies in retinal diseases involving RGC degeneration.}, }
@article {pmid39737993, year = {2024}, author = {Yuan, Q and Zeng, H and Daniel, TC and Liu, Q and Yang, Y and Osikpa, EC and Yang, Q and Peddi, A and Abramson, LM and Zhang, B and Xu, Y and Gao, X}, title = {Orthogonal and multiplexable genetic perturbations with an engineered prime editor and a diverse RNA array.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10868}, pmid = {39737993}, issn = {2041-1723}, support = {CBET-2143626//National Science Foundation (NSF)/ ; }, mesh = {Humans ; *Gene Editing/methods ; *Trans-Activators/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Homeodomain Proteins/genetics/metabolism ; Amyloid Neuropathies, Familial/genetics/therapy ; HEK293 Cells ; RNA/genetics ; Genome, Human ; CRISPR-Cas Systems ; Mutation ; RNA Interference ; Genetic Engineering/methods ; }, abstract = {Programmable and modular systems capable of orthogonal genomic and transcriptomic perturbations are crucial for biological research and treating human genetic diseases. Here, we present the minimal versatile genetic perturbation technology (mvGPT), a flexible toolkit designed for simultaneous and orthogonal gene editing, activation, and repression in human cells. The mvGPT combines an engineered compact prime editor (PE), a fusion activator MS2-p65-HSF1 (MPH), and a drive-and-process multiplex array that produces RNAs tailored to different types of genetic perturbation. mvGPT can precisely edit human genome via PE coupled with a prime editing guide RNA and a nicking guide RNA, activate endogenous gene expression using PE with a truncated single guide RNA containing MPH-recruiting MS2 aptamers, and silence endogenous gene expression via RNA interference with a short-hairpin RNA. We showcase the versatility of mvGPT by simultaneously correcting a c.3207C>A mutation in the ATP7B gene linked to Wilson's disease, upregulating the PDX1 gene expression to potentially treat Type I diabetes, and suppressing the TTR gene to manage transthyretin amyloidosis. In addition to plasmid delivery, we successfully utilize various methods to deliver the mvGPT payload, demonstrating its potential for future in vivo applications.}, }
@article {pmid39737984, year = {2024}, author = {Nagamura, R and Kujirai, T and Kato, J and Shuto, Y and Kusakizako, T and Hirano, H and Endo, M and Toki, S and Saika, H and Kurumizaka, H and Nureki, O}, title = {Structural insights into how Cas9 targets nucleosomes.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10744}, pmid = {39737984}, issn = {2041-1723}, support = {JP23fa627001//Japan Agency for Medical Research and Development (AMED)/ ; JP19am0401005//Japan Agency for Medical Research and Development (AMED)/ ; JP23ama121002//Japan Agency for Medical Research and Development (AMED)/ ; JP23ama121012//Japan Agency for Medical Research and Development (AMED)/ ; JP23ama121009//Japan Agency for Medical Research and Development (AMED)/ ; JP23H05475//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JPMJER1901//MEXT | JST | Exploratory Research for Advanced Technology (ERATO)/ ; }, mesh = {*Nucleosomes/metabolism/ultrastructure ; *Cryoelectron Microscopy ; *DNA/metabolism/chemistry ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; *RNA, Guide, CRISPR-Cas Systems/metabolism ; *Gene Editing ; Histones/metabolism/chemistry ; CRISPR-Cas Systems ; Chromatin/metabolism/chemistry ; DNA Cleavage ; Models, Molecular ; }, abstract = {The CRISPR-associated endonuclease Cas9 derived from prokaryotes is used as a genome editing, which targets specific genomic loci by single guide RNAs (sgRNAs). The eukaryotes, the target of genome editing, store their genome DNA in chromatin, in which the nucleosome is a basic unit. Despite previous structural analyses focusing on Cas9 cleaving free DNA, structural insights into Cas9 targeting of DNA within nucleosomes are limited, leading to uncertainties in understanding how Cas9 operates in the eukaryotic genome. In the present study, we perform native-polyacrylamide gel electrophoresis (PAGE) analyses and find that Cas9 targets the linker DNA and the entry-exit DNA region of the nucleosome but not the DNA tightly wrapped around the histone octamer. We further determine cryo-electron microscopy (cryo-EM) structure of the Cas9-sgRNA-nucleosome ternary complex that targets linker DNA in nucleosomes. The structure suggests interactions between Cas9 and nucleosomes at multiple sites. Mutants that reduce the interaction between nucleosomal DNA and Cas9 improve nucleosomal DNA cleavage activity in vitro, although inhibition by the interaction between Cas9 and nucleosomes is limited in vivo. These findings will contribute to the development of novel genome editing tools in chromatin.}, }
@article {pmid39737975, year = {2024}, author = {Wu, Y and Zhong, A and Sidharta, M and Kim, TW and Ramirez, B and Persily, B and Studer, L and Zhou, T}, title = {Robust and inducible genome editing via an all-in-one prime editor in human pluripotent stem cells.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10824}, pmid = {39737975}, issn = {2041-1723}, support = {P30 CA008748/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; *Gene Editing/methods ; *Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems ; Cell Differentiation/genetics ; Tumor Suppressor Protein p53/genetics/metabolism ; Genome, Human ; Mutation ; Cell Line ; }, abstract = {Prime editing (PE) allows for precise genome editing in human pluripotent stem cells (hPSCs), such as introducing single nucleotide modifications, small insertions or deletions at a specific genomic locus. Here, we systematically compare a panel of prime editing conditions in hPSCs and generate a potent prime editor, "PE-Plus", through co-inhibition of mismatch repair and p53-mediated cellular stress responses. We further establish an inducible prime editing platform in hPSCs by incorporating the PE-Plus into a safe-harbor locus and demonstrated temporal control of precise editing in both hPSCs and differentiated cells. By evaluating disease-associated mutations, we show that this platform allows efficient creation of both monoallelic and biallelic disease-relevant mutations in hPSCs. In addition, this platform enables the efficient introduction of single or multiple edits in one step, demonstrating potential for multiplex editing. Our method presents an efficient and controllable multiplex prime editing tool in hPSCs and their differentiated progeny.}, }
@article {pmid39737904, year = {2024}, author = {Li, D and Zhang, S and Lin, S and Xing, W and Yang, Y and Zhu, F and Su, D and Chen, C and Liu, JG}, title = {Cas12e orthologs evolve variable structural elements to facilitate dsDNA cleavage.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10727}, pmid = {39737904}, issn = {2041-1723}, support = {32150018//National Natural Science Foundation of China (National Science Foundation of China)/ ; 21877069//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22277063//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22061160466//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32101195//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2022M711848//China Postdoctoral Science Foundation/ ; }, mesh = {*CRISPR-Cas Systems ; *DNA/metabolism/genetics/chemistry ; *DNA Cleavage ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *Gene Editing/methods ; Evolution, Molecular ; Protein Domains ; }, abstract = {Exceptionally diverse type V CRISPR-Cas systems provide numerous RNA-guided nucleases as powerful tools for DNA manipulation. Two known Cas12e nucleases, DpbCas12e and PlmCas12e, are both effective in genome editing. However, many differences exist in their in vitro dsDNA cleavage activities, reflecting the diversity in Cas12e's enzymatic properties. To comprehensively understand the Cas12e family, we identify and characterize six unreported Cas12e members that vary in their CRISPR-locus architectures, PAM preferences, and cleavage efficacies. Interestingly, among all variants, PlmCas12e exhibits the most robust trans-cleavage activity and the lowest salt sensitivity in cis-cleavage. Further structural comparisons reveal that the unique NTSB domain in PlmCas12e is beneficial to DNA unwinding at high salt concentrations, while some NTSB-lacking Cas12e proteins rely on positively charged loops for dsDNA unwinding. These findings demonstrate how divergent evolution of structural elements shapes the nuclease diversity within the Cas12e family, potentially contributing to their adaptations to varying environmental conditions.}, }
@article {pmid39736572, year = {2024}, author = {Stelmach-Wityk, K and Szymonik, K and Grzebelus, E and Kiełkowska, A}, title = {Development of an optimized protocol for protoplast-to-plant regeneration of selected varieties of Brassica oleracea L.}, journal = {BMC plant biology}, volume = {24}, number = {1}, pages = {1279}, pmid = {39736572}, issn = {1471-2229}, mesh = {*Protoplasts ; *Brassica/genetics/physiology/growth &amp; development ; *Regeneration ; Plant Shoots/genetics/growth &amp; development ; Plant Breeding/methods ; Plant Roots/genetics/growth &amp; development/physiology ; }, abstract = {BACKGROUND: Brassica oleracea L. is a key plant in the Brassicaceae family, known for popular vegetables like cabbage, broccoli, kale and collard. Collard (B. oleracea var. viridis) is a non-heading leafy vegetable grown in urban farms and community gardens in the United States and Europe. Improving collard and other Brassica germplasm can benefit from both traditional and new plant breeding technologies (NPBTs), such as CRISPR-Cas mediated transformation. An efficient transformation or protoplast fusion can only be achieved with a robust and reproducible protocol for protoplast-to-plant regeneration. This research focuses on optimizing in vitro culture conditions to enhance cell divisions, microcallus formation, and the regeneration of shoots and roots in four Brassica oleracea varieties, including collard.<br><br>RESULTS: The protocol of protoplast release, purification and immobilization was optimized to obtain a suitable number and quality of protoplasts from seven cultivars of B. oleracea. The protoplast isolation efficiency after digestion of young leaves in optimized enzyme solution reached on average 2.5&#x2009;&#xd7;&#x2009;10[6] of cells per gram of fresh weight. Protoplasts were embedded in thin alginate layers and subjected to culture in three different media. Protoplasts of all studied cultivars were viable (88.2%), underwent cell wall resynthesis and re-entered mitotic divisions in the 5th day of culture. After 30 days of culture, protoplast-derived cells of all the tested cultivars formed microcallus. Six cultivars regenerated shoots, although the shoot formation efficiency strongly depended on the genotype and composition of the regeneration medium. The regeneration medium supplemented with 1 mg&#xa0;l[-1] of NAA, 1 mg&#xa0;l[-1] of 2iP, 0.02 mg&#xa0;l[-1] GA3 and with 2% of mannitol showed the highest shoot formation efficiency for five cultivars of B. oleracea.<br><br>CONCLUSIONS: The results of this research have led to the development of a robust protoplast-to-plant regeneration protocol for four varieties of B. oleracea that could be exploited as a tool for production of transformants and somatic hybrids. Furthermore, we present the first successful regeneration of protoplast-derived plants of collard, an overlooked but valuable variety of Brassica oleracea.}, }
@article {pmid39735189, year = {2024}, author = {Lu, Z and Liang, M and Li, C and Xu, Y and Weng, S and He, J and Guo, C}, title = {Rapid, sensitive, and visual detection of mandarin fish ranavirus and infectious spleen and kidney necrosis virus using an RPA-CRISPR/Cas12a system.}, journal = {Frontiers in microbiology}, volume = {15}, number = {}, pages = {1495777}, pmid = {39735189}, issn = {1664-302X}, abstract = {Iridoviruses are large cytoplasmic icosahedral viruses that contain dsDNA. Among them, mandarin fish ranavirus (MRV) and infectious spleen and kidney necrosis virus (ISKNV) are particularly notable due to their high contagiousness and pathogenicity. These viruses pose a significant threat to fish aquaculture, resulting in substantial annual economic losses for the fish farming industry. Therefore, the development of novel, rapid virus detection technologies is essential for the prevention and control of ISKNV and MRV diseases. In this study, we developed a rapid, sensitive, and visual detection method for MRV and ISKNV using the recombinase polymerase amplification (RPA)-CRISPR/Cas12a system. This method can detect as low as 1 copy/μL of MRV and 0.1 copy/μL of ISKNV, demonstrating excellent specificity and reproducibility. The detection can be performed at a constant temperature of 37-39°C, eliminating the need for complex equipment. A 30-min RPA amplification followed by a 15-min CRISPR/Cas reaction is sufficient for detecting most samples. For low-concentration samples, extending the CRISPR/Cas reaction time to 60 min improves result visibility. The designed RPA reaction system is capable of performing reverse transcription of RNA, allowing for the detection of mRNA transcribed from the MCP gene of MRV and ISKNV in the sample. Furthermore, two probes were identified that can be observed without the need for excitation light. In conclusion, a field-suitable detection method for ISKNV and MRV has been established, providing a powerful tool for the prompt diagnosis of these aquatic pathogens and aiding in the prevention and control of ISKNV and MRV diseases.}, }
@article {pmid39734219, year = {2024}, author = {Wen, Q and Chen, J and Li, J and Dharmasiddhi, IPW and Yang, M and Xing, J and Liu, Y}, title = {A single-plasmid-based, easily curable CRISPR/Cas9 system for rapid, iterative genome editing in Pseudomonas putida KT2440.}, journal = {Microbial cell factories}, volume = {23}, number = {1}, pages = {349}, pmid = {39734219}, issn = {1475-2859}, support = {2023YFC3403601//National Key Research and Development Program of China/ ; 2023YFC3403601//National Key Research and Development Program of China/ ; 2023YFC3403601//National Key Research and Development Program of China/ ; }, mesh = {*Pseudomonas putida/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Plasmids/genetics ; Genome, Bacterial ; }, abstract = {BACKGROUND: Pseudomonas putida KT2440, a non-pathogenic soil bacterium, is a key platform strain in synthetic biology and industrial applications due to its robustness and metabolic versatility. Various systems have been developed for genome editing in P. putida, including transposon modules, integrative plasmids, recombineering systems, and CRISPR/Cas systems. However, rapid iterative genome editing is limited by complex and lengthy processes.<br><br>RESULTS: We discovered that the pBBR1MCS2 plasmid carrying the CRISPR/Cas9 module could be easily cured in P. putida KT2440 at 30 [o]C. We then developed an all-in-one CRISPR/Cas9 system for yqhD and ech-vdh-fcs deletions, respectively, and further optimized the editing efficiency by varying homology arm lengths and target sites. Sequential gene deletions of vdh and vanAB were carried out rapidly using single-round processing and easy plasmid curing. This system's user-friendliness was validated by 3 researchers from two labs for 9 deletions, 3 substitutions, and 2 insertions. Finally, iterative genome editing was used to engineer P. putida for valencene biosynthesis, achieving a 10-fold increase in yield.<br><br>CONCLUSIONS: We developed and applied a rapid all-in-one plasmid CRISPR/Cas9 system for genome editing in P. putida. This system requires less than 1.5 days for one edit due to simplified plasmid construction, electroporation and curing processes, thus accelerating the cycle of genome editing. To our knowledge, this is the fastest iterative genome editing system for P. putida. Using this system, we rapidly engineered P. putida for valencene biosynthesis for the first time, showcasing the system's potential for expanding biotechnological applications.}, }
@article {pmid39730605, year = {2024}, author = {Sassone, F and Estay-Ahumada, C and Roux, MJ and Ciocca, D and Rossolillo, P and Birling, MC and Sparrow, JR and Montenegro, D and Hicks, D}, title = {Interruption of the visual cycle in a novel animal model induces progressive vision loss resembling Stargardts Disease.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {30880}, pmid = {39730605}, issn = {2045-2322}, support = {R01 EY012951/EY/NEI NIH HHS/United States ; NEI EY012951//UNADEV/ITMO Aviesan 2018-2021, Fondation de France (Association Berthe Fouassier) and USIAS (DH); and NEI EY012951 (JRS)./ ; }, mesh = {Animals ; *Disease Models, Animal ; *ATP-Binding Cassette Transporters/genetics/metabolism ; *Stargardt Disease ; Humans ; Retinal Cone Photoreceptor Cells/metabolism/pathology ; Macular Degeneration/pathology/genetics/metabolism ; Electroretinography ; CRISPR-Cas Systems ; Rats ; Retina/metabolism/pathology/physiopathology ; Tomography, Optical Coherence ; Retinal Degeneration/metabolism/pathology/genetics ; }, abstract = {Mutations in the gene ABCA4 coding for photoreceptor-specific ATP-binding cassette subfamily A member 4, are responsible for Stargardts Disease type 1 (STGD1), the most common form of inherited macular degeneration. STGD1 typically declares early in life and leads to severe visual handicap. Abca4 gene-deletion mouse models of STGD1 accumulate lipofuscin, a hallmark of the disease, but unlike the human disease show no or only moderate structural changes and no functional decline. The human macula is highly enriched in cones, and reasoning that the low cone percentage in mice retinas (< 3%) might compromise faithful modelling of human maculopathies, we performed sub-retinal injections of CRISPR/Cas9-abca4 Adeno-Associated Virus constructs into young Sand Rats (Psammomys obesus), a diurnal rodent containing > 30% cones. Compared to control injections of AAV-abca4-GFP, treated eyes exhibited extensive retinal degeneration by two months. Sanger sequencing of the CRISPR targeted sequence show a clear edition of Abca4 gene. Non-invasive fundus imaging showed widespread photoreceptor loss, confirmed by ocular coherence tomography. Functional recording by single flash and flicker electroretinography showed significant decline in photopic (cone) light responses. Post-mortem real-time PCR, immunohistochemistry and western blotting showed significant decrease of cone-specific (MW cone opsin) but not rod-specific (rhodopsin) markers. Transmission electron microscopy showed large numbers of lipid inclusions in treated but not control retinal pigmented epithelium. Finally, ultra-high performance liquid chromatography analysis of whole P. obesus eyes showed the presence of all-trans retinal-dimer, not detected in rod-rich rat eyes. In conclusion, Abca4 knockout in P. obesus results in a predominantly cone degeneration phenotype, more accurately reflecting the etiology of human STGD1, and should be valuable for characterizing pathogenic pathways and exploring treatment options.}, }
@article {pmid39730601, year = {2024}, author = {Nasrallah, A and Rezvani, HR and Kobaisi, F and Hammoud, A and Rambert, J and Smits, JPH and Sulpice, E and Rachidi, W}, title = {Generation and characterization of CRISPR-Cas9-mediated XPC gene knockout in human skin cells.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {30879}, pmid = {39730601}, issn = {2045-2322}, support = {ANR-18-CE17-0017//ANR grant PG2HEAL/ ; ANR-15-IDEX-02//the French National Research Agency in the framework of the "Investissements d'avenir" program (ANR-15-IDEX-02)./ ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *DNA-Binding Proteins/genetics/metabolism ; *DNA Repair ; *Xeroderma Pigmentosum/genetics ; *Gene Knockout Techniques ; *Ultraviolet Rays/adverse effects ; Skin/metabolism/pathology/radiation effects ; DNA Damage ; Keratinocytes/metabolism ; Fibroblasts/metabolism/radiation effects ; Melanocytes/metabolism/radiation effects ; }, abstract = {Xeroderma pigmentosum group C (XPC) is a versatile protein crucial for sensing DNA damage in the global genome nucleotide excision repair (GG-NER) pathway. This pathway is vital for mammalian cells, acting as their essential approach for repairing DNA lesions stemming from interactions with environmental factors, such as exposure to ultraviolet (UV) radiation from the sun. Loss-of-function mutations in the XPC gene confer a photosensitive phenotype in XP-C patients, resulting in the accumulation of unrepaired UV-induced DNA damage. This remarkable increase in DNA damage tends to elevate by 10,000-fold the risk of developing melanoma and non-melanoma skin cancers. To date, creating accurate and reproducible models to study human XP-C disease has been an important challenge. To tackle this, we used CRISPR-Cas9 technology in order to knockout the XPC gene in various human skin cells (keratinocytes, fibroblasts, and melanocytes). After validation of the knockout in these edited skin cells, we showed that they recapitulate the major phenotypes of XPC mutations: photosensitivity and the impairment of UV-induced DNA damage repair. Moreover, these knockout cells demonstrated a reduced proliferative capacity compared to their respective controls. Finally, to better mimic the disease environment, we built a 3D reconstructed skin using these XPC knockout skin cells. This model exhibited an abnormal behavior, showing an extensive remodeling of its extracellular matrix compared to normal skin. Analyzing the composition of the fibroblast secretome revealed a significant augmented shift in the inflammatory response following XPC knockout. Our innovative "disease on a dish" approach can provide valuable insights into the molecular mechanisms underlying XP-C disease, paving the way to design novel preventive and therapeutic strategies to alleviate the disease phenotype. Also, given the high risk of skin cancer onset in XP-C disease, our new approach can serve as a link to draw novel insights into this elusive field.}, }
@article {pmid39730554, year = {2024}, author = {Cheng, Y and Zhang, L and Ke, Y and Dang, X and Miki, D}, title = {Double step screening using endogenous marker improves relative gene targeting efficiency in Arabidopsis.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {30791}, pmid = {39730554}, issn = {2045-2322}, support = {20ZR1467000//Shanghai Science and Technology Innovation Plan/ ; G202201355L//Foreign Expert Project/ ; }, mesh = {*Arabidopsis/genetics ; *CRISPR-Cas Systems ; *Gene Targeting/methods ; Genetic Markers ; Plants, Genetically Modified/genetics ; Arabidopsis Proteins/genetics/metabolism ; Genome, Plant ; Gene Editing/methods ; }, abstract = {Gene targeting (GT) is a powerful tool for manipulating endogenous genomic sequences as intended. However, its efficiency is rather low, especially in seed plants. Numerous attempts have been made to improve the efficiency of GT via the CRISPR/Cas systems in plants, but these have not been sufficiently effective to be used routinely by everyone. Here, we report a surrogate screening method that improves the relative efficiency of CRISPR/Cas9-mediated GT in Arabidopsis. Our findings indicate that simultaneous mutagenesis of the endogenous MAR1 gene, which results in kanamycin resistance, can be employed to efficiently screen for precise and heritable GT events at multiple endogenous sites in the Arabidopsis genome. In this study, we demonstrate that a double-step screening strategy can achieve up to a four-fold increase in the efficiency of GT in Arabidopsis. The principle of this surrogate system has the potential to be widely applied.}, }
@article {pmid39729269, year = {2025}, author = {Qiu, M and Chen, N and Wang, Y}, title = {CRISPR/Cas9-Mediated Gene-Knockout and In Situ Complementation System for Phytophthora sojae.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2892}, number = {}, pages = {69-82}, pmid = {39729269}, issn = {1940-6029}, mesh = {*Phytophthora/genetics ; *CRISPR-Cas Systems ; *Gene Knockout Techniques/methods ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genetic Complementation Test ; Plasmids/genetics ; Genetic Vectors/genetics ; }, abstract = {The establishment of reliable and efficient systems for genome editing in Phytophthora is very important for studying gene functions. Here, step-by-step methods for CRISPR/Cas9-based gene knockout and in situ complementation for Phytophthora sojae are presented. These steps include the sgRNA design, Cas9-sgRNA plasmid construction, homologous replacement, complementation vector construction, P. sojae transformation, and detection of mutations for both gene knockout and in situ complementation. These methods may also potentially be adapted for other Phytophthora species.}, }
@article {pmid39729268, year = {2025}, author = {Mendoza, CS and Ah-Fong, AMV and Judelson, HS}, title = {Gene Editing and Protein Tagging in the Oomycete Phytophthora infestans Using CRISPR-Cas12a.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2892}, number = {}, pages = {49-67}, pmid = {39729268}, issn = {1940-6029}, mesh = {*Gene Editing/methods ; *Phytophthora infestans/genetics ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA End-Joining Repair/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Recombinational DNA Repair ; Genetic Vectors/genetics ; Endodeoxyribonucleases ; }, abstract = {Molecular genetic tools such as CRISPR-Cas gene editing systems are invaluable for understanding gene and protein function and revealing the details of a pathogen's life and disease cycles. Here we present protocols for genome editing in Phytophthora infestans, an oomycete with global importance as a pathogen of potato and tomato. Using a vector system that expresses variants of Cas12a from Lachnospiraceae bacterium and its guide RNA from a unified transcript, we first present a method for editing genes through the non-homologous end-joining (NHEJ) pathway. We then describe an application of homology-directed repair (HDR), in which Cas12a is used to fuse a protein-coding gene with a fluorescent or epitope tag. Both methods should be adaptable to many oomycetes other than P. infestans.}, }
@article {pmid39727993, year = {2024}, author = {Mascarenhas, MS and Nascimento, FDS and Schittino, LMP and Galinari, LB and Lino, LSM and Ramos, APS and Diniz, LEC and Mendes, TAO and Ferreira, CF and Santos-Serejo, JAD and Amorim, EP}, title = {Construction and Validation of CRISPR/Cas Vectors for Editing the PDS Gene in Banana (Musa spp.).}, journal = {Current issues in molecular biology}, volume = {46}, number = {12}, pages = {14422-14437}, pmid = {39727993}, issn = {1467-3045}, support = {OPP1093845//IITA - Accelerated Breeding of Better Bananas/ ; OPP1093845//Bill and Melinda Gates Foundation - Accelerated Breeding of Better Bananas/ ; }, abstract = {Bananas and plantains are important staple food crops affected by biotic and abiotic stresses. The gene editing technique via Clustered Regularly Interspaced Short Palindromic Repeats associated with the Cas protein (CRISPR/Cas) has been used as an important tool for development of cultivars with high tolerance to stresses. This study sought to develop a protocol for the construction of vectors for gene knockout. Here we use the phytoene desaturase (PDS) gene as a case study in Prata-Anã banana by the nonhomologous end junction (NHEJ) method. PDS is a key gene in the carotenoid production pathway in plants and its knockout leads to easily visualized phenotypes such as dwarfism and albinism in plants. Agrobacterium-mediated transformation delivered CRISPR/Cas9 constructs containing gRNAs were inserted into embryogenic cell suspension cultures. This is the first study to provide an effective method/protocol for constructing gene knockout vectors, demonstrating gene editing potential in a Brazilian banana variety. The constitutive (CaMV 35S) and root-specific vectors were successfully assembled and confirmed in transformed Agrobacterium by DNA extraction and PCR. The specificity of transformation protocols makes it possible to use the CRISPR-Cas9 technique to develop Prata-Anã banana plants with enhanced tolerance/resistance to major biotic and abiotic factors.}, }
@article {pmid39727898, year = {2024}, author = {Zhang, X and Huang, Z and Zhang, Y and Wang, W and Ye, Z and Liang, P and Sun, K and Kang, W and Tang, Q and Yu, X}, title = {Mitigating Antibiotic Resistance: The Utilization of CRISPR Technology in Detection.}, journal = {Biosensors}, volume = {14}, number = {12}, pages = {}, pmid = {39727898}, issn = {2079-6374}, support = {2022C02049//the "Pioneer" and "Leading Goose" R&amp;D Program of Zhejiang/ ; 2024SNJF044//Zhejiang Provincial Department of Agriculture and Rural Affairs Project/ ; 2023SNJF066//the Zhejiang Provincial Department of Agriculture and Rural Affairs Project/ ; }, mesh = {*CRISPR-Cas Systems ; *Anti-Bacterial Agents/pharmacology ; Humans ; Drug Resistance, Microbial ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Bacteria ; Biosensing Techniques ; Drug Resistance, Bacterial ; }, abstract = {Antibiotics, celebrated as some of the most significant pharmaceutical breakthroughs in medical history, are capable of eliminating or inhibiting bacterial growth, offering a primary defense against a wide array of bacterial infections. However, the rise in antimicrobial resistance (AMR), driven by the widespread use of antibiotics, has evolved into a widespread and ominous threat to global public health. Thus, the creation of efficient methods for detecting resistance genes and antibiotics is imperative for ensuring food safety and safeguarding human health. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) systems, initially recognized as an adaptive immune defense mechanism in bacteria and archaea, have unveiled their profound potential in sensor detection, transcending their notable gene-editing applications. CRISPR/Cas technology employs Cas enzymes and guides RNA to selectively target and cleave specific DNA or RNA sequences. This review offers an extensive examination of CRISPR/Cas systems, highlighting their unique attributes and applications in antibiotic detection. It outlines the current utilization and progress of the CRISPR/Cas toolkit for identifying both nucleic acid (resistance genes) and non-nucleic acid (antibiotic micromolecules) targets within the field of antibiotic detection. In addition, it examines the current challenges, such as sensitivity and specificity, and future opportunities, including the development of point-of-care diagnostics, providing strategic insights to facilitate the curbing and oversight of antibiotic-resistance proliferation.}, }
@article {pmid39727873, year = {2024}, author = {Lu, Z and Ye, Z and Li, P and Jiang, Y and Han, S and Ma, L}, title = {An MSRE-Assisted Glycerol-Enhanced RPA-CRISPR/Cas12a Method for Methylation Detection.}, journal = {Biosensors}, volume = {14}, number = {12}, pages = {}, pmid = {39727873}, issn = {2079-6374}, support = {WDZC20200821104802001//Universities Stable Funding Key Projects/ ; 2020YFA0908900//The National Key R&amp;D Program of China/ ; n/a//State Key Laboratory of Chemical Oncogenomics/ ; }, mesh = {*DNA Methylation ; *CRISPR-Cas Systems ; Humans ; *Glycerol ; DNA Restriction Enzymes ; Biosensing Techniques ; Nasopharyngeal Carcinoma/diagnosis/genetics ; Nucleic Acid Amplification Techniques ; Endodeoxyribonucleases ; }, abstract = {BACKGROUND: Nasopharyngeal carcinoma (NPC) is a malignant tumor with high prevalence in southern China. Aberrant DNA methylation, as a hallmark of cancer, is extensively present in NPC, the detection of which facilitates early diagnosis and prognostic improvement of NPC. Conventional methylation detection methods relying on bisulfite conversion have limitations such as time-consuming, complex processes and sample degradation; thus, a more rapid and efficient method is needed.<br><br>METHODS: We propose a novel DNA methylation assay based on methylation-sensitive restriction endonuclease (MSRE) HhaI digestion and Glycerol-enhanced recombinase polymerase amplification (RPA)-CRISPR/Cas12a detection (HGRC). MSRE has a fast digestion rate, and HhaI specifically cleaves unmethylated DNA at a specific locus, leaving the methylated target intact to trigger the downstream RPA-Cas12a detection step, generating a fluorescence signal. Moreover, the detection step was supplemented with glycerol for the separation of Cas12a-containing components and RPA- and template-containing components, which avoids over-consumption of the template and, thus, enhances the amplification efficiency and detection sensitivity.<br><br>RESULTS: The HGRC method exhibits excellent performance in the detection of a CNE2-specific methylation locus with a (limit of detection) LOD of 100 aM and a linear range of 100 aM to 100 fM. It also responds well to different methylation levels and is capable of distinguishing methylation levels as low as 0.1%. Moreover, this method can distinguish NPC cells from normal cells by detecting methylation in cellular genomes. This method provides a rapid and sensitive approach for NPC detection and also holds good application prospects for other cancers and diseases featuring DNA methylation as a biomarker.}, }
@article {pmid39727179, year = {2024}, author = {Lopez, SC and Lee, Y and Zhang, K and Shipman, SL}, title = {SspA is a transcriptional regulator of CRISPR adaptation in E. coli.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkae1244}, pmid = {39727179}, issn = {1362-4962}, support = {MCB 2137692//National Science Foundation/ ; //Pew Biomedical Scholars Program/ ; //Chan Zuckerberg Biohub - San Francisco/ ; //Berkeley Fellowship for Graduate Study/ ; }, abstract = {The CRISPR integrases Cas1-Cas2 create immunological memories of viral infection by storing phage-derived DNA in CRISPR arrays, a process known as CRISPR adaptation. A number of host factors have been shown to influence adaptation, but the full pathway from infection to a fully integrated, phage-derived sequences in the array remains incomplete. Here, we deploy a new CRISPRi-based screen to identify putative host factors that participate in CRISPR adaptation in the Escherichia coli Type I-E system. Our screen and subsequent mechanistic characterization reveal that SspA, through its role as a global transcriptional regulator of cellular stress, is required for functional CRISPR adaptation. One target of SspA is H-NS, a known repressor of CRISPR interference proteins, but we find that the role of SspA on adaptation is not H-NS-dependent. We propose a new model of CRISPR-Cas defense that includes independent cellular control of adaptation and interference by SspA.}, }
@article {pmid39726635, year = {2024}, author = {Brant, EJ and May, D and Eid, A and Altpeter, F}, title = {Comparison of genotyping assays for detection of targeted CRISPR/Cas mutagenesis in highly polyploid sugarcane.}, journal = {Frontiers in genome editing}, volume = {6}, number = {}, pages = {1505844}, pmid = {39726635}, issn = {2673-3439}, abstract = {Sugarcane (Saccharum spp.) is an important biofuel feedstock and a leading source of global table sugar. Saccharum hybrid cultivars are highly polyploid (2n = 100-130), containing large numbers of functionally redundant hom(e)ologs in their genomes. Genome editing with sequence-specific nucleases holds tremendous promise for sugarcane breeding. However, identification of plants with the desired level of co-editing within a pool of primary transformants can be difficult. While DNA sequencing provides direct evidence of targeted mutagenesis, it is cost-prohibitive as a primary screening method in sugarcane and most other methods of identifying mutant lines have not been optimized for use in highly polyploid species. In this study, non-sequencing methods of mutant screening, including capillary electrophoresis (CE), Cas9 RNP assay, and high-resolution melt analysis (HRMA), were compared to assess their potential for CRISPR/Cas9-mediated mutant screening in sugarcane. These assays were used to analyze sugarcane lines containing mutations at one or more of six sgRNA target sites. All three methods distinguished edited lines from wild type, with co-mutation frequencies ranging from 2% to 100%. Cas9 RNP assays were able to identify mutant sugarcane lines with as low as 3.2% co-mutation frequency, and samples could be scored based on undigested band intensity. CE was highlighted as the most comprehensive assay, delivering precise information on both mutagenesis frequency and indel size to a 1 bp resolution across all six targets. This represents an economical and comprehensive alternative to sequencing-based genotyping methods which could be applied in other polyploid species.}, }
@article {pmid39726634, year = {2024}, author = {Azeez, SS and Hamad, RS and Hamad, BK and Shekha, MS and Bergsten, P}, title = {Advances in CRISPR-Cas technology and its applications: revolutionising precision medicine.}, journal = {Frontiers in genome editing}, volume = {6}, number = {}, pages = {1509924}, pmid = {39726634}, issn = {2673-3439}, abstract = {CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated proteins) has undergone marked advancements since its discovery as an adaptive immune system in bacteria and archaea, emerged as a potent gene-editing tool after the successful engineering of its synthetic guide RNA (sgRNA) toward the targeting of specific DNA sequences with high accuracy. Besides its DNA editing ability, further-developed Cas variants can also edit the epigenome, rendering the CRISPR-Cas system a versatile tool for genome and epigenome manipulation and a pioneering force in precision medicine. This review explores the latest advancements in CRISPR-Cas technology and its therapeutic and biomedical applications, highlighting its transformative impact on precision medicine. Moreover, the current status of CRISPR therapeutics in clinical trials is discussed. Finally, we address the persisting challenges and prospects of CRISPR-Cas technology.}, }
@article {pmid39726403, year = {2024}, author = {Rasool, HMH and Chen, Q and Gong, X and Zhou, J}, title = {CRISPR/Cas system and its application in the diagnosis of animal infectious diseases.}, journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology}, volume = {38}, number = {24}, pages = {e70252}, pmid = {39726403}, issn = {1530-6860}, support = {2024BEG02029//The Key Research and Development Program of Ningxia Hui Autonomous Region/ ; 2022YFC2304001//MOST | National Key Research and Development Program of China (NKPs)/ ; CAAS-ZDRW202410//ASTIP | The Agricultural Science and Technology Innovation Program/ ; }, mesh = {Animals ; Humans ; *Communicable Diseases/diagnosis/genetics/veterinary ; *CRISPR-Cas Systems ; Molecular Diagnostic Techniques/methods ; Zoonoses/diagnosis/genetics ; }, abstract = {Infectious diseases are a serious threat to the existence of animals and humans' life. In the 21st century, the emergence and re-emergence of several zoonotic and non-zoonotic global pandemic diseases of socio-economic importance has affected billions of humans and animals. The need for expensive equipment and laboratories, non-availability of on-site testing abilities, with time-consuming and low sensitivity and specificity issues of currently available diagnostic techniques to identify these pathogenic micro-organisms on a large scale highlighted the need for developing cheap, portable environment friendly diagnostic methods. In recent years, these issues have been addressed by clustered regularly interspaced palindromic repeats (CRISPR)-based diagnostic platforms that have transformed the molecular diagnostic field due to their outstanding ultra-sensitive nucleic acid detecting capabilities. In this study, we highlight the types, potential of different Cas proteins, and amplification systems. We also illuminate the application of currently available CRISPR integrated setups on the diagnosis of infectious diseases, majorly in food-producing animals (pigs, ruminants, poultry, and aquaculture), domestic pets (dogs and cats), and diseases of zoonotic importance. We conclude the challenges and future perspectives of using these systems to rapidly diagnose and treat other infectious diseases and also develop control strategies to prevent the spread of pathogenic organisms.}, }
@article {pmid39725739, year = {2024}, author = {Pi, N and Xiang, R and Zhu, L and Li, Y and Wu, X}, title = {An HRP-integrated CRISPR/Cas12a biosensor towards chair-side diagnosis for Porphyromonas gingivalis.}, journal = {Biotechnology letters}, volume = {47}, number = {1}, pages = {15}, pmid = {39725739}, issn = {1573-6776}, support = {32060024//National Natural Science Foundation of China/ ; 32000088//National Natural Science Foundation of China/ ; 2022JJ40050//Natural Science Foundation of Hunan Province/ ; }, mesh = {*Porphyromonas gingivalis/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; Bacteroidaceae Infections/diagnosis/microbiology ; Periodontitis/microbiology/diagnosis ; }, abstract = {Rapid diagnostic tools for Porphyromonas gingivalis (Pg), the primary microorganism responsible for the development of periodontitis, particularly those designed for chair-side applications, could provide substantial health benefits to patients. To address this issue, we developed a CRISPR/Cas12a-based rapid Pg detection method. Dual-gRNA and hairpin reporter strategies were employed to enhance CRISPR/Cas12a reaction efficiency. By modifying the hairpin reporter with HRP, the pre-amplification-free HRP-CRISPR/Cas12a reaction was enabled to produce a colorimetric output, amplifying the detection signal. This method achieved high sensitivity (as low as 33 CFU) without the risk of aerosol contamination from pre-amplification. When testing clinical samples, the method showed high consistency with the reference RT-PCR. Furthermore, compared with RT-PCR, this method only requires room temperature operation, is simpler, and has a shorter detection time of about 35 min. In conclusion, the pre-amplification-free HRP-integrated CRISPR/Cas12a detection method requires no complex equipment, making it an ideal, end-user-friendly approach for chair-side Pg detection.}, }
@article {pmid39724853, year = {2024}, author = {Zhao, L and Zhao, Z and Li, N and Wang, X}, title = {The nucleic acid detection using CRISPR/Cas biosensing system with micro-nano modality for point-of-care applications.}, journal = {Talanta}, volume = {286}, number = {}, pages = {127457}, doi = {10.1016/j.talanta.2024.127457}, pmid = {39724853}, issn = {1873-3573}, abstract = {Nucleic acid detection is considered the golden standard for diagnosing infectious diseases caused by various pathogens, including viruses, bacteria, and parasites. PCR and other amplification-based technologies are highly sensitive and specific, allowing for accurate detection and identification of low-level causative pathogens by targeting and amplifying their unique genetic segment (DNA or RNA). However, it is important to recognize that machinery-dependent diagnostic methods may only sometimes be available or practical in resource-limited settings, where direct implementation can be challenging. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based diagnostics offer a promising alternative for nucleic acid detection. These methods provide gene sequence-specific targeting, multiplexing capability, rapid result disclosure, and ease of operation, making them suitable for point-of-care (POC) applications. CRISPR-Cas-based nucleic acid detection leverages the intrinsic gene-editing capabilities of CRISPR systems to detect specific DNA or RNA sequences with high precision, ensuring high specificity in identifying pathogens. When integrated with micro- and nano-technologies, CRISPR-based diagnostics gain additional benefits, including automated microfluidic processes, enhanced multiplexed detection, improved sensitivity through nanoparticle integration, and combined detection strategies. In this review, we analyze the motivations for tailoring the CRISPR-Cas system with microfluidic formats or nanoscale materials for nucleic acid biosensing and detection. We discuss and categorize current achievements in such systems, highlighting their differences, commonalities, and opportunities for addressing challenges, particularly for POC diagnostics. Micro- and nano-technologies can significantly enhance the practical utility of the CRISPR-Cas system, enabling more comprehensive diagnostic and surveillance capabilities. By integrating these technologies, CRISPR-based diagnostics can achieve higher levels of automation, sensitivity, and multiplexing, making them invaluable tools in the global effort to diagnose and control infectious diseases.}, }
@article {pmid39724343, year = {2025}, author = {Nakajima, T and Kuwasaki, Y and Yamamoto, S and Otabe, T and Sato, M}, title = {A Red Light-Activatable Endogenous Gene Transcription System with Red-CPTS.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2840}, number = {}, pages = {45-55}, pmid = {39724343}, issn = {1940-6029}, mesh = {Animals ; *Light ; Mice ; *CRISPR-Cas Systems ; *Optogenetics/methods ; Humans ; *Transcription, Genetic/radiation effects ; Red Light ; }, abstract = {Red light penetrates deep into mammalian tissues and has low phototoxicity. We developed a red light-activatable photoswitch (MagRed) for deep tissue optogenetics. Using MagRed, we developed a red light-activatable endogenous gene transcription system (Red-CPTS) based on CRISPR-Cas9. Here we provide a detailed protocol for endogenous gene activation using Red-CPTS in cultured mammalian cells and living mice in vivo.}, }
@article {pmid39724342, year = {2025}, author = {Renzl, C and Mayer, G}, title = {Optoribogenetic Modulation of Transcription.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2840}, number = {}, pages = {37-44}, pmid = {39724342}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems ; *Optogenetics/methods ; Humans ; Transcription, Genetic ; Light ; Transfection/methods ; HEK293 Cells ; Gene Expression Regulation ; Flow Cytometry/methods ; }, abstract = {Light can be used as a precise and reversible trigger for the activation of optogenetic tools with subcellular resolution. The interaction of the photoreceptor PAL and aptamer 53 was integrated into a CRISPR/dCas9 system, which can be applied for light-controlled activation of gene expression. Here, we describe a protocol for in vitro application of light-dependent overexpression using eBFP as a proof of concept. The experiment can be done in 3 days, which is split into cell seeding, transfection, and evaluation by flow cytometry. The method is broadly applicable including the upregulation of endogenous genes.}, }
@article {pmid39722550, year = {2025}, author = {Victor Atoki, A and Aja, PM and Shinkafi, TS and Ondari, EN and Adeniyi, AI and Fasogbon, IV and Dangana, RS and Shehu, UU and Akin-Adewumi, A}, title = {Exploring the versatility of Drosophila melanogaster as a model organism in biomedical research: a comprehensive review.}, journal = {Fly}, volume = {19}, number = {1}, pages = {2420453}, pmid = {39722550}, issn = {1933-6942}, mesh = {Animals ; *Drosophila melanogaster/genetics ; *Disease Models, Animal ; Biomedical Research ; Humans ; CRISPR-Cas Systems ; }, abstract = {Drosophila melanogaster is a highly versatile model organism that has profoundly advanced our understanding of human diseases. With more than 60% of its genes having human homologs, Drosophila provides an invaluable system for modelling a wide range of pathologies, including neurodegenerative disorders, cancer, metabolic diseases, as well as cardiac and muscular conditions. This review highlights key developments in utilizing Drosophila for disease modelling, emphasizing the genetic tools that have transformed research in this field. Technologies such as the GAL4/UAS system, RNA interference (RNAi) and CRISPR-Cas9 have enabled precise genetic manipulation, with CRISPR-Cas9 allowing for the introduction of human disease mutations into orthologous Drosophila genes. These approaches have yielded critical insights into disease mechanisms, identified novel therapeutic targets and facilitated both drug screening and toxicological studies. Articles were selected based on their relevance, impact and contribution to the field, with a particular focus on studies offering innovative perspectives on disease mechanisms or therapeutic strategies. Our findings emphasize the central role of Drosophila in studying complex human diseases, underscoring its genetic similarities to humans and its effectiveness in modelling conditions such as Alzheimer's disease, Parkinson's disease and cancer. This review reaffirms Drosophila's critical role as a model organism, highlighting its potential to drive future research and therapeutic advancements.}, }
@article {pmid39722520, year = {2024}, author = {Deng, K and Li, M and Zhang, H and Deng, Y and Qin, Y and Qin, C}, title = {[Characterization of host factors ARF4 and ARF5 upon Zika virus infection in vivo by construction of gene knockout mice].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {40}, number = {12}, pages = {4605-4615}, doi = {10.13345/j.cjb.240307}, pmid = {39722520}, issn = {1872-2075}, mesh = {Animals ; *ADP-Ribosylation Factors/genetics/metabolism ; *Zika Virus Infection/genetics ; Mice ; *Mice, Knockout ; *Zika Virus/genetics ; *Mice, Inbred C57BL ; *CRISPR-Cas Systems ; Viral Load ; Male ; Female ; }, abstract = {The effects of host factors ADP-ribosylation factor 4 (ARF4) and ADP-ribosylation factor 5 (ARF5) upon Zika virus (ZIKV) infection in vivo were characterized by construction of gene knockout mice via CRISPR-Cas9. Firstly, ARF5 and ARF4 genes were modified by the CRISPR-Cas9 system and then microinjected into the fertilized eggs of C57BL/6JGpt mice. Fertilized eggs were transplanted to obtain ARF4 or ARF5 knockout (ARF4KO or ARF5KO) mice, and ARF4/5 double knockout mice were achieved by the mating between ARF4KO and ARF5KO mice (ARF4KO/ARF5KO). Then, the mouse genotypes were identified by PCR to identify the positive knockout mice, and RT-qPCR was employed to examine the knockout efficiency. The mice were then infected with ZIKV and the blood and tissue samples were collected after 2, 4, and 6 days. RT-qPCR was then employed to determine the virus load, and hematoxylin-eosin staining was employed to observe the pathological changes in the tissue. The results showed that expected PCR bands were detected from ARF4KO[-/+], ARF5KO[-/-], and ARF4KO[-/+]/ARF5KO[-/-] mice, respectively. The results of mRNA transcription measurement indicated the significant knockdown of ARF4 by 37.8%-50.0% but not ARF5 in ARF4KO[-/+] compared with the wild-type mice. Meanwhile, complete knockout of ARF5 and no changes in ARF4 were observed in ARF5KO[-/-] mice. Additionally, completed knockout of ARF5 and down-regulated mRNA level of ARF4 in the lung, kidney, and testis were detected in ARF4KO[-/+]/ARF5KO[-/-]mice in comparison with the wild-type mice. The virus load in the serum decreased in ARF4KO[-/+] mice, while it showed no significant change in ARF5KO[-/-] or ARF4KO[-/+]/ARF5KO[-/-] mice compared with that in the wild type. Meanwhile, ARF4KO[-/+] mice showcased no significant difference in virus load in various tissues but attenuated pathological changes in the brain and testis compared with the wild-type mice. We successfully constructed ARF4KO and ARF5KO mice by CRISPR-Cas9 in this study. ARF4 rather than ARF5 is essential for ZIKV infection in vivo. This study provided animal models for studying the roles of ARF4 and ARF5 in ZIKV infection and developing antivirals.}, }
@article {pmid39722468, year = {2024}, author = {Lohani, N and Singh, MB and Bhalla, PL}, title = {Deciphering the Vulnerability of Pollen to Heat Stress for Securing Crop Yields in a Warming Climate.}, journal = {Plant, cell &amp; environment}, volume = {}, number = {}, pages = {}, doi = {10.1111/pce.15315}, pmid = {39722468}, issn = {1365-3040}, abstract = {Climate change is leading to more frequent and severe extreme temperature events, negatively impacting agricultural productivity and threatening global food security. Plant reproduction, the process fundamental to crop yield, is highly susceptible to heatwaves, which disrupt pollen development and ultimately affect seed-set and crop yields. Recent research has increasingly focused on understanding how pollen grains from various crops react to heat stress at the molecular and cellular levels. This surge in interest over the last decade has been driven by advances in genomic technologies, such as single-cell RNA sequencing, which holds significant potential for revealing the underlying regulatory reprogramming triggered by heat stress throughout the various stages of pollen development. This review focuses on how heat stress affects gene regulatory networks, including the heat stress response, the unfolded protein response, and autophagy, and discusses the impact of these changes on various stages of pollen development. It highlights the potential of pollen selection as a key strategy for improving heat tolerance in crops by leveraging the genetic variability among pollen grains. Additionally, genome-wide association studies and population screenings have shed light on the genetic underpinnings of traits in major crops that respond to high temperatures during male reproductive stages. Gene-editing tools like CRISPR/Cas systems could facilitate precise genetic modifications to boost pollen heat resilience. The information covered in this review is valuable for selecting traits and employing molecular genetic approaches to develop heat-tolerant genotypes.}, }
@article {pmid39721357, year = {2024}, author = {Petruškevičiūtė, A and Šimuliūnaitė, U and Polanco, CM and Rojas, B and Kuras, S and Valatkaitė-Rakštienė, B and Norvilas, R and Vijaya, AK and Muñoz, P and Neniškytė, U and Jakubauskas, A and Burokas, A and Nalvarte, I and Inzunza, J and Naumovas, D and Stoškus, M and Griškevičius, L and Baltriukienė, D and Arias, J}, title = {Generation of a genetically encoded voltage indicator MARINA reporter human iPS cell line using Cas9 (VULSCi002-A-2).}, journal = {Stem cell research}, volume = {82}, number = {}, pages = {103628}, doi = {10.1016/j.scr.2024.103628}, pmid = {39721357}, issn = {1876-7753}, abstract = {Fluorescent protein-based Genetically Encoded Voltage Indicators (GEVI) offer a remarkable system for high-throughput screening of membrane potential phenotypes. The GEVI MARINA is a derivative from ArcLight, which conversely to ArcLight increases its fluorescence intensity alongside depolarization. Here we created knock-in reporter human iPS cell lines carrying the MARINA reporter using SpCas9 programmable nuclease and characterize a heterozygous clone.}, }
@article {pmid39720725, year = {2024}, author = {Wi, T and Choi, Y and Kim, J and Choi, YS and Pipkin, ME and Choi, J}, title = {Efficient gene deletion of Integrin alpha 4 in primary mouse CD4 T cells using CRISPR RNA pair-mediated fragmentation.}, journal = {Frontiers in immunology}, volume = {15}, number = {}, pages = {1445341}, pmid = {39720725}, issn = {1664-3224}, mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Integrin alpha4/genetics/metabolism/immunology ; *Gene Deletion ; CD4-Positive T-Lymphocytes/immunology/metabolism ; Th1 Cells/immunology ; Cell Differentiation/genetics/immunology ; Mice, Inbred C57BL ; Lymphocytic Choriomeningitis/immunology/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Lymphocytic choriomeningitis virus/immunology ; }, abstract = {The functional specialization of CD4 T lymphocytes into various subtypes, including TH1 and TFH cells, is crucial for effective immune responses. TFH cells facilitate B cell differentiation within germinal centers, while TH1 cells are vital for cell-mediated immunity against intracellular pathogens. Integrin α4, a cell surface adhesion molecule, plays significant roles in cell migration and co-stimulatory signaling. In this study, we investigated the role of Integrin α4 in regulating TFH and TH1 cell populations during acute viral infection using CRISPR-Cas9 gene editing. To effectively delete the Itga4 in primary mouse CD4 T cells, we selected various combinations of crRNAs and generated ribonucleoprotein complexes with fluorochrome-conjugated tracrRNAs and Cas9 proteins. These crRNA pairs enhanced gene deletion by generating deletions in the gene. By analyzing the effects of Itga4 deficiency on TFH and TH1 cell differentiation during acute LCMV infection, we found that optimized crRNA pairs significantly increased the TH1 cell population. Our results highlight the importance of selecting and combining appropriate crRNAs for effective CRISPR-Cas9 gene editing in primary CD4 T cells. Additionally, our study demonstrates the role of Integrin α4 in regulating the differentiation of CD4 T cells, suggesting the potential molecular mechanisms driving T cell subset differentiation through integrin targeting.}, }
@article {pmid39719706, year = {2025}, author = {Zhang, AN and Gaston, JM and Cárdenas, P and Zhao, S and Gu, X and Alm, EJ}, title = {CRISPR-Cas spacer acquisition is a rare event in human gut microbiome.}, journal = {Cell genomics}, volume = {5}, number = {1}, pages = {100725}, doi = {10.1016/j.xgen.2024.100725}, pmid = {39719706}, issn = {2666-979X}, mesh = {Humans ; *Gastrointestinal Microbiome/genetics ; *CRISPR-Cas Systems/genetics ; Gene Transfer, Horizontal/genetics ; Bacteriophages/genetics ; Bifidobacterium longum/genetics ; Metagenome/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome, Bacterial/genetics ; }, abstract = {Host-parasite relationships drive the evolution of both parties. In microbe-phage dynamics, CRISPR functions as an adaptive defense mechanism, updating immunity via spacer acquisition. Here, we investigated these interactions within the human gut microbiome, uncovering low frequencies of spacer acquisition at an average rate of one spacer every ∼2.9 point mutations using isolates' whole genomes and ∼2.7 years using metagenome time series. We identified a highly prevalent CRISPR array in Bifidobacterium longum spreading via horizontal gene transfer (HGT), with six spacers found in various genomic regions in 15 persons from the United States and Europe. These spacers, targeting two prominent Bifidobacterium phages, comprised 76% of spacer occurrence of all spacers targeting these phages in all B. longum populations. This result suggests that HGT of an entire CRISPR-Cas system introduced three times more spacers than local CRISPR-Cas acquisition in B. longum. Overall, our findings identified key ecological and evolutionary factors in prokaryote adaptive immunity.}, }
@article {pmid39719647, year = {2024}, author = {Yan, L and Song, YS and Zhou, J and Zhu, L and Shi, TW and Yu, HQ and Dong, ZQ and Wang, W and Long, T and Liu, HY and Shi, ZY and Li, JG}, title = {Expression of nicastrin, NICD1, and Hes1 in NCSTN knockout mice: implications for hidradenitis suppurativa, Alzheimer's, and liver cancer.}, journal = {European journal of medical research}, volume = {29}, number = {1}, pages = {622}, pmid = {39719647}, issn = {2047-783X}, support = {81773344//National Natural Science Foundation of China/ ; }, mesh = {Animals ; *Transcription Factor HES-1/genetics/metabolism ; *Amyloid Precursor Protein Secretases/genetics/metabolism ; *Mice, Knockout ; *Alzheimer Disease/genetics/metabolism ; Mice ; *Hidradenitis Suppurativa/genetics ; Female ; Male ; *Liver Neoplasms/genetics/pathology ; *Membrane Glycoproteins/genetics/metabolism ; Mice, Inbred C57BL ; Disease Models, Animal ; Receptor, Notch1/genetics ; }, abstract = {BACKGROUND: Nicastrin, a subunit of the γ-secretase complex, is encoded by the NCSTN gene and regulates notch signaling, it is involved in the pathogenesis of hidradenitis suppurativa (HS), Alzheimer disease (AD), and liver cancer. However, the animal models for studying HS are relatively scarce.<br><br>METHODS: CRISPR/Cas-mediated genetic engineering was used to generate targeted knockout offspring mice (C57BL/6J). Different doses (10&#xa0;mg/kg, 20&#xa0;mg/kg, and 30&#xa0;mg/kg) and injection methods (subcutaneous/intraperitoneal/gavage injection) of tamoxifen were used to induce the construction of NCSTN knockout mice (mice model). The expressions of nicastrin, NICD1, hes1 in skin, brain, and liver tissue in mice model and wild-type (WT) mice were measured by qRT-PCR and IHC.<br><br>RESULTS: The construction of mice model was successfully induced by tamoxifen, knockout efficiency was 93%, there was no difference in knockout efficiency among three doses, injection methods, genders (P&#x2009;>&#x2009;0.05). HS-like lesions appeared on the skin of NCSTN knockout mice after 1&#xa0;month of treatment with tamoxifen, male mice had a higher number of skin lesions compared to female mice (male vs female&#x2009;=&#x2009;76.5% vs 41.7%, P&#x2009;=&#x2009;0.027). Compared with WT mice, the expressions of nicastrin (skin P&#x2009;=&#x2009;0.0009, brain P&#x2009;=&#x2009;0.0194, liver P&#x2009;=&#x2009;0.0066), NICD1 (skin P&#x2009;=&#x2009;0.0115, brain P&#x2009;=&#x2009;0.0307, liver P&#x2009;=&#x2009;0.008), hes1 (skin P&#x2009;=&#x2009;0.0476, brain P&#x2009;=&#x2009;0.0143, liver P&#x2009;=&#x2009;0.0003) in mice model all decreased.<br><br>CONCLUSIONS: The NCSTN knockout mouse might be employed as HS animal model; Reducing nicastrin may affect the expression of notch1-hes1 pathway molecules in skin, brain, and liver tissues; low dose (10&#xa0;mg/kg/d) tamoxifen could be used to induce the deletion of the target gene in mice.}, }
@article {pmid39719558, year = {2024}, author = {Nadeem, I and Han, Z and Xiaoliang, H and Adzraku, SY and Kambey, PA and Kanwore, K and Peipei, M and Adekunle, AO and Adu-Amankwaah, J and Ayanlaja, AA and Zheng, Y and Dianshuai, G and Liu, X and Song, Y}, title = {Doublecortin regulates the mitochondrial-dependent apoptosis in glioma via Rho-A/Net-1/p38-MAPK signaling.}, journal = {Molecular medicine (Cambridge, Mass.)}, volume = {30}, number = {1}, pages = {272}, pmid = {39719558}, issn = {1528-3658}, support = {82273250//National Natural Science Foundation of China/ ; KYCX22_2868//Postgraduate Research &amp; Practice Innovation Program of Jiangsu Province/ ; }, mesh = {Humans ; *Glioma/metabolism/pathology/genetics ; *Apoptosis ; *Mitochondria/metabolism ; Animals ; *Doublecortin Domain Proteins ; *Doublecortin Protein ; Cell Line, Tumor ; *p38 Mitogen-Activated Protein Kinases/metabolism ; *Microtubule-Associated Proteins/metabolism/genetics ; Mice ; *Neuropeptides/metabolism/genetics ; Cell Proliferation ; Brain Neoplasms/metabolism/pathology/genetics ; Temozolomide/pharmacology ; Signal Transduction ; MAP Kinase Signaling System ; Reactive Oxygen Species/metabolism ; }, abstract = {Doublecortin (DCX) is a microtubule-associated protein known to be a key regulator of neuronal migration and differentiation during brain development. However, the role of DCX, particularly in regulating the survival and growth of glioma cells, remains unclear. In this study, we utilized CRISPR/Cas9 technology to knock down DCX in the human glioma cell line (U251). DCX depletion suppressed cell proliferation and enhanced the pro-apoptotic effects of temozolomide (TMZ) and γ-radiation treatment. DCX knockdown led to the translocation of Bax to the mitochondria and mitochondria dysfunction. Furthermore, DCX deficiency-induced apoptosis took place along with the generation of reactive oxygen species (ROS), which is crucial in triggering mitochondrial membrane depolarization, the release of cytochrome c (Cyt-c), and caspase activation. Importantly, the transcriptional inhibition of DCX downregulated Rho-A, Net-1, and activated p38-MAPK cue, critical for cell survival and proliferation. Subsequent treatment with TMZ and γ-radiation further increased p38-MAPK activity through the decreased expression of Rho-A/Net-1, resulting in a significant reduction in glioma cell migration and invasion. Additionally, intracranial xenograft tumors of DCX-modified U251 cells in nude mice demonstrated inhibited tumor growth. Tumor sections treated with TMZ and γ-radiation exhibited a higher number of TUNEL-positive cells compared to the control group, indicating increased apoptosis. Our finding suggests that DCX depletion reduces glioma cell proliferation and promotes mitochondria-dependent apoptosis by enhancing the chemo and radiotherapy response. Targeting DCX represents a potential therapeutic target for glioma treatment.}, }
@article {pmid39718242, year = {2024}, author = {Dong, J and Hou, C and Deng, L and Gu, T and Zhu, S and Hou, J and Huo, D}, title = {CRISPR/Cas12a-Powered Electrochemical Platform for Dual-miRNA Detection via an AND Logic Circuit.}, journal = {Analytical chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.analchem.4c06256}, pmid = {39718242}, issn = {1520-6882}, abstract = {The CRISPR/Cas technology shows great potential in molecular detection and diagnostics. However, it is still challenging to detect multiple targets simultaneously using the CRISPR-Cas system. Herein, we ingeniously leverage the synergistic effect of two short single-stranded DNA activators to construct a CRISPR/Cas12a-driven electrochemical sensing platform based on an AND logic circuit ("AND" LC-CRISPR) for the simultaneous detection of dual miRNAs. Specifically, the exponential amplification reaction products triggered by the dual-specific miRNAs are designed as binary inputs to bind with Cas12a/crRNA, forming an AND logic circuit and activating the trans-cleavage ability of the CRISPR-Cas12a system. Subsequently, the hairpin probe biogate on the surface of the functionalized electrochemical signal probe (MB@HP-Fe-MOF) is cleaved by activated Cas12a, leading to the release of the encapsulated electroactive signal molecule methylene blue, thereby generating a strong electrochemical signal. As a result, this "AND" LC-CRISPR sensing platform, requiring only a single crRNA assembled with Cas12a, achieves simultaneous detection of miRNA-155 and miRNA-21 at concentrations as low as 3.2 fM. Moreover, the platform allows easy adjustment of the AND logic circuit inputs according to different detection targets, allowing it to be easily expanded for the analysis and diagnosis of other multibiomarkers. This approach demonstrates promising potential for future applications in intelligent diagnostic medicine.}, }
@article {pmid39717575, year = {2024}, author = {Ahmad, Z and Niyazi, S and Firdoos, A and Wang, C and Manzoor, MA and Ramakrishnan, M and Upadhyay, A and Ding, Y}, title = {Enhancing plant resilience: Nanotech solutions for sustainable agriculture.}, journal = {Heliyon}, volume = {10}, number = {23}, pages = {e40735}, pmid = {39717575}, issn = {2405-8440}, abstract = {The global population growth is driving up the demand for agricultural products, while traditional farming methods like those from the Green Revolution are becoming unsustainable due to climate change. To address these challenges and ensure agricultural sustainability, innovative techniques, such as nanotechnology, are essential to meet rising food demands and enhance agricultural sustainability. Nanotechnology, which promotes a more sustainable and resilient agricultural system while enhancing food security, is a key catalyst for the Agri-tech revolution. This review offers a progressive analysis of nanotechnology's role in managing plant stress. It explores how precision agriculture, particularly via nanosensors, is enhancing our comprehension of plant stress conditions. The integration of nanotechnology with genetic engineering methods, notably CRISPR-Cas technology, is also examined. Furthermore, the review considers the potential toxicological effects of nanoparticles (NPs) on both the environment and plants. Our review has the potential to make a significant impact on human food security by enhancing food production and availability while promoting sustainable agricultural practices. By tackling these challenges, we can contribute to a more reliable and sustainable food supply for the global population.}, }
@article {pmid39716183, year = {2024}, author = {Cohen, S and Bergman, S and Lynn, N and Tuller, T}, title = {A tool for CRISPR-Cas9 sgRNA evaluation based on computational models of gene expression.}, journal = {Genome medicine}, volume = {16}, number = {1}, pages = {152}, pmid = {39716183}, issn = {1756-994X}, mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Software ; Computational Biology/methods ; Gene Editing/methods ; Gene Knockout Techniques ; Computer Simulation ; Models, Genetic ; Gene Expression ; Gene Expression Regulation ; }, abstract = {BACKGROUND: CRISPR is widely used to silence genes by inducing mutations expected to nullify their expression. While numerous computational tools have been developed to design single-guide RNAs (sgRNAs) with high cutting efficiency and minimal off-target effects, only a few tools focus specifically on predicting gene knockouts following CRISPR. These tools consider factors like conservation, amino acid composition, and frameshift likelihood. However, they neglect the impact of CRISPR on gene expression, which can dramatically affect the success of CRISPR-induced gene silencing attempts. Furthermore, information regarding gene expression can be useful even when the objective is not to silence a gene. Therefore, a tool that considers gene expression when predicting CRISPR outcomes is lacking.<br><br>RESULTS: We developed EXPosition, the first computational tool that combines models predicting gene knockouts after CRISPR with models that forecast gene expression, offering more accurate predictions of gene knockout outcomes. EXPosition leverages deep-learning models to predict key steps in gene expression: transcription, splicing, and translation initiation. We showed our tool performs better at predicting gene knockout than existing tools across 6 datasets, 4 cell types and ~207k sgRNAs. We also validated our gene expression models using the ClinVar dataset by showing enrichment of pathogenic mutations in high-scoring mutations according to our models.<br><br>CONCLUSIONS: We believe EXPosition will enhance both the efficiency and accuracy of genome editing projects, by directly predicting CRISPR's effect on various aspects of gene expression. EXPosition is available at http://www.cs.tau.ac.il/~tamirtul/EXPosition .&#xa0;The source code is available at https://github.com/shaicoh3n/EXPosition .}, }
@article {pmid39715238, year = {2024}, author = {Qiu, M and Tian, Y and Wang, H and Yang, J and Qu, B and Jiang, Y and Zhao, Q and Zhang, X and Man, C}, title = {CRISPR/Cas System Meets CLICK-17 DNAzyme: A Click Chemistry-Based Fluorescence Biosensing Platform Designed for Highly Sensitive Detection of Salmonella.}, journal = {Analytical chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.analchem.4c05316}, pmid = {39715238}, issn = {1520-6882}, abstract = {Salmonella is one of the most dangerous and contagious foodborne pathogens, posing a significant threat to public health and food safety. In this study, we developed a click chemistry-based fluorescence biosensing platform for highly sensitive detection of Salmonella enterica (S. enterica) by integrating the trans-cleavage activity of CRISPR/Cas12a with the CLICK17-mediated copper(II)-dependent azide-alkyne cycloaddition (Cu(II)AAC) click reaction. Herein, CLICK-17 can provide binding sites for Cu ions and high redox stability for one or much catalytically vital Cu[+] within its active sites, which facilitate the click reaction. With the existence of only Cu[2+], CLICK17 still can catalyze the click reaction between 3-butyn-1-ol and 3-azido-7-hydroxycoumarin to produce a fluorescence signal. By integrating the recombinase polymerase amplification (RPA), specific recognition, and trans-cleavage ability of the CRISPR/Cas12a system and the CLICK17-catalyzed Cu(II)AAC click reaction, the established biosensor obtained high detection sensitivity. This CLICK17-assisted CRISPR/Cas12a fluorescence biosensor was used for the detection of S. enterica with a limit of detection (LOD) as low as 1 cfu/mL in a wide linear detection range of 6 × 10[1]-6 × 10[7] cfu/mL. Moreover, the developed biosensor exhibited high specificity and anti-interference capability and had a recovery of 93%-104% in detection of S. enterica in spiked milk, infant formula, orange juice, and meat samples. This study provides a promising CRISPR/Cas12a-based fluorescence biosensor for the detection of foodborne pathogens.}, }
@article {pmid39714464, year = {2024}, author = {Böck, D and Wilhelm, M and Mumenthaler, J and Carpanese, DF and Kulcsár, PI and d'Aquin, S and Cremonesi, A and Rassi, A and Häberle, J and Patriarchi, T and Schwank, G}, title = {Base editing of Ptbp1 in neurons alleviates symptoms in a mouse model of Parkinson's disease.}, journal = {eLife}, volume = {13}, number = {}, pages = {}, pmid = {39714464}, issn = {2050-084X}, support = {310030_185293/SNSF_/Swiss National Science Foundation/Switzerland ; 310030_196455/SNSF_/Swiss National Science Foundation/Switzerland ; FN20-0000000203//Novartis Foundation for Medical-Biological Research/ ; 196287/SNSF_/Swiss National Science Foundation/Switzerland ; 891959/ERC_/European Research Council/International ; }, mesh = {Animals ; *Polypyrimidine Tract-Binding Protein/metabolism/genetics ; Mice ; *Disease Models, Animal ; *Heterogeneous-Nuclear Ribonucleoproteins/metabolism/genetics ; *Parkinson Disease/genetics/metabolism ; *Astrocytes/metabolism ; Dopaminergic Neurons/metabolism ; Gene Editing ; Male ; Corpus Striatum/metabolism ; Tyrosine 3-Monooxygenase/metabolism/genetics ; Mice, Inbred C57BL ; }, abstract = {Parkinson's disease (PD) is a multifactorial disease caused by irreversible progressive loss of dopaminergic neurons (DANs). Recent studies have reported the successful conversion of astrocytes into DANs by repressing polypyrimidine tract binding protein 1 (PTBP1), which led to the rescue of motor symptoms in a chemically-induced mouse model of PD. However, follow-up studies have questioned the validity of this astrocyte-to-DAN conversion model. Here, we devised an adenine base editing strategy to downregulate PTBP1 in astrocytes and neurons in a chemically-induced PD mouse model. While PTBP1 downregulation in astrocytes had no effect, PTBP1 downregulation in neurons of the striatum resulted in the expression of the DAN marker tyrosine hydroxylase (TH) in non-dividing neurons, which was associated with an increase in striatal dopamine concentrations and a rescue of forelimb akinesia and spontaneous rotations. Phenotypic analysis using multiplexed iterative immunofluorescence imaging further revealed that most of these TH-positive cells co-expressed the dopaminergic marker DAT and the pan-neuronal marker NEUN, with the majority of these triple-positive cells being classified as mature GABAergic neurons. Additional research is needed to fully elucidate the molecular mechanisms underlying the expression of the observed markers and understand how the formation of these cells contributes to the rescue of spontaneous motor behaviors. Nevertheless, our findings support a model where downregulation of neuronal, but not astrocytic, PTBP1 can mitigate symptoms in PD mice.}, }
@article {pmid39714446, year = {2024}, author = {Petazzi, P and Ventura, T and Luongo, FP and McClafferty, H and May, A and Taylor, HA and Shipston, MJ and Romanò, N and Forrester, LM and Menendez, P and Fidanza, A}, title = {A novel human pluripotent stem cell gene activation system identifies IGFBP2 as a mediator in the production of haematopoietic progenitors in vitro.}, journal = {eLife}, volume = {13}, number = {}, pages = {}, pmid = {39714446}, issn = {2050-084X}, support = {S002219/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; EHA RAG 2021//European Hematology Association/ ; Research Global Award//American Society for Hematology/ ; Precision Medicine PhD scholarship/MRC_/Medical Research Council/United Kingdom ; Tissue Repair PhD studentship//College of Medicine and Veterinary Medicine, University of Edinburgh/ ; Traineeship Program 2016/2017//Erasmus +/ ; PERIS program//Catalan Government/ ; RTC-2018-4603-1//MINECO/ ; }, mesh = {*Insulin-Like Growth Factor Binding Protein 2/metabolism/genetics ; Humans ; *Cell Differentiation ; *Hematopoietic Stem Cells/metabolism ; Induced Pluripotent Stem Cells/metabolism ; Transcriptional Activation ; CRISPR-Cas Systems ; Transcription Factors/metabolism/genetics ; Pluripotent Stem Cells/metabolism ; }, abstract = {A major challenge in the stem cell biology field is the ability to produce fully functional cells from induced pluripotent stem cells (iPSCs) that are a valuable resource for cell therapy, drug screening, and disease modelling. Here, we developed a novel inducible CRISPR-mediated activation strategy (iCRISPRa) to drive the expression of multiple endogenous transcription factors (TFs) important for in vitro cell fate and differentiation of iPSCs to haematopoietic progenitor cells. This work has identified a key role for IGFBP2 in developing haematopoietic progenitors. We first identified nine candidate TFs that we predicted to be involved in blood cell emergence during development, then generated tagged gRNAs directed to the transcriptional start site of these TFs that could also be detected during single-cell RNA sequencing (scRNAseq). iCRISPRa activation of these endogenous TFs resulted in a significant expansion of arterial-fated endothelial cells expressing high levels of IGFBP2, and our analysis indicated that IGFBP2 is involved in the remodelling of metabolic activity during in vitro endothelial to haematopoietic transition. As well as providing fundamental new insights into the mechanisms of haematopoietic differentiation, the broader applicability of iCRISPRa provides a valuable tool for studying dynamic processes in development and for recapitulating abnormal phenotypes characterised by ectopic activation of specific endogenous gene expression in a wide range of systems.}, }
@article {pmid39714100, year = {2024}, author = {Cui, Q and Zhang, Z and Qin, L and Teng, Z and Wang, Z and Wu, W and Fan, L and Su, J and Hao, Y and Qin, J and Zhang, L and Wang, Q and Zhuang, Y and Zheng, H and Zhang, S and Geng, X and Zhu, L and Chen, Y and Lu, B and Li, Y and Zhu, X}, title = {Interleukin-37 promotes wound healing in diabetic mice by inhibiting the MAPK/NLRP3 pathway.}, journal = {Journal of diabetes investigation}, volume = {}, number = {}, pages = {}, doi = {10.1111/jdi.14389}, pmid = {39714100}, issn = {2040-1124}, support = {3030294002//Multidisciplinary Diagnosis and Treatment (MDT) Construction Project of Diabetic Foot/ ; 82400978//National Natural Science Foundation of China/ ; 82000790//National Natural Science Foundation of China/ ; 82200949//National Natural Science Foundation of China/ ; DGF501069/015//Multidisciplinary Diagnosis and Treatment (MDT) demonstration project in research hospital, Shanghai Medical College, Fudan University/ ; }, abstract = {AIMS/INTRODUCTION: Diabetic foot ulcer (DFU) is a prevalent complication of diabetes characterized by heightened inflammation and impaired wound-healing processes. Interleukin-37 (IL-37) is a natural suppressor of innate inflammation. Here, we aim to investigate the potential of IL-37 in enhancing the healing process of diabetic wounds.<br><br>MATERIALS AND METHODS: The skin samples of DFU and non-diabetic patients during foot and ankle orthopedic surgery were collected. The IL-37 transgenic mice (IL-37Tg) were created using CRISPR/Cas-mediated genome engineering. Mice were administered streptozotocin (STZ, 150&#x2009;mg/kg) to induce a diabetic model. After 4&#x2009;weeks, an equidistant full-thickness excisional wound measuring 8&#x2009;mm was created on the central back of each mouse and allowed to heal naturally. Body weight and blood glucose levels were measured weekly. The wound area was measured, and skin samples were collected on Day 10 for further Quantitative polymerase chain reaction (qPCR) and WB detection and RNA sequencing analysis.<br><br>RESULTS: The proinflammation cytokines such as TNF-&#x3b1; and IL-1&#x3b2; and the MAPK signaling pathway were significantly increased in the wound margin of DFU patients. Compared with diabetic mice, diabetic IL-37Tg mice showed a significantly accelerated healing process. The enriched signaling pathways in RNA sequencing included cytokine-cytokine receptor interaction, TNF signaling pathway, and NOD-like receptor signaling pathway. Through QPCR and WB detection, we found that IL-37 could inhibit the activated MAPK and NOD-like signaling pathway, reducing TNF-&#x3b1;, IL-1&#x3b2;, and NLRP3 expression in the diabetic wound.<br><br>CONCLUSIONS: IL-37 promotes skin wound healing in diabetic mice, providing a new possible target for treating diabetic wounds.}, }
@article {pmid39713824, year = {2024}, author = {Karesh, WB}, title = {Shifting from wildlife disease threats to wildlife health.}, journal = {Revue scientifique et technique (International Office of Epizootics)}, volume = {Special Edition}, number = {}, pages = {141-144}, doi = {10.20506/rst.SE.3568}, pmid = {39713824}, issn = {0253-1933}, mesh = {Animals ; *Animals, Wild ; Humans ; Zoonoses/prevention &amp; control ; Conservation of Natural Resources ; Animal Diseases/prevention &amp; control/epidemiology ; }, abstract = {The evolution of wildlife disease management and surveillance, as documented in the World Organisation for Animal Health's Scientific and Technical Review, reflects a deepening understanding of the links between wildlife health, ecosystem integrity and human well-being. Early work, beginning with the World Assembly of Delegates in 1954, primarily focused on diseases like rabies. This focus expanded over time to include broader concerns such as the impacts of climate change, habitat loss and increased human-wildlife interactions on wildlife health. By the late 20th century, the emphasis had shifted towards improved practices for wildlife disease control and the development of advanced diagnostic methods and vaccines. Articles in the Review highlight the growing complexity of wildlife diseases and the need for holistic management strategies. The adoption in recent years of cutting-edge technologies like CRISPR-Cas systems and metagenomics points to a future of more proactive and integrated approaches to wildlife disease management. There is still a need to address not just the consequences of wildlife diseases but also their anthropogenic drivers. The latest perspectives advocate for nature-based solutions, expanded partnerships and systems-level thinking to effectively tackle 21st-century challenges in wildlife and biodiversity conservation.}, }
@article {pmid39712894, year = {2024}, author = {Wang, X and Yang, R and Tang, T and Zhou, Y and Chen, H and Jiang, Y and Zhang, S and Qin, S and Wang, M and Wang, C}, title = {One-pot MCDA-CRISPR-Cas-based detection platform for point-of-care testing of severe acute respiratory syndrome coronavirus 2.}, journal = {Frontiers in microbiology}, volume = {15}, number = {}, pages = {1503356}, pmid = {39712894}, issn = {1664-302X}, abstract = {Compared to quantitative real-time PCR (q-PCR), CRISPR-Cas-mediated technology is more suitable for point-of-care testing (POCT) and has potential for wider application in the future. Generally, the operational procedure of CRISPR-Cas-mediated diagnostic method consists of two independent steps, the reaction of signal amplification and the CRISPR-Cas-mediated signal detection. Complex multi-step procedures can easily lead to cross-contamination. To develop a convenient and rapid method for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection, we propose a MCTOP method (Multiple cross displacement amplification-CRISPR-Cas12b-based testing in one-pot), which targets the open reading frame 1ab (ORF1ab) and nucleocapsid protein (N) gene of SARS-CoV-2. This method combines MCDA isothermal amplification and CRISPR-Cas-mediated sequence-specific detection into a one-pot reaction. The optimal reaction was achieved with isothermal amplification of 40 min and CRISPR-Cas-based detection of 15 min, both at 64°C. Then, the results can be visualized by the real-time fluorescence instrument and also lateral flow biosensor. The lowest detection limit of the proposed method is 10 copies of each of target sequences, and it has no cross-reactivity with non-SARS-CoV-2 templates. In a clinical test of 70 pharyngeal swab samples, MCTOP assay showed a specificity of 100% and sensitivities of 98 and 96% for the real-time fluorescence instrument and lateral flow biosensor, respectively. The MCTOP developed in this study is a rapid, convenient, highly sensitive, and specific method for SARS-CoV-2 nucleic acid detection. It can be used as an effective point-of-care testing (POCT) tool for clinical diagnosis and epidemiologic surveillance of SARS-CoV-2 infections, especially suitable for the basic, field and clinical laboratory.}, }
@article {pmid39710679, year = {2024}, author = {Jia, X and Yuan, B and Wang, W and Wang, K and Ling, D and Wei, M and Hu, Y and Guo, W and Chen, Z and Du, L and Jin, Y}, title = {Gene editing tool-loaded biomimetic cationic vesicles with highly efficient bacterial internalization for in vivo eradication of pathogens.}, journal = {Journal of nanobiotechnology}, volume = {22}, number = {1}, pages = {787}, pmid = {39710679}, issn = {1477-3155}, support = {7232261//Beijing Natural Science Foundation of China/ ; }, mesh = {*Gene Editing/methods ; *Pseudomonas aeruginosa/drug effects/genetics ; *CRISPR-Cas Systems ; *Plasmids/genetics ; *Acinetobacter baumannii/drug effects/genetics ; Animals ; *Cations/chemistry ; Mice ; Humans ; Pseudomonas Infections/drug therapy ; Anti-Bacterial Agents/pharmacology/chemistry ; Drug Resistance, Multiple, Bacterial/drug effects/genetics ; Acinetobacter Infections/drug therapy ; COVID-19 ; Female ; Mice, Inbred BALB C ; Biomimetics/methods ; }, abstract = {In the post-COVID-19 era, drug-resistant bacterial infections emerge as one of major death causes, where multidrug-resistant Acinetobacter baumannii (MRAB) and drug-resistant Pseudomonas aeruginosa (DRPA) represent primary pathogens. However, the classical antibiotic strategy currently faces the bottleneck of drug resistance. We develop an antimicrobial strategy that applies the selective delivery of CRISPR/Cas9 plasmids to pathogens with biomimetic cationic hybrid vesicles (BCVs), irrelevant to bacterial drug resistance. CRISPR/Cas9 plasmids were constructed, replicating in MRAB or DRPA and expressing ribonucleic proteins, leading to irreparable chromosomal lesions; however, delivering the negatively charged plasmids with extremely large molecular weight to the pathogens at the infection site became a huge challenge. We found that the BCVs integrating the bacterial out membrane vesicles and cationic lipids efficiently delivered the plasmids in vitro/in vivo to the pathogens followed by effective internalization. The BCVs were used by intratracheal or topical hydrogel application against MRAB pulmonary infection or DRPA wound infection, and both of the two pathogens were eradicated from the lung or the wound. CRISPR/Cas9 plasmid-loaded BCVs become a promising medication for drug-resistant bacteria infections.}, }
@article {pmid39709563, year = {2024}, author = {Shepelev, MV and Komkov, DS and Golubev, DS and Borovikova, SE and Mazurov, DV and Kruglova, NA}, title = {[Donor DNA Modification with Cas9 Targeting Sites Improves the Efficiency of MTC34 Knock-in into the CXCR4 Locus].}, journal = {Molekuliarnaia biologiia}, volume = {58}, number = {4}, pages = {590-600}, pmid = {39709563}, issn = {0026-8984}, mesh = {Humans ; *CRISPR-Cas Systems ; *Receptors, CXCR4/genetics/metabolism ; *Gene Knock-In Techniques/methods ; Gene Editing/methods ; Plasmids/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; DNA/genetics/metabolism ; Cell Line ; DNA Breaks, Double-Stranded ; HIV-1/genetics/metabolism ; }, abstract = {To successfully apply the genome editing technology using the CRISPR/Cas9 system in the clinic, it is necessary to achieve a high efficiency of knock-in, which is insertion of a genetic construct into a given locus of the target cell genome. One of the approaches to increase the efficiency of knock-in is to modify donor DNA with the same Cas9 targeting sites (CTS) that are used to induce double-strand breaks (DSBs) in the cell genome (the double-cut donor method). Another approach is based on introducing truncated CTS (tCTS), including a PAM site and 16 proximal nucleotides, into the donor DNA. Presumably, tCTS sites do not induce cleavage of the donor plasmid, but can support its transport into the nucleus by Cas9. However, the exact mechanisms whereby these two donor DNA modifications increase the knock-in level are unknown. In this study, the modifications were tested for effect on the knock-in efficiency of the MTC34 genetic construct encoding the HIV-1 fusion inhibitory peptide MT-C34 into the CXCR4 locus of the CEM/R5 T-cell line. When full-length CTSs were introduced into the donor plasmid DNA, the knock-in level was doubled regardless of the CTS number or position relative to the donor sequence. Modifications with tCTSs did not affect the knock-in levels. In vitro, both CTS and tCTS were efficiently cleaved by Cas9. To understand the mechanism of action of these modifications in detail, it is necessary to evaluate their cleavage both in vitro and in vivo.}, }
@article {pmid39709562, year = {2024}, author = {Golubev, DS and Komkov, DS and Shepelev, MV and Mazurov, DV and Kruglova, NA}, title = {[Methods to Increase the Efficiency of Knock-in of a Construct Encoding the HIV-1 Fusion Inhibitor, MT-C34 Peptide, into the CXCR4 Locus in the CEM/R5 T Cell Line].}, journal = {Molekuliarnaia biologiia}, volume = {58}, number = {4}, pages = {575-589}, pmid = {39709562}, issn = {0026-8984}, mesh = {*Receptors, CXCR4/genetics/metabolism ; Humans ; *Gene Knock-In Techniques ; *HIV-1/genetics/drug effects ; HIV Fusion Inhibitors/pharmacology ; CRISPR-Cas Systems ; Cell Line ; DNA End-Joining Repair/drug effects/genetics ; Gene Editing/methods ; Nuclear Localization Signals/genetics ; Plasmids/genetics/metabolism ; T-Lymphocytes/metabolism/drug effects ; NF-kappa B/metabolism/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; HIV Infections/genetics/virology/drug therapy ; }, abstract = {The low knock-in efficiency, especially in primary human cells, limits the use of the genome editing technology for therapeutic purposes, rendering it important to develop approaches for increasing the knock-in levels. In this work, the efficiencies of several approaches were studied using a model of knock-in of a construct coding for the peptide HIV fusion inhibitor MT-C34 into the human CXCR4 locus in the CEM/R5 T cell line. First, donor DNA modification was evaluated as a means to improve the efficiency of plasmid transport into the nucleus. The donor plasmid was modified to include the simian virus 40 (SV40) DNA nuclear targeting sequence (DTS) or binding sites for the transcription factor NF-κB, whose effects on the knock-in levels have not been described. The modification was ineffective in the model of MT-C34 knock-in into the CXCR4 locus. A second approach consisted in modification of Cas9 nuclease by introducing two additional nuclear localization signals (NLSs) and increased the knock-in level by 30%. Finally, blocking DNA repair via the nonhomologous end joining (NHEJ) pathway with DNA-dependent protein kinase inhibitors caused a 1.8-fold increase in knock-in. A combination of the last two approaches caused an additive effect. Thus, increasing the number of NLSs in the Cas9 protein and inhibiting DNA repair via the NHEJ pathway significantly increased the level of knock-in of the HIV-1 fusion inhibitory peptide into the clinically relevant locus CXCR4. The finding can be used to develop effective gene therapy approaches for treating HIV infection.}, }
@article {pmid39709560, year = {2024}, author = {Averina, OA and Kuznetsova, SA and Permyakov, OA and Sergiev, PV}, title = {[How to Shift the Equilibrium of DNA Break Repair in Favor of Homologous Recombination].}, journal = {Molekuliarnaia biologiia}, volume = {58}, number = {4}, pages = {525-548}, pmid = {39709560}, issn = {0026-8984}, mesh = {Humans ; Animals ; *DNA Breaks, Double-Stranded ; *DNA End-Joining Repair ; *Recombinational DNA Repair ; CRISPR-Cas Systems ; Gene Editing/methods ; Cell Cycle/genetics ; Homologous Recombination ; }, abstract = {The CRISPR/Cas technology of targeted genome editing made it possible to carry out genetic engineering manipulations with eukaryotic genomes with a high efficiency. Targeted induction of site-specific DNA breaks is one of the key stages of the technology. The cell repairs the breaks via one of the two pathways, nonhomologous end joining (NHEJ) and homology-driven repair (HDR). The choice of the DNA repair pathway is determined by the architecture of the DNA break region formed as a result of terminal resection and depends on the cell cycle phase. NHEJ is the main pathway of double-strand break (DSB) repair in mammalian cells and involves a nonspecific ligation reaction. The reaction accuracy depends on the structure of break ends, and various insertions or deletions may arise as a result in the target genome region. Integration of a necessary sequence into the genome occurs via HDR, which requires a template with homology regions flanking a DSB. Introducing a genetic construct into a particular genomic locus is an important task, but is currently intricate and laborious to perform. However, the choice of the repair pathway can be of principal importance for basic research of gene functions and construction of animal models of human diseases to develop therapies. The review summarizes and systematizes the available information on strategies designed to increase the HDR efficiency. The strategies that most efficiently shift the balance towards HDR include use of NHEJ inhibitors, regulation of the key factors of homologous recombination, control of the cell cycle and chromatin status, and construction of HDR templates.}, }
@article {pmid39709559, year = {2024}, author = {Averina, OA and Kuznetsova, SA and Permyakov, OA and Sergiev, PV}, title = {[Current Knowledge of Base Editing and Prime Editing].}, journal = {Molekuliarnaia biologiia}, volume = {58}, number = {4}, pages = {508-524}, pmid = {39709559}, issn = {0026-8984}, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; Animals ; Genetic Engineering/methods ; DNA/genetics ; }, abstract = {Modern genetic engineering technologies, such as base editing and prime editing (PE), have proven to provide the efficient and reliable genome editing tools that obviate the need for donor templates and double-strand breaks (DSBs) introduced in DNA. Relatively new, they quickly gained recognition for their accuracy, simplicity, and multiplexing capabilities. The review summarizes the new literature on the technologies and considers their architecture, methods to create editors, specificity, efficiency, and versatility. Advantages and disadvantages of the editors are discussed along with their prospective use in basic and applied research. The review may be useful for planning genome editing studies and analyzing their results to solve various problems of fundamental biology, biotechnology, medicine, and agriculture.}, }
@article {pmid39709015, year = {2025}, author = {Bigot, S and Ouameur, AA and Roy, G and Fakhfakh, R and Ritt, JF and Légaré, D and Ouellette, M}, title = {Studies of the FBT family transporters in Leishmania infantum by gene deletion and protein localization.}, journal = {Experimental parasitology}, volume = {268}, number = {}, pages = {108880}, doi = {10.1016/j.exppara.2024.108880}, pmid = {39709015}, issn = {1090-2449}, mesh = {*Leishmania infantum/genetics/drug effects/metabolism ; *Gene Deletion ; Cell Membrane/metabolism ; Membrane Transport Proteins/genetics/metabolism ; Protozoan Proteins/genetics/metabolism ; Methotrexate/pharmacology ; Folic Acid Transporters/genetics/metabolism ; CRISPR-Cas Systems ; Animals ; Green Fluorescent Proteins/genetics/metabolism ; }, abstract = {The protozoan parasite Leishmania has a large family of major facilitator membrane proteins part of the Folate Biopterin Transporter (FBT) family. The chromosome 10 of Leishmania has a cluster of 7 FBT genes including the S-Adenosyl methionine (AdoMet) transporter and the functionally characterized folate transporters FT1 and FT5. Six of the 7 FBT proteins coded by this locus are located at the plasma membrane as determined by gene fusions with the green fluorescent protein. We deleted the whole locus of 7 genes (>30 kb) using CRISPR-Cas9 genome editing as a first step in studying the potential function of the four uncharacterized FBT genes from the locus. This knock out strain was viable, highly resistant to sinefungin (an AdoMet analogue) and to methotrexate (a folate analogue) but not to allopurinol, pentamidine or 5-fluorouracil. We similarly studied another FBT family member whose gene is encoded on chromosome 19. The protein was also located at the plasma membrane and its gene was dispensable for growth and not associated to any of the drug tested. Our work has indicated that large diploid deletion is achievable in Leishmania and the cell lines produced here will serve to better understand the function and putative substrates of these FBT proteins yet to be characterized.}, }
@article {pmid39708170, year = {2024}, author = {Feng, R and Mao, K and Zhang, H and Zhu, H and Du, W and Yang, Z and Wang, S}, title = {Portable microfluidic devices for monitoring antibiotic resistance genes in wastewater.}, journal = {Mikrochimica acta}, volume = {192}, number = {1}, pages = {19}, pmid = {39708170}, issn = {1436-5073}, support = {42377456//the National Natural Science Foundation of China/ ; 2023415//the Youth Innovation Promotion Association CAS/ ; CAS-ANSO-FS-2024-34//CAS-ANSO Fellowship/ ; Qiankehe Jichu-ZK [2022] Yiban 565, Qiankehe Platform Talents-GCC [2023] 046//Guizhou Provincial Science and Technology Projects/ ; }, mesh = {*Wastewater/microbiology ; *Lab-On-A-Chip Devices ; Nucleic Acid Amplification Techniques/methods ; Drug Resistance, Microbial/genetics ; Genes, Bacterial ; CRISPR-Cas Systems/genetics ; Anti-Bacterial Agents ; Microfluidic Analytical Techniques/instrumentation/methods ; Drug Resistance, Bacterial/genetics ; }, abstract = {Antibiotic resistance genes (ARGs) pose serious threats to environmental and public health, and monitoring ARGs in wastewater is a growing need because wastewater is an important source. Microfluidic devices can integrate basic functional units involved in sample assays on a small chip, through the precise control and manipulation of micro/nanofluids in micro/nanoscale spaces, demonstrating the great potential of ARGs detection in wastewater. Here, we (1) summarize the state of the art in microfluidics for recognizing ARGs, (2) determine the strengths and weaknesses of portable microfluidic chips, and (3) assess the potential of portable microfluidic chips to detect ARGs in wastewater. Isothermal nucleic acid amplification and CRISPR/Cas are two commonly used identification elements for the microfluidic detection of ARGs. The former has better sensitivity due to amplification, but false positives due to inappropriate primer design and contamination; the latter has better specificity. The combination of the two can achieve complementarity to a certain extent. Compared with traditional microfluidic chips, low-cost and biocompatible paper-based microfluidics is a very attractive test for ARGs, whose fluid flow in paper does not require external force, but it is weaker in terms of repeatability and high-throughput detection. Due to that only a handful of portable microfluidics detect ARGs in wastewater, fabricating high-throughput microfluidic chips, developing and optimizing recognition techniques for the highly selective and sensitive identification and quantification of a wide range of ARGs in complex wastewater matrices are needed.}, }
@article {pmid39707688, year = {2024}, author = {Garay-Novillo, JN and Ruiz-Masó, J&#xc1; and Del Solar, G and Barra, JL}, title = {Easy-Curing and pH-Regulated CRISPR-Cas9 Plasmids for Gene Editing and Plasmid Curing in Lactococcus cremoris.}, journal = {Microbial biotechnology}, volume = {17}, number = {12}, pages = {e70060}, pmid = {39707688}, issn = {1751-7915}, support = {CSIC 2022AEP028//Consejo Superior de Investigaciones Cient&#xed;ficas/ ; 2023/ResMi-00000018//Secretar&#xed;a de Ciencia y Tecnolog&#xed;a - Universidad Nacional de C&#xf3;rdoba and Ministerio de Ciencia y Tecnolog&#xed;a - C&#xf3;rdoba/ ; PICT Start-Up 2018-00811//Agencia Nacional de Promoci&#xf3;n Cient&#xed;fica y Tecnol&#xf3;gica/ ; CONSOLIDAR RESOL-2023-258-E-UNC-SECYT#ACTIP//Secretar&#xed;a de Ciencia y Tecnolog&#xed;a - Universidad Nacional de C&#xf3;rdoba/ ; MHE200027-EMHE-CSIC-2016-program//Consejo Superior de Investigaciones Cient&#xed;ficas/ ; }, mesh = {*Plasmids/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Genetic Vectors/genetics ; *Lactococcus/genetics ; Hydrogen-Ion Concentration ; }, abstract = {In this work, we developed a plasmid-based CRISPR-Cas9 strategy for editing Lactococcus cremoris, which allows easy generation of plasmid-free strains with the desired modification. We constructed versatile shuttle vectors based on the theta-type pAMβ1 promiscuous replicon and p15A ori, expressing both the Cas9 nuclease gene (under pH-regulated promoters derived from P170) and a single-guide RNA for specific targeting (under a strong constitutive promoter). The vectors designed for plasmid targeting were very effective for low- and high-copy-number plasmid curing in L. cremoris, and their targeting efficiency was shown to be tunable by regulating cas9 expression. For chromosome editing, we implemented a host-independent method that enhances double-homologous recombination events using plasmids expressing the genes encoding λRed-phage Redβ recombinase and Escherichia coli single-stranded DNA binding protein (EcSSB). By coupling either the endogenous recombination machinery or the Redβ-EcSSB-assisted recombination system with our novel chromosome-targeting CRISPR-Cas9 plasmids, we efficiently generated and selected thousands of gene-edited cells. Examination of the impact of the constructed CRISPR-Cas9 vectors on host fitness revealed no Cas9-associated toxicity, and, remarkably, these vectors exhibited a very high loss rate when growing the bacterial host cells in the absence of selective pressure.}, }
@article {pmid39707395, year = {2024}, author = {Xu, W and Zhang, S and Qin, H and Yao, K}, title = {From bench to bedside: cutting-edge applications of base editing and prime editing in precision medicine.}, journal = {Journal of translational medicine}, volume = {22}, number = {1}, pages = {1133}, pmid = {39707395}, issn = {1479-5876}, support = {82471107//National Natural Science Foundation of China/ ; 31970930//National Natural Science Foundation of China/ ; 2020CFA069//Natural Science Foundation of Hubei Province/ ; 2018CFB434//Natural Science Foundation of Hubei Province/ ; }, mesh = {Humans ; *Precision Medicine/methods ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Translational Research, Biomedical ; Animals ; Genetic Therapy/methods ; }, abstract = {CRISPR-based gene editing technology theoretically allows for precise manipulation of any genetic target within living cells, achieving the desired sequence modifications. This revolutionary advancement has fundamentally transformed the field of biomedicine, offering immense clinical potential for treating and correcting genetic disorders. In the treatment of most genetic diseases, precise genome editing that avoids the generation of mixed editing byproducts is considered the ideal approach. This article reviews the current progress of base editors and prime editors, elaborating on specific examples of their applications in the therapeutic field, and highlights opportunities for improvement. Furthermore, we discuss the specific performance of these technologies in terms of safety and efficacy in clinical applications, and analyze the latest advancements and potential directions that could influence the future development of genome editing technologies. Our goal is to outline the clinical relevance of this rapidly evolving scientific field and preview a roadmap for successful DNA base editing therapies for the treatment of hereditary or idiopathic diseases.}, }
@article {pmid39706269, year = {2024}, author = {Li, X and Wei, Y and Wang, SY and Wang, SG and Xia, PF}, title = {One-for-all gene inactivation via PAM-independent base editing in bacteria.}, journal = {The Journal of biological chemistry}, volume = {}, number = {}, pages = {108113}, doi = {10.1016/j.jbc.2024.108113}, pmid = {39706269}, issn = {1083-351X}, abstract = {Base editing is preferable for bacterial gene inactivation without generating double strand breaks, requiring homology recombination or highly efficient DNA delivery capability. However, the potential of base editing is limited by the adjoined dependence on the editing window and protospacer adjacent motif (PAM). Herein, we report an unconstrained base editing system to enable the inactivation of any genes of interest (GOIs) in bacteria. We employed a dCas9 derivative, dSpRY, and activation-induced cytidine deaminase to build a PAM-independent base editor. Then, we programmed the base editor to exclude the START codon of a GOI instead of introducing premature STOP codons to obtain a universal approach for gene inactivation, namely XSTART, with an overall efficiency approaching 100%. By using XSTART, we successfully manipulated the amino acid metabolisms in Escherichia coli, generating glutamine, arginine, and aspartate auxotrophic strains. While we observed a high frequency of off-target events as a trade-off for increased efficiency, refining the regulatory system of XSTART to limit expression levels reduced off-target events by over 60% without sacrificing efficiency, aligning our results with previously reported levels. Finally, the effectiveness of XSTART was also demonstrated in probiotic E. coli Nissle 1917 and photoautotrophic cyanobacterium Synechococcus elongatus, illustrating its potential in reprogramming diverse bacteria.}, }
@article {pmid39706164, year = {2024}, author = {Cenik, BK and Aoi, Y and Iwanaszko, M and Howard, BC and Morgan, MA and Andersen, GD and Bartom, ET and Shilatifard, A}, title = {TurboCas: A method for locus-specific labeling of genomic regions and isolating their associated protein interactome.}, journal = {Molecular cell}, volume = {84}, number = {24}, pages = {4929-4944.e8}, doi = {10.1016/j.molcel.2024.11.007}, pmid = {39706164}, issn = {1097-4164}, mesh = {Humans ; *Promoter Regions, Genetic ; *Transcription Factors/metabolism/genetics ; *Chromatin/metabolism/genetics ; HEK293 Cells ; RNA Polymerase II/metabolism/genetics ; Protein Binding ; CRISPR-Cas Systems ; Cell Cycle Proteins/metabolism/genetics ; Positive Transcriptional Elongation Factor B/metabolism/genetics ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; Proto-Oncogene Proteins c-fos/genetics/metabolism ; Gene Expression Regulation ; Heat-Shock Response/genetics ; Protein Interaction Maps ; Bromodomain Containing Proteins ; }, abstract = {Regulation of gene expression during development and stress response requires the concerted action of transcription factors and chromatin-binding proteins. Because this process is cell-type specific and varies with cellular conditions, mapping of chromatin factors at individual regulatory loci is crucial for understanding cis-regulatory control. Previous methods only characterize static protein binding. We present "TurboCas," a method combining a proximity-labeling (PL) enzyme, miniTurbo, with CRISPR-dCas9 that allows for efficient and site-specific labeling of chromatin factors in mammalian cells. Validating TurboCas at the FOS promoter, we identify proteins recruited upon heat shock, cross-validated via RNA polymerase II and P-TEFb immunoprecipitation. These methodologies reveal canonical and uncharacterized factors that function to activate expression of heat-shock-responsive genes. Applying TurboCas to the MYC promoter, we identify two P-TEFb coactivators, the super elongation complex (SEC) and BRD4, as MYC co-regulators. TurboCas provides a genome-specific targeting PL, with the potential to deepen our molecular understanding of transcriptional regulatory pathways in development and stress response.}, }
@article {pmid39705367, year = {2024}, author = {Su, Z and Zhang, W and Shi, Y and Cui, T and Xu, Y and Yang, R and Huang, M and Zhou, C and Zhang, H and Lu, T and Qu, J and He, ZG and Gan, J and Feng, Y}, title = {A bacterial methyltransferase that initiates biotin synthesis, an attractive anti-ESKAPE druggable pathway.}, journal = {Science advances}, volume = {10}, number = {51}, pages = {eadp3954}, pmid = {39705367}, issn = {2375-2548}, mesh = {*Biotin/biosynthesis ; *Methyltransferases/metabolism/chemistry ; *Acinetobacter baumannii/enzymology ; *Klebsiella pneumoniae/enzymology ; Bacterial Proteins/metabolism/genetics/chemistry ; Adenosine/analogs &amp; derivatives/metabolism ; Anti-Bacterial Agents/pharmacology ; Models, Molecular ; Humans ; CRISPR-Cas Systems ; }, abstract = {The covalently attached cofactor biotin plays pivotal roles in central metabolism. The top-priority ESKAPE-type pathogens, Acinetobacter baumannii and Klebsiella pneumoniae, constitute a public health challenge of global concern. Despite the fact that the late step of biotin synthesis is a validated anti-ESKAPE drug target, the primary stage remains fragmentarily understood. We report the functional definition of two BioC isoenzymes (AbBioC for A. baumannii and KpBioC for K. pneumoniae) that act as malonyl-ACP methyltransferase and initiate biotin synthesis. The physiological requirement of biotin is diverse within ESKAPE pathogens. CRISPR-Cas9-based inactivation of bioC rendered A. baumannii and K. pneumoniae biotin auxotrophic. The availability of soluble AbBioC enabled the in vitro reconstitution of DTB/biotin synthesis. We solved two crystal structures of AbBioC bound to SAM cofactor (2.54 angstroms) and sinefungin (SIN) inhibitor (1.72 angstroms). Structural and functional study provided molecular basis for SIN inhibition of BioC. We demonstrated that BioC methyltransferase plays dual roles in K. pneumoniae infection and A. baumannii colistin resistance.}, }
@article {pmid39705306, year = {2024}, author = {Hou, J and Guo, P and Wang, J and Han, D and Tan, W}, title = {Artificial dynamic structure ensemble-guided rational design of a universal RNA aptamer-based sensing tag.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {52}, pages = {e2414793121}, pmid = {39705306}, issn = {1091-6490}, mesh = {*Aptamers, Nucleotide/chemistry ; Humans ; *Biosensing Techniques/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Gene Editing/methods ; Nucleic Acid Conformation ; RNA/chemistry ; Fluorescent Dyes/chemistry ; }, abstract = {Artificially functional RNAs, such as fluorogenic RNA aptamer (FRApt)-based biosensing tag, represent significant advancements in various biological applications but are limited by the lack of insight into dynamic structure ensembles and universal design concepts. Through the development of an artificial RNA structure ensemble, we rationally established an RNA reconstitution model, "SSPepper-Apt," to generate a universal fluorogenic RNA biosensing tag. By utilizing various target-recognizing RNA motifs, SSPepper-Apt enables the modular generation of sensing tags for low-background, highly selective imaging of metabolites, peptides, and proteins in living cells. Additionally, by employing single guide RNA (sgRNA) as the recognition RNA motif, SSPepper-Apt generates fluorescence in both CRISPR-mediated imaging and gene editing only when the Cas9-sgRNA complex is successfully assembled; therefore, it can be an effective sgRNA screening tool for gene editing. Our fluorogenic RNA-sensing tag provides a universal approach for constructing functional RNA systems, avoiding the laborious and time-consuming process of sequence combination, and expanding the application of synthetic biological tools.}, }
@article {pmid39704190, year = {2024}, author = {Fan, Z and Xu, L and Cao, Y and Liu, T and Tian, Y and Pan, Z and Mo, Y and Wang, X and Zhu, X and Gao, Y and Zhang, X and Pan, CQ and Wang, L and Ren, F}, title = {One-Pot Assay Based on CRISPR/Cas13a Technology for HEV RNA Point-of-Care Testing.}, journal = {Journal of medical virology}, volume = {96}, number = {12}, pages = {e70115}, pmid = {39704190}, issn = {1096-9071}, support = {//This study was supported by the National Natural Science Foundation of China (82002243, 82100653), Key Projects of the Beijing Municipal Education Commission's Science and Technology Plan (KZ202010025035), Chinese Institutes for Medical Research, Beijing (Grant No. CX24PY23), Beijing Hospitals Authority Youth Programme (QML20201702), Talent Cultivation Plan of Climbing the Peak of Beijing Municipal Hospital Administration (DFL20221503), Beijing Natural Science Foundation-Changping Innovation Joint Fund (L234046), Training Fund for Open Projects at Clinical Institutes and Departments of Capital Medical University (CCMU2023ZKYXZ003), High-Level Public Health Technical Talents Project of Beijing (Subject Leaders-02-13, xuekegugan-03-48)./ ; }, mesh = {*Hepatitis E virus/genetics/isolation &amp; purification ; *Point-of-Care Testing ; *Hepatitis E/diagnosis/virology ; *RNA, Viral/genetics ; Humans ; *CRISPR-Cas Systems ; *Sensitivity and Specificity ; Animals ; Limit of Detection ; Reverse Transcriptase Polymerase Chain Reaction/methods ; Genotype ; Nucleic Acid Amplification Techniques/methods ; }, abstract = {Hepatitis E virus (HEV) poses a serious threat to both public health and animal food safety, thereby highlighting the demands for rapid, sensitive, and easy-to-use detection. This study aimed to develop a One-Pot assay using CRISPR/Cas13a for detecting HEV RNA, suitable for point-of-care testing (POCT) in resource-limited settings. CRISPR/Cas13a combined with reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription recombinase-aided amplification (RT-RAA) was applied to a One-Pot assay device. Additionally, a large cohort of HEV-infected patient (154) and animal (104) specimens was utilized for validation. The RT-PCR/RT-RAA + CRISPR/Cas13a assays for HEV RNA detection (genotypes: HEV-1, HEV-3, and HEV-4) were established, optimized, and validated, achieving a limit of detection (LoD) of 1 copy/μL and 100% specificity. In the application validation for HEV infection, the positive rates of the RT-PCR + CRISPR and RT-RAA + CRISPR assays were 98.6% and 89.6% for patients, and 96.6% and 88.8% for animals, respectively, which were superior to those of RT-qPCR. Furthermore, sample rapid lysis, reagent lyophilization, and the One-Pot device were integrated to construct a One-Pot assay with an LoD of 10[2] copies/μL. Despite slight decreases in sensitivity, the One-Pot assay significantly reduces the assay time to 35 min, making it easy to perform, minimizing contamination, and meeting the requirements for screening. We developed a One-Pot assay of HEV RNA using the CRISPR/Cas13a which effectively realizes a POCT test and maximizes the impetus for POCT implementation and shows potential as a valuable tool for detecting and monitoring HEV infection.}, }
@article {pmid39703747, year = {2024}, author = {Fallah, T and Shafiei, M}, title = {Comprehensive Analysis of CRISPR-Cas Systems and Their Influence on Antibiotic Resistance in Salmonella enterica Strains.}, journal = {Bioinformatics and biology insights}, volume = {18}, number = {}, pages = {11779322241307984}, pmid = {39703747}, issn = {1177-9322}, abstract = {Salmonella enterica is a gram-negative bacterium that demonstrates a remarkable ability to acquire antibiotic resistance genes (ARGs). The role of the CRISPR-Cas system in influencing antibiotic resistance in S. enterica is still under investigation. This study explores the distribution and impact of CRISPR-Cas systems on antibiotic resistance by analyzing 316 S. enterica genomes. We conducted sequence alignments, phylogenetic analyses, and conservation studies on Cas genes, direct repeats (DRs), and leader sequences. Promoter predictions and RNA secondary structure analyses were also performed. ARGs were identified, and their correlation with Cas gene clusters was evaluated. Our findings revealed that 82.33% of strains possess complete CRISPR-Cas systems, while 17.66% have orphan CRISPRs. We identified 290 distinct DRs, most of which formed stable stem-loop structures, although no promoter regions were detected within the leader sequences. Most spacers were chromosome-targeting, with a smaller proportion homologous to phages and plasmids. Importantly, strains with complete CRISPR-Cas systems showed a higher incidence of ARGs compared with those with orphan or no CRISPR systems. Specifically, the incidence of ARGs was 54.3% higher in strains with complete CRISPR-Cas systems than in strains without CRISPR-Cas systems, and 15.1% higher than in strains with orphan CRISPRs. Spearman's correlation analysis confirmed a statistically significant but weak correlation between the presence of Cas genes and the frequency of ARGs (P-value = 3.892e-06). These results suggest that CRISPR-Cas systems may play a role in the acquisition of ARGs, potentially through mutations under antibiotic pressure. Future studies should investigate mutations, particularly in Cas3-the signature protein of type I CRISPR-Cas systems. In addition, experimental validation, such as culturing S. enterica strains with complete CRISPR-Cas systems under different antibiotic conditions, followed by sequencing to assess the uptake or absence of newly acquired ARGs, would help clarify the potential role of CRISPR-Cas systems in bacterial adaptation to antimicrobial pressures.}, }
@article {pmid39702810, year = {2024}, author = {Bhagat, M and Kamal, R and Sharma, J and Kaur, K and Sharma, A and Thakur, GS and Bhatia, R and Awasthi, A}, title = {Gene Therapy: Towards a New Era of Medicine.}, journal = {AAPS PharmSciTech}, volume = {26}, number = {1}, pages = {17}, pmid = {39702810}, issn = {1530-9932}, mesh = {Humans ; *Genetic Therapy/methods/trends ; Animals ; *Genetic Vectors ; CRISPR-Cas Systems ; Gene Editing/methods ; Gene Transfer Techniques ; }, abstract = {Over the past years, many significant advances have been made in the field of gene therapy and shown promising results in clinical trials conducted. Gene therapy aims at modifying or replacing a defective, inefficient, or nonfunctional gene with a healthy, functional gene by administration of genome material into the cell to cure genetic diseases. Various methods have been devised to do this by using several viral and non-viral vectors which are either administered by in vivo or ex vivo technique. Viral vectors are best suitable for this therapy due to their potential to invade cells and deliver their genetic material whereas non-viral vectors are less efficient than viral vectors but possess some advantages such as less immunogenic response and large gene carrying capacity. Recent advances in biotechnology such as CRISPR-Cas9 mediated genome engineering and Cancer treatment with Chimeric antigen receptor (CAR) T-cell therapy are addressed in this review. This review article also delves into some recent research studies, gene therapy trials, and its applications, laying out future hopes for gene therapy in the treatment of various diseases namely haemophilia, Muscular dystrophy, SCID, Sickle cell disease, Familial Hypercholesterolemia, Cystic Fibrosis. Additionally, it also includes various nanoformulations and clinical trial data related to gene therapy.}, }
@article {pmid39702692, year = {2024}, author = {Du, W and Meister, LL and van Grinsven, T and Branco Dos Santos, F}, title = {Efficient Multiplex Genome Editing of the Cyanobacterium Synechocystis sp. PCC6803 via CRISPR-Cas12a.}, journal = {Biotechnology and bioengineering}, volume = {}, number = {}, pages = {}, doi = {10.1002/bit.28910}, pmid = {39702692}, issn = {1097-0290}, abstract = {Cyanobacteria have been genetically modified to convert CO2 into biochemical products, but efficient genetic engineering tools, including CRISPR-Cas systems, remain limited. This is primarily due to the polyploid nature of cyanobacteria, which hinders their effectiveness. Here, we address the latter by specifically (i) modifying the RSF1010-based replicative plasmid to simplify cloning efforts while maintaining high conjugation efficiency; (ii) improving the design of the guide RNA (gRNA) to facilitate chromosomal cleavage; (iii) introducing template DNA fragments as pure plasmids via natural transformation; and (iv) using sacB to facilitate replicative plasmid curing. With this system, the replicative plasmid containing both Cas12a and gRNA is introduced to Synechocystis sp. PCC6803 cells via conjugation to cleave the circular chromosomes. Template DNA plasmid that has meanwhile been assimilated will then repair it achieving the desired genetic modifications. This system was validated by successfully deleting various "neutral" chromosomal loci, both individually and collectively, as well as targeting an essential gene, sll1797. With the sacB-sucrose counter-selection, all deletions were simultaneously made markerless in < 4 weeks. Moreover, we also integrate YFP with various protein degradation tags into the chromosome, allowing for their characterization at the chromosomal level. We foresee this system will greatly facilitate future genome engineering in cyanobacteria.}, }
@article {pmid39702666, year = {2024}, author = {Kou, Z and Wang, S and Luo, X and Xu, J and Tomberlin, JK and Huang, Y}, title = {Wingless strain created using binary transgenic CRISPR/Cas9 alleviates concerns about mass rearing of Hermetia illucens.}, journal = {Communications biology}, volume = {7}, number = {1}, pages = {1652}, pmid = {39702666}, issn = {2399-3642}, support = {32021001//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32100381//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {Animals ; *CRISPR-Cas Systems ; *Animals, Genetically Modified ; *Diptera/genetics ; Female ; Male ; }, abstract = {Larvae of the black soldier fly Hermetia illucens have potential as a natural waste recycler and subsequent use as protein-rich feed for livestock. A common question about the insect-farming processes is, what about the concerns of mass escape of insects from large populations? Here, we present a binary transgenic CRISPR/Cas9 system to generate wingless strain with the potential to address this issue. We identified gonad-specific promoters in vivo and evaluated use of the two strongest promoters, nanos and exuperantia, to drive Cas9 expression. We found that crossing the Hiexu-Cas9 with transgenic sgRNA-expressing insects resulted in higher knockout efficiency of the marker gene white. The Hiexu-Cas9 strain exhibited a maternal deposition of Cas9 that caused more effective knockout in the progeny of female Cas9-expressing individuals. Using this system, we generated wingless mutants lacking mating ability, which can be maintained in colony through a genetic cross of two single strain. These insects are less likely to escape and would be unable to successfully mate if they did escape. Taken together, this study validates effective genetic tools that can be used for gene function studies and industrial applications in black soldier fly and provides an approach to alleviate the concern about massive rearing.}, }
@article {pmid39702333, year = {2024}, author = {Liang, QZ and Chen, W and Liu, RC and Fu, QL and Fu, GH and Cheng, LF and Chen, HM and Jiang, NS and Zhu, T and Huang, Y}, title = {CRISPR/Cas12a and recombinase polymerase amplification-based rapid on-site nucleic acid detection of duck circovirus.}, journal = {Virology journal}, volume = {21}, number = {1}, pages = {322}, pmid = {39702333}, issn = {1743-422X}, support = {ZYTS202423//the Freedom Explore Program of Fujian Academy of Agricultural Sciences/ ; 2023J01363//the Natural Science Foundation Project of Fujian Province/ ; CARS-42//the National Waterfowl Industry Technology System of Modern Agriculture for China/ ; }, mesh = {*Circovirus/genetics/isolation &amp; purification ; Animals ; *Ducks/virology ; *Circoviridae Infections/veterinary/diagnosis/virology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Sensitivity and Specificity ; Poultry Diseases/virology/diagnosis ; Recombinases/metabolism/genetics ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {BACKGROUND: Duck circovirus (DuCV) infections commonly induce immunosuppression and secondary infections in ducks, resulting in significant economic losses in the duck breeding industry. Currently, effective vaccines and treatments for DuCV have been lacking. Therefore, rapid, specific, and sensitive detection methods are crucial for preventing and controlling DuCV.<br><br>METHODS: A lateral flow strip (LFS) detection method was developed using recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a). The RPA-CRISPR/Cas12a-LFS targeted the DuCV replication protein (Rep) and was operated at 37 &#x2103; and allowed for visual interpretation without requiring sophisticated equipment.<br><br>RESULTS: The results revealed that the reaction time of RPA-CRISPR/Cas12a-LFS is only 45&#xa0;min. This method achieved a low detection limit of 2.6 gene copies. Importantly, this method demonstrated high specificity and no cross-reactivity with six other avian viruses. In a study involving 97 waterfowl samples, the Rep RPA-CRISPR/Cas12a-LFS showed 100% consistency and agreement with real-time quantitative polymerase chain reaction.<br><br>CONCLUSION: These findings underscored the potential of this user-friendly, rapid, sensitive, and accurate detection method for on-site DuCV detection.}, }
@article {pmid39701024, year = {2024}, author = {Lee, J and Jeong, C}, title = {Single-molecule perspectives of CRISPR/Cas systems: target search, recognition, and cleavage.}, journal = {BMB reports}, volume = {}, number = {}, pages = {}, pmid = {39701024}, issn = {1976-670X}, abstract = {CRISPR/Cas systems have emerged as powerful tools for gene editing, nucleic acid detection, and therapeutic applications. Recent advances in single-molecule techniques have provided new insights into the DNA-targeting mechanisms of CRISPR/Cas systems, in particular, Types I, II, and V. Here, we review how single-molecule approaches have expanded our understanding of key processes, namely target search, recognition, and cleavage. Furthermore, we focus on the dynamic behavior of Cas proteins, including PAM site recognition and R-loop formation, which are crucial to ensure specificity and efficiency in gene editing. Additionally, we discuss the conformational changes and interactions that drive precise DNA cleavage by different Cas proteins. This mini review provides a comprehensive overview of CRISPR/Cas molecular dynamics, offering conclusive insights into their broader potential for genome editing and biotechnological applications.}, }
@article {pmid39700796, year = {2024}, author = {Wang, X and Ding, H and Sun, Y and Ma, Y and Wang, G and Chen, J and Choo, J and Chen, L}, title = {CRISPR/HCR-powered ratiometric fluorescence aptasensor for ochratoxin A detection.}, journal = {Food chemistry}, volume = {468}, number = {}, pages = {142437}, doi = {10.1016/j.foodchem.2024.142437}, pmid = {39700796}, issn = {1873-7072}, abstract = {To address the need for highly sensitive and reliable detection of trace ochratoxin A (OTA) in food matrices, we developed a ratiometric fluorescent aptasensor by integrating CRISPR/Cas12a, hybridization chain reaction (HCR), and horseradish peroxidase (HRP)-induced inner filter effect (IFE). The mechanism involves OTA releasing an activator that initiates CRISPR/Cas12a trans-cleavage, blocking HCR assembly. This reduces HRP levels, limiting the conversion of o-phenylenediamine (OPD) to fluorescent 2,3-diaminophenazine (DAP) (emitting at 562 nm) while maintaining strong emission from 2-amino terephthalic acid (BDC-NH2) at 426 nm. The F426/F562 ratio serves as a "signal-on" indicator, enabling sensitive OTA detection over 0.1 pM to 10 nM, with a detection limit of 0.0417 pM. The method exhibits excellent reproducibility, with intra-day and inter-day relative standard deviations (RSDs) of 1.91 %-3.87 % and 1.79 %, respectively, along with recovery rates of 90.1 %-110.6 % in real samples. These advantages highlight its significant potential for CRISPR/Cas-based OTA detection.}, }
@article {pmid39700179, year = {2024}, author = {Bowland, K and Lai, J and Skaist, A and Zhang, Y and Teh, SSK and Roberts, NJ and Thompson, E and Wheelan, SJ and Hruban, RH and Karchin, R and Bailey, MH and Iacobuzio-Donahue, CA and Eshleman, JR}, title = {Islands of genomic stability in the face of genetically unstable metastatic cancer.}, journal = {PloS one}, volume = {19}, number = {12}, pages = {e0298490}, pmid = {39700179}, issn = {1932-6203}, support = {P30 CA006973/CA/NCI NIH HHS/United States ; P50 CA062924/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; *Genomic Instability ; *Pancreatic Neoplasms/genetics/pathology ; *CRISPR-Cas Systems/genetics ; *Neoplasm Metastasis/genetics ; Carcinoma, Pancreatic Ductal/genetics/pathology/secondary ; Mutation ; Male ; Whole Genome Sequencing ; Female ; }, abstract = {INTRODUCTION: Metastatic cancer affects millions of people worldwide annually and is the leading cause of cancer-related deaths. Most patients with metastatic disease are not eligible for surgical resection, and current therapeutic regimens have varying success rates, some with 5-year survival rates below 5%. Here, we test the hypothesis that metastatic cancer can be genetically targeted by exploiting single base substitution mutations unique to individual cells that occur as part of normal aging prior to transformation. These mutations are targetable because ~10% of them form novel tumor-specific "NGG" protospacer adjacent motif (PAM) sites targetable by CRISPR-Cas9.<br><br>METHODS: Whole genome sequencing was performed on five rapid autopsy cases of patient-matched primary tumor, normal and metastatic tissue from pancreatic ductal adenocarcinoma decedents. CRISPR-Cas9 PAM targets were determined by bioinformatic tumor-normal subtraction for each patient and verified in metastatic samples by high-depth capture-based sequencing.<br><br>RESULTS: We found that 90% of PAM targets were maintained between primary carcinomas and metastases overall. We identified rules that predict PAM loss or retention, where PAMs located in heterozygous regions in the primary tumor can be lost in metastases (private LOH), but PAMs occurring in regions of loss of heterozygosity (LOH) in the primary tumor were universally conserved in metastases.<br><br>CONCLUSIONS: Regions of truncal LOH are strongly retained in the presence of genetic instability and, therefore, represent genetic vulnerabilities in pancreatic adenocarcinomas. A CRISPR-based gene therapy approach targeting these regions may be a novel way to genetically target metastatic cancer.}, }
@article {pmid39700052, year = {2024}, author = {Li, X and Chen, B and Xie, Y and Luo, Y and Zhu, D and Wang, L and Su, S}, title = {Polyvalent Aptamers Structure-Mediated Fluorescent Aptasensor for the Early Diagnosis of Alzheimer's Disease by Coupling with HCR and CRISPR-Cas System.}, journal = {Analytical chemistry}, volume = {}, number = {}, pages = {}, doi = {10.1021/acs.analchem.4c05329}, pmid = {39700052}, issn = {1520-6882}, abstract = {The early diagnosis of Alzheimer's disease (AD) plays a vital role in slowing the progression of AD and improving the quality of human life. However, it is still a challenge in the medical field. Herein, an ultrasensitive fluorescent aptasensor was designed for the detection of special phosphorylated tau181 (P-tau181) by coupling with polyvalent aptamers (PAs) structure, hybridization chain reaction (HCR), and the CRISPR-Cas system. Coupling with the signal amplification strategy, the specific recognition ability of the aptamer, and the high cleavage activity of Cas12a protein, the designed aptasensor showed a wide linear range (0.1-10[6] pg/mL), a low detection limit (0.069 pg/mL), high selectivity, and excellent anti-interference ability for the detection of P-tau181. Moreover, the aptasensor can efficiently analyze P-tau181 in artificial cerebro spinal fluid (aCSF) and serum, proving that it has a promising application in the early diagnosis of AD.}, }
@article {pmid39699588, year = {2025}, author = {Yang, Q and Dong, MJ and Xu, J and Xing, Y and Wang, Y and Yang, J and Meng, X and Xie, T and Li, Y and Dong, H}, title = {CRISPR/RNA Aptamer System Activated by an AND Logic Gate for Biomarker-Driven Theranostics.}, journal = {Journal of the American Chemical Society}, volume = {147}, number = {1}, pages = {169-180}, doi = {10.1021/jacs.4c08719}, pmid = {39699588}, issn = {1520-5126}, mesh = {Humans ; *Aptamers, Nucleotide/chemistry ; *MicroRNAs/analysis ; *Theranostic Nanomedicine ; Photosensitizing Agents/chemistry/pharmacology ; Photochemotherapy ; CRISPR-Cas Systems ; Cell Line, Tumor ; Biomarkers, Tumor ; Nanoparticles/chemistry ; }, abstract = {The development of an engineered RNA device capable of detecting multiple biomarkers to evaluate pathological states and autonomously implement responsive therapies is urgently needed. Here, we report InCasApt, an integrated nano CRISPR Cas13a/RNA aptamer theranostic platform capable of achieving both biomarker detection and biomarker-driven therapy. Within this system, a Cas13a/crRNA complex, a hairpin reporter (HR), a dinitroaniline caged Ce6 photosensitizer (Ce6-DN), and a DN-binding RNA aptamer precursor (DNBApt) are coloaded onto dendritic mesoporous silicon nanoparticles (DMSN) in a controlled manner. While InCasApt remains inert in normal cells, its programmable theranostic capabilities are activated in tumor cells that have elevated expression of carcinogenic miRNA-155 and miRNA-21. These miRNAs act as an AND logic gate, generating fluorescence for disease condition evaluation and ROS for photodynamic therapy. This process also upregulates antioncogene BRG1 and suppresses tumor migration by inhibiting the function of miRNA-155 and miRNA-21. These effects underscore the versatility of InCasApt as an miRNA-targeting strategy for bridging the gap between diagnosis and therapy.}, }
@article {pmid39699265, year = {2024}, author = {Li, X and Wang, L and Lin, J and Gu, Y and Liu, Z and Hu, J}, title = {Detection of CRISPR‒Cas and type I R-M systems in Klebsiella pneumoniae of human and animal origins and their relationship to antibiotic resistance and virulence.}, journal = {Microbiology spectrum}, volume = {}, number = {}, pages = {e0000924}, doi = {10.1128/spectrum.00009-24}, pmid = {39699265}, issn = {2165-0497}, abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)‒CRISPR-associated protein (Cas) and restriction‒modification (R-M) systems are important immune systems in bacteria. Information about the distributions of these two systems in Klebsiella pneumoniae from different hosts and their mutual effect on antibiotic resistance and virulence is still limited. In this study, the whole genomes of 520 strains of K. pneumoniae from GenBank, including 325 from humans and 195 from animals, were collected for CRISPR‒Cas systems and type I R-M systems, virulence genes, antibiotic resistance genes, and multilocus sequence typing detection. The results showed that host origin had no obvious influence on the distributions of the two systems (CRISPR‒Cas systems in 29.8% and 24.1%, type I R-M systems in 9.8% and 11.8% of human-origin and animal-origin strains, respectively) in K. pneumoniae. Identical spacer sequences from different hosts demonstrated there was a risk of human-animal transmission. All virulence genes (yersiniabactin, colibactin, aerobactin, salmochelin, rmpADC, and rmpA2) detection rates were higher when only the CRISPR‒Cas systems were present but were all reduced when coexisting with type I R-M systems. However, a lower prevalence of most antibiotic-resistance genes was found when the CRISPR‒Cas systems were alone, and when type I R-M systems were coexisting, some of the antibiotic resistance gene incidence rates were even lower (quinolones, macrolides, tetracyclines and carbapenems), and some of them were higher instead (aminoglycosides, clindamycins, rifampicin-associated, sulfonamides, methotrexates, beta-lactamases and ultrabroad-spectrum beta-lactamases). The synergistic and opposed effects of the two systems on virulence and antibiotic-resistance genes need further study.IMPORTANCEK. pneumoniae is an important opportunistic pathogen responsible for both human and animal infections, and the emergence of hypervirulent and multidrug-resistant K. pneumoniae has made it difficult to control this pathogen worldwide. Here, we find that CRISPR‒Cas and restriction-modification systems, which function as adaptive and innate immune systems in bacteria, have synergistic and opposed effects on virulence and antibiotic resistance genes in K. pneumoniae. Moreover, this study provides insights into the distributions of the two systems in K. pneumoniae from different hosts, and there is no significant difference in the prevalence of the two systems among K. pneumoniae spp. In addition, this study also characterizes the CRISPR arrays of K. pneumoniae from different hosts, suggesting that the strains sharing the same spacer sequences have the potential to spread between humans and animals.}, }
@article {pmid39699033, year = {2024}, author = {Paggi, RA and Ferrari, MC and Cerletti, M and Giménez, MI and Schwarz, TS and Marchfelder, A and De Castro, RE}, title = {Practical laboratory class to assess gene silencing using CRISPR interference (CRISPRi) technology in the archaeon Haloferax volcanii.}, journal = {Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology}, volume = {}, number = {}, pages = {}, doi = {10.1002/bmb.21872}, pmid = {39699033}, issn = {1539-3429}, support = {//Universidad Nacional de Mar del Plata/ ; PICT 02228//Fondo para la Investigaci&#xf3;n Cient&#xed;fica y Tecnol&#xf3;gica (FONCYT)/ ; }, abstract = {Perturbation of gene expression using RNA interference (RNAi) or CRISPR interference (CRISPRi) is a useful strategy to explore the function of essential genes. In the archaeon Haloferax volcanii, the CRISPR-Cas system has been adapted as a CRISPRi tool to silence the expression of specific genes. We developed a laboratory class (LC) to conceptualize gene silencing through inactivation of the H. volcanii LonB protease gene, a negative regulator of carotenoid pigments biosynthesis, using CRISPRi. This LC has been successfully applied in the Biology and Biochemistry of Microorganisms course for undergraduate students of Biology in 2022 and 2023. The following objectives were proposed: (a) generate H. volcanii mutant strains with reduced expression of the lonB gene using CRISPRi; (b) examine the effect of lonB gene silencing on cell pigmentation and growth rate; (c) assess lonB gene repression by Western blotting (WB). This LC allows students to obtain and screen CRISPRi silenced-mutants by means of simple procedures using a non-pathogenic organism as well as handle basic microbiology, biochemistry and molecular biology protocols. Additionally, the LC fosters social actions through collaborative work (experimental work), the interpretation and discussion of data and the ability to communicate outcomes orally and in a written format (scientific report).}, }
@article {pmid39698811, year = {2024}, author = {Soczek, KM and Cofsky, JC and Tuck, OT and Shi, H and Doudna, JA}, title = {CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkae1192}, pmid = {39698811}, issn = {1362-4962}, support = {U19AI171110/NH/NIH HHS/United States ; U19NS132303/NS/NINDS NIH HHS/United States ; R21HL173710/HB/NHLBI NIH HHS/United States ; 2334028//National Science Foundation/ ; /HHMI/Howard Hughes Medical Institute/United States ; //Panattoni Family Foundation/ ; //National Science Foundation Graduate Research Fellowship/ ; //Helen Hay Whitney Foundation/ ; //HHMI Fellow of The Jane Coffin Childs Fund for Medical Research/ ; DE-AC02-05CH11231//Department of Energy/ ; 24&#x2009;180//Apple Tree Partners/ ; //Lawrence Livermore National Laboratory/ ; //UCB-Hampton University Summer Program/ ; //Mr. Li Ka Shing/ ; //Koret-Berkeley-TAU/ ; //Innovative Genomics Institute/ ; }, abstract = {RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ∼20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA interference begins with local helix distortion by transient CRISPR-Cas protein binding.}, }
@article {pmid39698318, year = {2024}, author = {Cao, S and Ma, D and Xie, J and Wu, Z and Yan, H and Ji, S and Zhou, M and Zhu, S}, title = {Point-of-care testing diagnosis of African swine fever virus by targeting multiple genes with enzymatic recombinase amplification and CRISPR/Cas12a System.}, journal = {Frontiers in cellular and infection microbiology}, volume = {14}, number = {}, pages = {1474825}, pmid = {39698318}, issn = {2235-2988}, mesh = {*African Swine Fever Virus/genetics/isolation &amp; purification ; Animals ; *CRISPR-Cas Systems ; Swine ; *African Swine Fever/diagnosis/virology ; *Point-of-Care Testing ; *Sensitivity and Specificity ; *Recombinases/metabolism/genetics ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/genetics ; Endodeoxyribonucleases/genetics ; Molecular Diagnostic Techniques/methods ; Bacterial Proteins ; }, abstract = {African swine fever virus (ASFV) infection is causing devastating outbreaks globally; pig farming has suffered severe economic losses due to the ASFV. Currently, strict biosecurity control measures can mitigate the incidence of ASF. Rapid, cost-effective, and sensitive detection of ASFV can significantly reduce disease transmission and mortality. CRISPR/Cas-associated proteins can detect polymorphisms with high specificity and sensitivity, making them ideal for detecting pathogens. In this study, based on CRISPR/Cas12a integrated with enzymatic recombinase amplification (ERA) technology, a CRISPR/Cas12a detection system capable of identifying ASFV E183L, K205R, and C962R gene sequences has been developed. The ERA-CRISPR/Cas12a detection system detected ASFV precisely without cross-reactivity with other porcine pathogen templates and with a sensitivity detection limit of 10 copies per reaction; it takes 60 minutes to complete the detection process. In combination with this integrated ERA pre-amplification and Cas12a/crRNA cutting assay, it provides a rapid, straightforward, sensitive, and specific method for ASFV detection in the field.}, }
@article {pmid39696686, year = {2024}, author = {Izadifar, M and Massumi, M and Prentice, KJ and Oussenko, T and Li, B and Elbaz, J and Puri, M and Wheeler, MB and Nagy, A}, title = {Microfluidic chip systems for characterizing glucose-responsive insulin-secreting cells equipped with FailSafe kill-switch.}, journal = {Stem cell research &amp; therapy}, volume = {15}, number = {1}, pages = {486}, pmid = {39696686}, issn = {1757-6512}, support = {143231//Canadian Institutes of Health Research Foundation Grant/ ; 950-230422//Canada Research Chairs/ ; }, mesh = {*Insulin-Secreting Cells/metabolism/drug effects ; Animals ; Mice ; *Glucose/metabolism ; *Thymidine Kinase/genetics/metabolism ; Ganciclovir/pharmacology ; CRISPR-Cas Systems ; CDC2 Protein Kinase/metabolism/genetics ; Lab-On-A-Chip Devices ; Insulin/metabolism ; Homeodomain Proteins/metabolism/genetics ; Trans-Activators ; }, abstract = {BACKGROUND: Pluripotent cell-derived islet replacement therapy offers promise for treating Type 1 diabetes (T1D), but concerns about uncontrolled cell proliferation and tumorigenicity present significant safety challenges. To address the safety concern, this study aims to establish a proof-of-concept for a glucose-responsive, insulin-secreting cell line integrated with a built-in FailSafe kill-switch.<br><br>METHOD: We generated &#x3b2; cell-induced progenitor-like cells (&#x3b2;iPLCs) from primary mouse pancreatic &#x3b2; cells through interrupted reprogramming. Then, we transcriptionally linked our FailSafe (FS) kill-switch, HSV-thymidine kinase (TK), to Cdk1 gene using&#xa0;a CRISPR/Cas9 knock-in strategy, resulting in a FailSafe &#x3b2;iPLC line, designated as FS&#x3b2;iPLCs. Subsequently we evaluated and confirmed the functionality of the drug-inducible kill-switch in FS&#x3b2;iPLCs at different ganciclovir (GCV) concentrations using our PDMS-based transcapillary microfluidic system. Finally, we assessed the functionality of FS&#x3b2;iPLCs by characterizing the dynamics of insulin secretion in response to changes in glucose concentration using our microfluidic perfusion glucose-stimulated insulin secretion (GSIS) assay-on- chip.<br><br>RESULTS: The &#x3b2;iPLCs exhibited Ins1, Pdx1 and Nkx6.1 expression, and glucose responsive insulin secretion, the essential properties of pancreatic beta cells. The &#x3b2;iPLCs were amenable to genome editing which allowed for the insertion of the kill-switch into the 3'UTR of Cdk1, confirmed by PCR genotyping. Our transcapillary microfluidic system confirmed the functionality of the drug-inducible kill-switch in FS&#x3b2;iPLCs, showing an effective cell ablation of dividing cells from a heterogeneous cell population at different ganciclovir (GCV) concentrations. The Ki67 expression assessment further confirmed that slow- or non-dividing cells in the FS&#x3b2;iPLC population were resistant to GCV. Our perfusion glucose-stimulated insulin secretion (GSIS) assay-on-chip revealed that the resistant non-dividing FS&#x3b2;iPLCs exhibited higher levels of insulin secretion and glucose responsiveness compared to their proliferating counterparts.<br><br>CONCLUSIONS: This study establishes a proof-of-concept for the integration of a FailSafe kill-switch system into a glucose-responsive, insulin-secreting cell line to address the safety concerns in stem cell-derived cell replacement treatment for T1D. The microfluidic systems provided valuable insights into the functionality and safety of these engineered cells, demonstrating the potential of the kill-switch to reduce the risk of tumorigenicity in pluripotent cell-derived insulin-secreting cells.}, }
@article {pmid39696608, year = {2024}, author = {Lin, Z and Yao, Q and Lai, K and Jiao, K and Zeng, X and Lei, G and Zhang, T and Dai, H}, title = {Cas12f1 gene drives propagate efficiently in herpesviruses and induce minimal resistance.}, journal = {Genome biology}, volume = {25}, number = {1}, pages = {311}, pmid = {39696608}, issn = {1474-760X}, mesh = {*CRISPR-Cas Systems ; *Herpesvirus 1, Human/genetics ; Gene Drive Technology/methods ; Animals ; CRISPR-Associated Protein 9/metabolism ; CRISPR-Associated Proteins/metabolism/genetics ; Humans ; }, abstract = {BACKGROUND: Synthetic CRISPR-Cas9 gene drive has been developed to control harmful species. However, resistance to Cas9 gene drive can be acquired easily when DNA repair mechanisms patch up the genetic insults introduced by Cas9 and incorporate mutations to the sgRNA target. Although many strategies to reduce the occurrence of resistance have been developed so far, they are difficult to implement and not always effective.<br><br>RESULTS: Here, Cas12f1, a recently developed CRISPR-Cas system with minimal potential for causing mutations within target sequences, has been explored as a potential platform for yielding low-resistance in gene drives. We construct Cas9 and Cas12f1 gene drives in a fast-replicating DNA virus, HSV1. Cas9 and Cas12f1 gene drives are able to spread among the HSV1 population with specificity towards their target sites, and their transmission among HSV1 viruses is not significantly affected by the reduced fitness incurred by the viral carriers. Cas12f1 gene drives spread similarly as Cas9 gene drives at high introduction frequency but transmit more slowly than Cas9 gene drives at low introduction frequency. However, Cas12f1 gene drives outperform Cas9 gene drives because they reach higher penetration and induce lower resistance than Cas9 gene drives in all cases.<br><br>CONCLUSIONS: Due to lower resistance and higher penetration, Cas12f1 gene drives could potentially supplant Cas9 gene drives for population control.}, }
@article {pmid39696488, year = {2024}, author = {Koonin, EV and Makarova, KS}, title = {CRISPR in mobile genetic elements: counter-defense, inter-element competition and RNA-guided transposition.}, journal = {BMC biology}, volume = {22}, number = {1}, pages = {295}, pmid = {39696488}, issn = {1741-7007}, mesh = {*DNA Transposable Elements/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems ; Interspersed Repetitive Sequences ; RNA, Guide, CRISPR-Cas Systems/genetics ; Bacteria/genetics/virology ; Plasmids/genetics ; }, abstract = {CRISPR are adaptive immunity systems that protect bacteria and archaea from viruses and other mobile genetic elements (MGE) via an RNA-guided interference mechanism. However, in the course of the host-parasite co-evolution, CRISPR systems have been recruited by MGE themselves for counter-defense or other functions. Some bacteriophages encode fully functional CRISPR systems that target host defense systems, and many others recruited individual components of CRISPR systems, such as single repeat units that inhibit host CRISPR systems and CRISPR mini-arrays that target related viruses contributing to inter-virus competition. Many plasmids carry type IV or subtype V-M CRISPR systems that appear to be involved in inter-plasmid competition. Numerous Tn7-like and Mu-like transposons encode CRISPR-associated transposases (CASTs) in which interference-defective CRISPR systems of type I or type V mediate RNA-guided, site-specific transposition. The recruitment of CRISPR systems and their components by MGE is a manifestation of extensive gene shuttling between host immune systems and MGE, a major trend in the coevolution of MGE with their hosts.}, }
@article {pmid39696352, year = {2024}, author = {Zheng, S and Luo, M and Huang, H and Huang, X and Peng, Z and Zheng, S and Tan, J}, title = {New insights into the role of mitophagy related gene affecting the metastasis of osteosarcoma through scRNA-seq and CRISPR-Cas9 genome editing.}, journal = {Cell communication and signaling : CCS}, volume = {22}, number = {1}, pages = {592}, pmid = {39696352}, issn = {1478-811X}, support = {82460548//National Natural Science Foundation of China/ ; 20242BAB20368//Natural Science Foundation of Jiangxi Province of China/ ; }, mesh = {*Osteosarcoma/genetics/pathology ; *Mitophagy/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Animals ; *Bone Neoplasms/genetics/pathology/metabolism ; Neoplasm Metastasis/genetics ; Cell Line, Tumor ; Mice ; Single-Cell Analysis ; Single-Cell Gene Expression Analysis ; Mitochondrial Precursor Protein Import Complex Proteins ; }, abstract = {BACKGROUND: Osteosarcoma (OSA), the most common primary bone malignancy, poses significant challenges due to its aggressive nature and propensity for metastasis, especially in adolescents. Mitophagy analysis can help identify new therapeutic targets and combined treatment strategies.<br><br>METHODS: This study integrates single-cell sequencing (scRNA-seq) data and bulk-seq to identify mitophagy-related genes (MRGs) associated with the progression of OSA metastasis and analyze their clinical significance. scRNA-seq data elucidates the relationship between mitophagy and OSA metastasis, employing "CellChat" R package to explore intercellular communications and report on hundreds of ligand-receptor interactions. Subsequently, the combination of bulk-seq and CRISPR-Cas9 gene editing identifies mitophagy-related biomarker associated with metastatic prognosis. Finally, validation of the relationship between mitophagy and OSA metastasis is achieved through cellular biology experiments and animal studies.<br><br>RESULTS: The distinct mitophagy activity of various mitochondria manifests in diverse spatial localization, cellular developmental trajectories, and intercellular interactions. OSA tissue exhibits notable heterogeneity in mitophagy within osteoblastic OSA cells. However, high mitophagy activity correlates consistently with high metastatic potential. Subsequently, we identified three critical genes associated with mitophagy in OSA, namely RPS27A, TOMM20 and UBB. According to the aforementioned queue of genes, we have constructed a mitophagy_score (MIP_score). We observed that it consistently predicts patient prognosis in both internal and external datasets, demonstrating strong robustness and stability. Furthermore, we have found that MIP_score can also guide chemotherapy, with varying sensitivities to chemotherapeutic agents based on different MIP_score. It is noteworthy that, through the integration of CRISPR-Cas9 genome-wide screening and validation via cellular and animal experiments, we have identified RPS27A as a potential novel biomarker for OSA.<br><br>CONCLUSIONS: Our comprehensive analysis elucidated the profile of mitophagy throughout the OSA metastasis process, forming the basis for a mitophagy-related prognostic model that addresses clinical outcomes and drug sensitivity following OSA metastasis. Additionally, an online interactive platform was established to assist clinicians in decision-making (https://mip-score.shinyapps.io/labtan/). These findings lay the groundwork for developing targeted therapies aimed at improving the prognosis of OSA patients.}, }
@article {pmid39695426, year = {2024}, author = {Prado, MB and Coelho, BP and Iglesia, RP and Alves, RN and Boccacino, JM and Fernandes, CFL and Melo-Escobar, MI and Ayyadhury, S and Cruz, MC and Santos, TG and Beraldo, FH and Fan, J and Ferreira, FM and Nakaya, HI and Prado, MAM and Prado, VF and Duennwald, ML and Lopes, MH}, title = {Prion protein regulates invasiveness in glioblastoma stem cells.}, journal = {BMC cancer}, volume = {24}, number = {1}, pages = {1539}, pmid = {39695426}, issn = {1471-2407}, support = {2017/26158&#x2011;0//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2019/14952&#x2011;0//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2020/03714-8//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2022/08198-3//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2020/07450&#x2011;5//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2019/14741&#x2011;9//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2019/11097&#x2011;1//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 2017/20271&#x2011;0//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; 101796/2020&#x2011;0//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; 409941/2021&#x2011;2//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; 03592-2021 RGPIN//Natural Sciences and Engineering Research Council of Canada/ ; }, mesh = {*Glioblastoma/pathology/metabolism/genetics ; Humans ; *Neoplastic Stem Cells/metabolism/pathology ; Cell Line, Tumor ; *Neoplasm Invasiveness ; *Brain Neoplasms/pathology/metabolism/genetics ; Cell Proliferation ; Gene Knockout Techniques ; PrPC Proteins/metabolism/genetics ; Prion Proteins/metabolism/genetics ; Gene Expression Regulation, Neoplastic ; Cell Movement ; CRISPR-Cas Systems ; }, abstract = {BACKGROUND: Glioblastoma (GBM) is an aggressive brain tumor driven by glioblastoma stem cells (GSCs), which represent an appealing target for therapeutic interventions. The cellular prion protein (PrP[C]), a scaffold protein involved in diverse cellular processes, interacts with various membrane and extracellular matrix molecules, influencing tumor biology. Herein, we investigate the impact of PrP[C] expression on GBM.<br><br>METHODS: To address this goal, we employed CRISPR-Cas9 technology to generate PrP[C] knockout (KO) glioblastoma cell lines, enabling detailed loss-of-function studies. Bulk RNA sequencing followed by differentially expressed gene and pathway enrichment analyses between U87 or U251 PrP[C]-wild-type (WT) cells and PrP[C]-knockout (KO) cells were used to identify pathways regulated by PrP[C]. Immunofluorescence assays were used to evaluate cellular morphology and protein distribution. For assessment of protein levels, Western blot and flow cytometry assays were employed. Transwell and growth curve assays were used to determine the impact of loss-of-PrP[C] in GBM invasiveness and proliferation, respectively. Single-cell RNA sequencing analysis of data from patient tumors from The Cancer Genome Atlas (TCGA) and the Broad Institute of Single-Cell Data Portal were used to evaluate the correspondence between our in vitro results and patient samples.<br><br>RESULTS: Transcriptome analysis of PrP[C]-KO GBM cell lines revealed altered expression of genes associated with crucial tumor progression pathways, including migration, proliferation, and stemness. These findings were corroborated by assays that revealed impaired invasion, migration, proliferation, and self-renewal in PrP[C]-KO GBM cells, highlighting its critical role in sustaining tumor growth. Notably, loss-of-PrP[C] disrupted the expression and localization of key stemness markers, particularly CD44. Additionally, the modulation of PrP[C] levels through CD44 overexpression further emphasizes their regulatory role in these processes.<br><br>CONCLUSIONS: These findings establish PrP[C] as a modulator of essential molecules on the cell surface of GSCs, highlighting its potential as a therapeutic target for GBM.}, }
@article {pmid39694744, year = {2024}, author = {Zhu, Y and Yu, X and Wu, J}, title = {CRISPR/Cas: a toolkit for plant disease diagnostics.}, journal = {Trends in plant science}, volume = {}, number = {}, pages = {}, doi = {10.1016/j.tplants.2024.11.011}, pmid = {39694744}, issn = {1878-4372}, abstract = {Genetic factors and infectious pathogens that cause plant diseases have a major impact on agricultural production. In recent years, the potential of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system in nucleic acid analysis and plant disease diagnostics has been demonstrated. We highlight progress of CRISPR/Cas technology that is significant for monitoring plant growth and preventing diseases.}, }
@article {pmid39694477, year = {2024}, author = {Harding, KR and Malone, LM and Kyte, NAP and Jackson, SA and Smith, LM and Fineran, PC}, title = {Genome-wide identification of bacterial genes contributing to nucleus-forming jumbo phage infection.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkae1194}, pmid = {39694477}, issn = {1362-4962}, support = {//Marsden Fund/ ; //Royal Society of New Zealand/ ; //Bioprotection Aotearoa/ ; //James Cook Research Fellowship/ ; //University of Otago Doctoral Scholarships/ ; //Division of Health Sciences Career Development Postdoctoral Fellowship/ ; //EMBO Postdoctoral Fellowship/ ; }, abstract = {The Chimalliviridae family of bacteriophages (phages) form a proteinaceous nucleus-like structure during infection of their bacterial hosts. This phage 'nucleus' compartmentalises phage DNA replication and transcription, and shields the phage genome from DNA-targeting defence systems such as CRISPR-Cas and restriction-modification. Their insensitivity to DNA-targeting defences makes nucleus-forming jumbo phages attractive for phage therapy. However, little is known about the bacterial gene requirements during the infectious cycle of nucleus-forming phages or how phage resistance may emerge. To address this, we used the Serratia nucleus-forming jumbo phage PCH45 and exploited a combination of high-throughput transposon mutagenesis and deep sequencing (Tn-seq), and CRISPR interference (CRISPRi). We identified over 90 host genes involved in nucleus-forming phage infection, the majority of which were either involved in the biosynthesis of the primary receptor, flagella, or influenced swimming motility. In addition, the bacterial outer membrane lipopolysaccharide contributed to PCH45 adsorption. Other unrelated Serratia-flagellotropic phages used similar host genes as the nucleus-forming phage, indicating that phage resistance can lead to cross-resistance against diverse phages. Our findings demonstrate that resistance to nucleus-forming jumbo phages can readily emerge via bacterial surface receptor mutation and this should be a major factor when designing strategies for their use in phage therapy.}, }
@article {pmid39693940, year = {2025}, author = {Baranova, SV and Zhdanova, PV and Golyshev, VM and Lomzov, AA and Pestryakov, PE and Chernonosov, AA and Koval, VV}, title = {Thermodynamic parameters obtained for the formation of the Cas12a-RNA/DNA complex.}, journal = {Biochemical and biophysical research communications}, volume = {743}, number = {}, pages = {151176}, doi = {10.1016/j.bbrc.2024.151176}, pmid = {39693940}, issn = {1090-2104}, mesh = {*Thermodynamics ; *Molecular Dynamics Simulation ; *DNA/chemistry/metabolism ; *CRISPR-Associated Proteins/chemistry/metabolism ; *DNA, Single-Stranded/chemistry/metabolism ; *RNA/chemistry/metabolism ; Protein Binding ; Calorimetry ; Nucleic Acid Conformation ; Endodeoxyribonucleases/chemistry/metabolism ; Bacterial Proteins/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; }, abstract = {The thermodynamics of interactions between Cas12a, RNA, and DNA are important to understanding the molecular mechanisms governing CRISPR-Cas12a's specificity and function. In this study, we employed isothermal titration calorimetry (ITC) and molecular dynamics (MD) simulations to investigate the binding properties and energetic contributions of Cas12a-crRNA complexes with single-stranded (ssDNA) and double-stranded (dsDNA) DNA substrates. ITC analyses revealed significant thermal effects during the interaction of Cas12a-crRNA with ssDNA but no detectable effects with dsDNA. The binding to ssDNA was characterized by an enthalpy change (ΔH°) of -243 ± 18 kcal/mol and a stoichiometry of ∼0.3, indicating partial binding due to structural hindrances such as intramolecular secondary structures in RNA and DNA. MD simulations further supported these findings, highlighting the stability and dynamic behavior of Cas12a-crRNA complexes with both DNA substrates. Binding free energy calculations (MM-GBSA) revealed stronger stabilization of the Cas12a-crRNA complex by dsDNA compared to ssDNA, likely driven by additional electrostatic interactions and protein-DNA contacts. However, these interactions did not produce measurable heat effects in ITC experiments. The combined experimental and computational findings demonstrate that the CRISPR-Cas12a system's interactions with nucleic acids are predominantly governed by their structural characteristics and conformational flexibility. These results deepen our understanding of the thermodynamic and structural principles underlying Cas12a-mediated target recognition and cleavage.}, }
@article {pmid39693439, year = {2024}, author = {Wang, Q and Xu, X and Chen, S and Lu, R and Li, L and Lo, CH and Liu, Z and Ning, K and Li, T and Kowal, TJ and Wang, B and Hartnett, ME and Wang, S and Qi, LS and Sun, Y}, title = {dCasMINI-mediated therapy rescues photoreceptors degeneration in a mouse model of retinitis pigmentosa.}, journal = {Science advances}, volume = {10}, number = {51}, pages = {eadn7540}, pmid = {39693439}, issn = {2375-2548}, mesh = {Animals ; *Retinitis Pigmentosa/therapy/genetics/pathology/metabolism ; *Disease Models, Animal ; *Cyclic Nucleotide Phosphodiesterases, Type 6/genetics/metabolism ; Mice ; *Genetic Therapy/methods ; *Dependovirus/genetics ; Electroretinography ; Retinal Degeneration/therapy/genetics/pathology ; CRISPR-Cas Systems ; Humans ; Retinal Rod Photoreceptor Cells/metabolism/pathology ; Eye Proteins/metabolism/genetics ; Retinal Cone Photoreceptor Cells/metabolism/pathology ; }, abstract = {Retinitis pigmentosa (RP) is characterized by degeneration of rod and cone photoreceptors that progresses to irreversible blindness. Now, there are no mutation-agnostic approaches to treat RP. Here, we utilized a single adeno-associated virus (AAV)-based CRISPR activation system to activate phosphodiesterase 6B (Pde6b) to mitigate the severe degeneration in Pde6a[nmf363] mice. We demonstrate that transcriptional activation of Pde6b can rescue the loss of Pde6a, with preservation of retinal structure, restoration of electroretinography responses, and improvement of visual function as assessed by optokinetic response and looming-induced escape behaviors. These findings demonstrate the therapeutic potential of a dCasMINI-mediated activation strategy that provides a mutation-independent treatment for retinal degeneration. This study offers a promising therapeutic approach for RP and potentially other forms of genetic diseases.}, }
@article {pmid39693429, year = {2024}, author = {Shembrey, C and Yang, R and Casan, J and Hu, W and Chen, H and Singh, GJ and Sadras, T and Prasad, K and Shortt, J and Johnstone, RW and Trapani, JA and Ekert, PG and Fareh, M}, title = {Principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with single-base precision.}, journal = {Science advances}, volume = {10}, number = {51}, pages = {eadl0731}, pmid = {39693429}, issn = {2375-2548}, mesh = {*CRISPR-Cas Systems ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Point Mutation ; Gene Silencing ; Gene Editing/methods ; Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins p21(ras)/genetics ; Base Pair Mismatch ; Membrane Proteins/genetics ; Oncogenes ; RNA, Neoplasm/genetics/metabolism ; GTP Phosphohydrolases ; Proto-Oncogene Proteins B-raf ; }, abstract = {Single-nucleotide variants (SNVs) are extremely prevalent in human cancers, although most of these remain clinically unactionable. The programmable RNA nuclease CRISPR-Cas13 has been deployed to specifically target oncogenic RNAs. However, silencing oncogenic SNVs with single-base precision remains extremely challenging due to the intrinsic mismatch tolerance of Cas13. Here, we show that introducing synthetic mismatches at precise positions of the spacer sequence enables de novo design of guide RNAs [CRISPR RNAs (crRNAs)] with strong preferential silencing of point-mutated transcripts. We applied these design principles to effectively silence the oncogenic KRAS G12 hotspot, NRAS G12D and BRAF V600E transcripts with minimal off-target silencing of the wild-type transcripts, underscoring the adaptability of this platform to silence various SNVs. Unexpectedly, the SNV-selective crRNAs harboring mismatched nucleotides reduce the promiscuous collateral activity of the RfxCas13d ortholog. These findings demonstrate that the CRISPR-Cas13 system can be reprogrammed to target mutant transcripts with single-base precision, showcasing the tremendous potential of this tool in personalized transcriptome editing.}, }
@article {pmid39693343, year = {2024}, author = {Casagrande Raffi, G and Chen, J and Feng, X and Chen, Z and Lieftink, C and Deng, S and Mo, J and Zeng, C and Steur, M and Wang, J and Bleijerveld, OB and Hoekman, L and van der Wel, N and Wang, F and Beijersbergen, R and Zheng, J and Bernards, R and Wang, L}, title = {An antibiotic that mediates immune destruction of senescent cancer cells.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {52}, pages = {e2417724121}, pmid = {39693343}, issn = {1091-6490}, support = {787925//EC | ERC | HORIZON EUROPE European Research Council (ERC)/ ; LSH-TKI-LSHM20083//Health Holland/ ; 19-051-ASP//Mark Foundation For Cancer Research (The Mark Foundation for Cancer Research)/ ; 12539//Dutch Cancer Society KWF/ ; W2432050; 82372695//National Natural Science Foundation of China-Guangdong Joint Fund (NSFC)/ ; 2024A04J6484//Guangdong Basic and Applied Basic Research Foundation/ ; }, mesh = {Humans ; *Cellular Senescence/drug effects/immunology ; *Pyrans/pharmacology ; *Killer Cells, Natural/immunology/drug effects/metabolism ; Cell Line, Tumor ; Reactive Oxygen Species/metabolism ; Necroptosis/drug effects ; Neoplasms/immunology/drug therapy/metabolism ; CD8-Positive T-Lymphocytes/immunology/drug effects/metabolism ; Anti-Bacterial Agents/pharmacology ; Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism/genetics ; Pyroptosis/drug effects ; Apoptosis/drug effects ; Animals ; CRISPR-Cas Systems ; Polyether Polyketides ; }, abstract = {Drugs that eliminate senescent cells, senolytics, can be powerful when combined with prosenescence cancer therapies. Using a CRISPR/Cas9-based genetic screen, we identify here SLC25A23 as a vulnerability of senescent cancer cells. Suppressing SLC25A23 disrupts cellular calcium homeostasis, impairs oxidative phosphorylation, and interferes with redox signaling, leading to death of senescent cells. These effects can be replicated by salinomycin, a cation ionophore antibiotic. Salinomycin prompts a pyroptosis-apoptosis-necroptosis (PAN)optosis-like cell death in senescent cells, including apoptosis and two forms of immunogenic cell death: necroptosis and pyroptosis. Notably, we observed that salinomycin treatment or SLC25A23 suppression elevates reactive oxygen species, upregulating death receptor 5 via Jun N-terminal protein kinase (JNK) pathway activation. We show that a combination of a death receptor 5 (DR5) agonistic antibody and salinomycin is a robust senolytic cocktail. We provide evidence that this drug combination provokes a potent natural killer (NK) and CD8+ T cell-mediated immune destruction of senescent cancer cells, mediated by the pyroptotic cytokine interleukin 18 (IL18).}, }
@article {pmid39693337, year = {2024}, author = {Li, LL and Xiao, Y and Wang, B and Zhuang, Y and Chen, Y and Lu, J and Lou, Y and Li, R}, title = {A frameshift mutation in JAZ10 resolves the growth versus defense dilemma in rice.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {52}, pages = {e2413564121}, pmid = {39693337}, issn = {1091-6490}, support = {32372551//MOST | National Natural Science Foundation of China (NSFC)/ ; 32402385//MOST | National Natural Science Foundation of China (NSFC)/ ; 2021YFD1401100//MOST | National Key Research and Development Program of China (NKPs)/ ; NA//Max Planck Partner Group Program/ ; 226-2024-00159//MOE | Fundamental Research Funds for the Central Universities (Fundamental Research Fund for the Central Universities)/ ; }, mesh = {*Oryza/genetics/growth &amp; development/parasitology/metabolism ; *Frameshift Mutation ; *Cyclopentanes/metabolism ; *Plant Proteins/genetics/metabolism ; *Oxylipins/metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; Animals ; Plant Diseases/parasitology/genetics ; Hemiptera/genetics/growth &amp; development/metabolism ; Gene Editing ; Signal Transduction ; Disease Resistance/genetics ; Plants, Genetically Modified/genetics ; Gibberellins/metabolism ; }, abstract = {CRISPR-Cas9 genome editing systems have revolutionized plant gene functional studies by enabling the targeted introduction of insertion-deletions (INDELs) via the nonhomologous end-joining (NHEJ) pathway. Frameshift-inducing INDELs can introduce a premature termination codon and, in other instances, can lead to the appearance of new proteins. Here, we found that mutations in the rice jasmonate (JA) signaling gene OsJAZ10 by CRISPR-Cas9-based genome editing did not affect canonical JA signaling. However, a type of mutant with an INDEL that yielded a novel frameshift protein named FJ10 (Frameshift mutation of JAZ10), exhibited enhanced rice growth and increased resistance to brown planthopper attacks. Overexpression of FJ10 in wild-type plants phenocopies OsJAZ10 frameshift mutants. Further characterization revealed that FJ10 interacts with Slender Rice 1 (OsSLR1) and F-box/Kelch 16 (OsFBK16). These interactions disrupt the function of OsSLR1 in suppressing gibberellin-mediated growth and the function of OsFBK16 in repressing lignin-mediated defense responses, respectively. Field experiments with FJ10-expressing plants demonstrate that this protein uncouples the growth-defense tradeoff, opening broad avenues to obtain cultivars with enhanced yield without compromised defenses.}, }
@article {pmid39693330, year = {2024}, author = {Perampalam, P and McDonald, JI and Dick, FA}, title = {GO-CRISPR: A highly controlled workflow to discover gene essentiality in loss-of-function screens.}, journal = {PloS one}, volume = {19}, number = {12}, pages = {e0315923}, pmid = {39693330}, issn = {1932-6203}, mesh = {Humans ; *Genes, Essential ; *CRISPR-Cas Systems ; *Workflow ; Ovarian Neoplasms/genetics/pathology ; Female ; Software ; Cell Line, Tumor ; RNA, Guide, CRISPR-Cas Systems/genetics ; Loss of Function Mutation ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Genome-wide CRISPR screens are an effective discovery tool for genes that underlie diverse cellular mechanisms that can be scored through cell fitness. Loss-of-function screens are particularly challenging compared to gain-of-function because of the limited dynamic range of decreased sgRNA sequence detection. Here we describe Guide-Only control CRISPR (GO-CRISPR), an improved loss-of-function screening workflow, and its companion software package, Toolset for the Ranked Analysis of GO-CRISPR Screens (TRACS). We demonstrate a typical GO-CRISPR workflow in a non-proliferative 3D spheroid model of dormant high grade serous ovarian cancer and demonstrate superior performance to standard screening methods. The unique integration of the pooled sgRNA library quality and guide-only controls allows TRACS to identify novel molecular pathways that were previously unidentified in tumor dormancy and undetectable to analysis packages that lack the guide only controls. Together, GO-CRISPR and TRACS can robustly improve the discovery of essential genes in challenging biological scenarios such as growth arrested cells.}, }
@article {pmid39692063, year = {2024}, author = {Yuan, P and Usman, M and Liu, W and Adhikari, A and Zhang, C and Njiti, V and Xia, Y}, title = {Advancements in Plant Gene Editing Technology: From Construct Design to Enhanced Transformation Efficiency.}, journal = {Biotechnology journal}, volume = {19}, number = {12}, pages = {e202400457}, pmid = {39692063}, issn = {1860-7314}, support = {OHO01511//USDA Hatch Project/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Plants, Genetically Modified/genetics ; Genome, Plant/genetics ; Transformation, Genetic ; Biotechnology/methods ; Plants/genetics ; }, abstract = {Plant gene editing technology has significantly advanced in recent years, thereby transforming both biotechnological research and agricultural practices. This review provides a comprehensive summary of recent advancements in this rapidly evolving field, showcasing significant discoveries from improved transformation efficiency to advanced construct design. The primary focus is on the maturation of the Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)9 system, which has emerged as a powerful tool for precise gene editing in plants. Through a detailed exploration, we elucidate the intricacies of integrating genetic modifications into plant genomes, shedding light on transport mechanisms, transformation techniques, and optimization strategies specific to CRISPR constructs. Furthermore, we explore the initiatives aimed at extending the frontiers of gene editing to nonmodel plant species, showcasing the growing scope of this technology. Overall, this comprehensive review highlights the significant impact of recent advancements in plant gene editing, illuminating its transformative potential in driving agricultural innovation and biotechnological progress.}, }
@article {pmid39690340, year = {2024}, author = {Dixit, Y and Yadav, P and Asnani, H and Sharma, AK}, title = {CRISPR/Cas9-Engineering for Increased Amylolytic Potential of Microbes for Sustainable Wastewater Treatment: A Review.}, journal = {Current microbiology}, volume = {82}, number = {1}, pages = {44}, pmid = {39690340}, issn = {1432-0991}, mesh = {*Amylases/biosynthesis/genetics ; Bacteria/enzymology/genetics ; *CRISPR-Cas Systems ; Gene Editing/methods ; Genetic Engineering/methods ; *Wastewater/chemistry/microbiology ; Water Purification/methods ; }, abstract = {Amylases are pivotal enzymes with extensive industrial applications, including food processing, textile manufacturing, pharmaceuticals, and biofuel production. Traditional methods for enhancing amylase production in microbial strains often lack precision and efficiency. The advent of CRISPR/Cas9 technology has revolutionized genetic engineering, offering precise and targeted modifications to microbial genomes. This review explores the potential of CRISPR/Cas9 for improving amylase production, highlighting its advantages over conventional methods. This review discusses the mechanism of CRISPR/Cas9, the identification and targeting of key genes involved in amylase synthesis and regulation, and the optimization of expression systems. Additionally, current review examines case studies demonstrating successful CRISPR/Cas9 applications in various microbial hosts. The review also delves into the integration of CRISPR/Cas9 in wastewater treatment, where genetically engineered amylolytic strains enhance the degradation of complex organic pollutants. Despite the promising prospects, challenges such as off-target effects and regulatory considerations remain. This review provides a comprehensive overview of the current advancements, challenges, and future directions in the application of CRISPR/Cas9 technology for amylase production and environmental biotechnology.}, }
@article {pmid39690326, year = {2024}, author = {Barber, HM and Pater, AA and Gagnon, KT and Damha, MJ and O'Reilly, D}, title = {Chemical engineering of CRISPR-Cas systems for therapeutic application.}, journal = {Nature reviews. Drug discovery}, volume = {}, number = {}, pages = {}, pmid = {39690326}, issn = {1474-1784}, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) technology has transformed molecular biology and the future of gene-targeted therapeutics. CRISPR systems comprise a CRISPR-associated (Cas) endonuclease and a guide RNA (gRNA) that can be programmed to guide sequence-specific binding, cleavage, or modification of complementary DNA or RNA. However, the application of CRISPR-based therapeutics is challenged by factors such as molecular size, prokaryotic or phage origins, and an essential gRNA cofactor requirement, which impact efficacy, delivery and safety. This Review focuses on chemical modification and engineering approaches for gRNAs to enhance or enable CRISPR-based therapeutics, emphasizing Cas9 and Cas12a as therapeutic paradigms. Issues that chemically modified gRNAs seek to address, including drug delivery, physiological stability, editing efficiency and off-target effects, as well as challenges that remain, are discussed.}, }
@article {pmid39688838, year = {2024}, author = {Xu, X and Zhang, Y and Liu, J and Wei, S and Li, N and Yao, X and Wang, M and Su, X and Jing, G and Xu, J and Liu, Y and Lu, Y and Cheng, J and Xu, Y}, title = {Concurrent Detection of Protein and miRNA at the Single Extracellular Vesicle Level Using a Digital Dual CRISPR-Cas Assay.}, journal = {ACS nano}, volume = {}, number = {}, pages = {}, doi = {10.1021/acsnano.4c13557}, pmid = {39688838}, issn = {1936-086X}, abstract = {The simultaneous detection of proteins and microRNA (miRNA) at the single extracellular vesicle (EV) level shows great promise for precise disease profiling, owing to the heterogeneity and scarcity of tumor-derived EVs. However, a highly reliable method for multiple-target analysis of single EVs remains to be developed. In this study, a digital dual CRISPR-Cas-powered Single EV Evaluation (ddSEE) system was proposed to enable the concurrent detection of surface protein and inner miRNA of EVs at the single-molecule level. By optimizing simultaneous reaction conditions of CRISPR-Cas12a and CRISPR-Cas13a, the surface protein of EVs was detected by Cas12a using antibody-DNA conjugates to transfer the signal of the protein to DNA, while the inner miRNA was analyzed by Cas13a through EV-liposome fusion. A microfluidic chip containing 188,000 microwells was used to convert the CRISPR-Cas system into a digital assay format to enable the absolute quantification of miRNA/protein-positive EVs without bias through fluorescence imaging, which can detect as few as 214 EVs/μL. Finally, a total of 31 blood samples, 21 from breast cancer patients and 10 from healthy donors, were collected and tested, achieving a diagnostic accuracy of 92% in distinguishing patients with breast cancer from healthy donors. With its absolute quantification, ease of use, and multiplexed detection capability, the ddSEE system demonstrates its great potential for both EV research and clinical applications.}, }
@article {pmid39687096, year = {2024}, author = {Karpenko, A and Shelenkov, A and Petrova, L and Gusarov, V and Zamyatin, M and Mikhaylova, Y and Akimkin, V}, title = {Two multidrug-resistant Proteus mirabilis clones carrying extended spectrum beta-lactamases revealed in a single hospital department by whole genome sequencing.}, journal = {Heliyon}, volume = {10}, number = {23}, pages = {e40821}, pmid = {39687096}, issn = {2405-8440}, abstract = {Proteus mirabilis bacteria is a component of normal intestinal microflora of humans and animals, but can also be found in hospital settings causing urinary tract infections and sepsis. The problem of treating such infections is complicated by multidrug-resistant isolates producing extended spectrum beta-lactamases (ESBL), and the number of ESBL-carrying P. mirabilis strains has significantly increased recently. This study presents a detailed analysis of 12 multidrug-resistant P. mirabilis isolates obtained from the wounds of different patients in one surgical department of a multidisciplinary hospital in Moscow, Russia, using the short- and long-read whole genome sequencing. The isolates under investigation divided into two clusters (clones) C1 and C2 based on their genomic profiles and carried antimicrobial resistance (AMR) genes corresponding well with phenotypic profiles, which was the first case of reporting two different P. mirabilis clones obtained simultaneously from the same specimens at one hospital, to the best of our knowledge. Some genes, including ESBL encoding ones, were specific for either C1 or C2 (aac(6')-Ib10, ant(2″)-Ia, qnrA1, bla VEB-6 and fosA3, bla CTX -M-65 , correspondingly). Additionally, the Salmonella genomic islands 1 were found that differed in composition of multiple antibiotic resistance regions between C1 and C2 groups. CRISPR-Cas system type I-E was revealed only in C2 isolates, while the same set of virulence factors was determined for both P. mirabilis clones. Diversity of all genetic factors found in case of simultaneous existence of two clones collected from the same source at one department indicates high pathogenic potential of P. mirabilis and poses a requirement of proper spreading monitoring. The data obtained will facilitate the understanding of AMR transfer and dynamics within clinical P. mirabilis isolates and contribute to epidemiological surveillance of this pathogen.}, }
@article {pmid39686519, year = {2024}, author = {Hernandez, FJ}, title = {Nucleases: From Primitive Immune Defenders to Modern Biotechnology Tools.}, journal = {Immunology}, volume = {}, number = {}, pages = {}, doi = {10.1111/imm.13884}, pmid = {39686519}, issn = {1365-2567}, support = {2021-05641//Vetenskapsr&#xe5;det/ ; //HORIZON-MSCA-2022-COFUND-101126600-SmartBRAIN3/ ; }, abstract = {The story of nucleases begins on the ancient battlefields of early Earth, where simple bacteria fought to survive against viral invaders. Nucleases are enzymes that degrade nucleic acids, with restriction endonucleases emerging as some of the earliest defenders, cutting foreign DNA to protect their bacteria hosts. However, bacteria sought more than just defence. They evolved the CRISPR-Cas system, an adaptive immune mechanism capable of remembering past invaders. The now-famous Cas9 nuclease, a key player in this system, has been harnessed for genome editing, revolutionising biotechnology. Over time, nucleases evolved from basic viral defence tools into complex regulators of immune function in higher organisms. In humans, DNases and RNases maintain immune balance by clearing cellular debris, preventing autoimmunity, and defending against pathogens. These enzymes have transformed from simple bacterial defenders to critical players in both human immunity and biotechnology. This review explores the evolutionary history of nucleases and their vital roles as protectors in the story of life's defence mechanisms.}, }
@article {pmid39684611, year = {2024}, author = {Pavlova, SV and Shulgina, AE and Minina, JM and Zakian, SM and Dementyeva, EV}, title = {Generation of Isogenic iPSC Lines for Studying the Effect of the p.N515del (c.1543_1545delAAC) Variant on MYBPC3 Function and Hypertrophic Cardiomyopathy Pathogenesis.}, journal = {International journal of molecular sciences}, volume = {25}, number = {23}, pages = {}, pmid = {39684611}, issn = {1422-0067}, support = {22-15-00271//Russian Science Foundation/ ; }, mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; *Cardiomyopathy, Hypertrophic/genetics/pathology/metabolism ; Humans ; *Myocytes, Cardiac/metabolism/cytology/pathology ; *Carrier Proteins/genetics/metabolism ; *Cell Differentiation/genetics ; *CRISPR-Cas Systems ; Cell Line ; Gene Editing ; }, abstract = {The clinical significance of numerous cardiovascular gene variants remains to be determined. CRISPR/Cas9 allows for the introduction and/or correction of a certain variant in induced pluripotent stem cells (iPSCs). The resulting isogenic iPSC lines can be differentiated into cardiomyocytes and used as a platform to assess the pathogenicity of the variant. In this study, isogenic iPSC lines were generated for a variant of unknown significance found previously in a patient with hypertrophic cardiomyopathy (HCM), p.N515del (c.1543_1545delAAC) in MYBPC3. The deletion was corrected with CRISPR/Cas9 in the patient-specific iPSCs. The iPSC lines with the corrected deletion in MYBPC3 maintained pluripotency and a normal karyotype and showed no off-target CRISPR/Cas9 activity. The isogenic iPSC lines, together with isogenic iPSC lines generated earlier via introducing the p.N515del (c.1543_1545delAAC) variant in MYBPC3 of iPSCs of a healthy donor, were differentiated into cardiomyocytes. The cardiomyocytes derived from both panels of the isogenic iPSCs showed an increased size in the presence of the deletion in MYBPC3, which is one of the HCM traits at the cellular level. This finding indicates the effectiveness of these iPSC lines for studying the impact of the variant on HCM development.}, }
@article {pmid39684477, year = {2024}, author = {Borovikova, SE and Shepelev, MV and Mazurov, DV and Kruglova, NA}, title = {Efficient Genome Editing Using 'NanoMEDIC' AsCas12a-VLPs Produced with Pol II-Transcribed crRNA.}, journal = {International journal of molecular sciences}, volume = {25}, number = {23}, pages = {}, pmid = {39684477}, issn = {1422-0067}, support = {22-15-00381//Russian Science Foundation/ ; 075-15-2019-1661//Ministry of Science and Higher Education of the Russian Federation/ ; }, mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; HEK293 Cells ; *RNA Polymerase II/metabolism/genetics ; Jurkat Cells ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; }, abstract = {Virus-like particles (VLPs) are an attractive vehicle for the delivery of Cas nuclease and guide RNA ribonucleoprotein complexes (RNPs). Most VLPs are produced by packaging SpCas9 and its sgRNA, which is expressed from the RNA polymerase III (Pol III)-transcribed U6 promoter. VLPs assemble in the cytoplasm, but U6-driven sgRNA is localized in the nucleus, which hinders the efficient formation and packaging of RNPs into VLPs. In this study, using the nuclease packaging mechanism of 'NanoMEDIC' VLPs, we produced VLPs with AsCas12a and exploited its ability to process pre-crRNA. This allowed us to direct crRNA in the cytoplasm as part of a Pol II-driven transcript where AsCas12a excised mature crRNA, thus boosting RNP incorporation into VLPs. CMV-driven crRNA increased Venus and CCR5 transgene knockout levels in 293 cells from 30% to 50-90% and raised the level of endogenous CXCR4 knockout in Jurkat T cells from 1% to 20%. Changing a single crRNA to an array of three or six identical crRNAs improved CXCR4 knockout rates by up to 60-70%. Compared to SpCas9-VLPs, the editing efficiencies of AsCas12a-VLPs were higher, regardless of promoter usage. Thus, we showed that AsCas12a and CMV-driven crRNA could be efficiently packaged into VLPs and mediate high levels of gene editing. AsCas12a-VLPs are a new and promising tool for the delivery of RNPs into mammalian cells that will allow efficient target genome editing and may be useful for gene therapy applications.}, }
@article {pmid39684395, year = {2024}, author = {Xuan, Q and Wang, J and Nie, Y and Fang, C and Liang, W}, title = {Research Progress and Application of Miniature CRISPR-Cas12 System in Gene Editing.}, journal = {International journal of molecular sciences}, volume = {25}, number = {23}, pages = {}, pmid = {39684395}, issn = {1422-0067}, support = {32201248//National Natural Science Foundation of China/ ; 242102111164//Key R&amp;D and Promotion Projects in Henan Province/ ; 222301420106//Natural Science Foundation of Henan/ ; 222300420202//Natural Science Foundation of Henan/ ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; Plants/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; Humans ; }, abstract = {CRISPR-Cas system, a natural acquired immune system in prokaryotes that defends against exogenous DNA invasion because of its simple structure and easy operation, has been widely used in many research fields such as synthetic biology, crop genetics and breeding, precision medicine, and so on. The miniature CRISPR-Cas12 system has been an emerging genome editing tool in recent years. Compared to the commonly used CRISPR-Cas9 and CRISPR-Cas12a, the miniature CRISPR-Cas12 system has unique advantages, such as rich PAM sites, higher specificity, smaller volume, and cytotoxicity. However, the application of miniature Cas12 proteins and the methods to improve its editing efficiency have not been systematically summarized. In this review, we introduce the classification of CRISPR-Cas system and summarize the structural characteristics of type V CRISPR-Cas system and the cleavage mechanism of five miniature Cas12 proteins. The application of a miniature CRISPR-Cas12 system in the gene editing of animals, plants, and microorganisms is summarized, and the strategies to improve the editing efficiency of the miniature CRISPR-Cas12 system are discussed, aiming to provide reference for further understanding the functional mechanism and engineering modification of the miniature CRISPR-Cas12 system.}, }
@article {pmid39684387, year = {2024}, author = {Aksoy, MO and Bilinska, A and Stachowiak, M and Flisikowska, T and Szczerbal, I}, title = {Deciphering the Role of the SREBF1 Gene in the Transcriptional Regulation of Porcine Adipogenesis Using CRISPR/Cas9 Editing.}, journal = {International journal of molecular sciences}, volume = {25}, number = {23}, pages = {}, pmid = {39684387}, issn = {1422-0067}, support = {2018/29/B/NZ2/00956//National Science Center/ ; }, mesh = {Animals ; *Adipogenesis/genetics ; *Sterol Regulatory Element Binding Protein 1/genetics/metabolism ; *CRISPR-Cas Systems ; Swine ; *Gene Editing/methods ; Adipocytes/metabolism/cytology ; Gene Expression Regulation ; Lipid Metabolism/genetics ; }, abstract = {Sterol regulatory element-binding protein 1 (SREBP1) is an important transcription factor that controls lipid metabolism and adipogenesis. Two isoforms, SREBP1a and SREBP1c, are generated by alternative splicing of the first exon of the SREBF1 gene. The porcine SREBF1 gene has mainly been studied for its role in lipid metabolism in adipose tissues, but little is known about its involvement, and the role of its two isoforms, in adipogenesis. The aim of the present study was to introduce a deletion in the 5'-regulatory region of the SREBF1c gene, considered crucial for adipogenesis, using the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) method. This approach allows for the evaluation of how inhibiting SREBF1c transcription affects the expression of other genes essential for adipocyte differentiation, particularly PPARG, CEBPA, CEBPB, CEBPD, GATA2, and FABP4. It was observed that disrupting the SREBF1c isoform had no effect on the GATA2 gene but did result in a decrease in the expression of the CEBPA and CEBPD genes, an increase in the expression of CEBPB, and an inhibition in the expression of the PPARG and FABP4 genes. These changes in gene expression blocked adipogenesis, as could be seen by the failure of lipid droplets to accumulate. Our results provide evidence highlighting the pivotal role of the SREBP1c isoform in the regulation of porcine adipogenesis.}, }
@article {pmid39684256, year = {2024}, author = {Yang, P and Zhang, S and Hu, D and Li, X and Guo, Y and Guo, H and Zhang, L and Ding, X}, title = {Research Progress on the Mechanism and Application of the Type I CRISPR-Cas System.}, journal = {International journal of molecular sciences}, volume = {25}, number = {23}, pages = {}, pmid = {39684256}, issn = {1422-0067}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Archaea/genetics ; Bacteria/genetics ; Genetic Engineering/methods ; }, abstract = {The CRISPR-Cas system functions as an adaptive immune mechanism in archaea and bacteria, providing defense against the invasion of foreign nucleic acids. Most CRISPR-Cas systems are classified into class 1 or class 2, with further subdivision into several subtypes. The primary distinction between class 1 and class 2 systems lies in the assembly of their effector modules. In class 1 systems, the effector complex consists of multiple proteins with distinct functions, whereas in class 2 systems, the effector is associated with a single protein. Class 1 systems account for approximately 90% of the CRISPR-Cas repertoire and are categorized into three types (type I, type IV, and type III) and 12 subtypes. To date, various CRISPR-Cas systems have been widely employed in the field of genetic engineering as essential tools and techniques for genome editing. Type I CRISPR-Cas systems remain a valuable resource for developing sophisticated application tools. This review provides a comprehensive review of the characteristics, mechanisms of action, and applications of class 1 type I CRISPR-Cas systems, as well as transposon-associated systems, offering effective approaches and insights for future research on the mechanisms of action, as well as the subsequent development and application of type I CRISPR-Cas systems.}, }
@article {pmid39684244, year = {2024}, author = {Wattad, H and Molcho, J and Manor, R and Weil, S and Aflalo, ED and Chalifa-Caspi, V and Sagi, A}, title = {Roadmap and Considerations for Genome Editing in a Non-Model Organism: Genetic Variations and Off-Target Profiling.}, journal = {International journal of molecular sciences}, volume = {25}, number = {23}, pages = {}, pmid = {39684244}, issn = {1422-0067}, mesh = {*Gene Editing/methods ; Animals ; *CRISPR-Cas Systems ; Palaemonidae/genetics ; Genetic Variation ; Polymorphism, Single Nucleotide ; High-Throughput Nucleotide Sequencing ; Molecular Sequence Annotation ; Sex Chromosomes/genetics ; }, abstract = {The CRISPR/Cas genome editing approach in non-model organisms poses challenges that remain to be resolved. Here, we demonstrated a generalized roadmap for a de novo genome annotation approach applied to the non-model organism Macrobrachium rosenbergii. We also addressed the typical genome editing challenges arising from genetic variations, such as a high frequency of single nucleotide polymorphisms, differences in sex chromosomes, and repetitive sequences that can lead to off-target events. For the genome editing of M. rosenbergii, our laboratory recently adapted the CRISPR/Cas genome editing approach to embryos and the embryonic primary cell culture. In this continuation study, an annotation pipeline was trained to predict the gene models by leveraging the available genomic, transcriptomic, and proteomic data, and enabling accurate gene prediction and guide design for knock-outs. A next-generation sequencing analysis demonstrated a high frequency of genetic variations in genes on both autosomal and sex chromosomes, which have been shown to affect the accuracy of editing analyses. To enable future applications based on the CRISPR/Cas tool in non-model organisms, we also verified the reliability of editing efficiency and tracked off-target frequencies. Despite the lack of comprehensive information on non-model organisms, this study provides an example of the feasibility of selecting and editing specific genes with a high degree of certainty.}, }
@article {pmid39683106, year = {2024}, author = {Zhan, X and Zhang, F and Li, N and Xu, K and Wang, X and Gao, S and Yin, Y and Yuan, W and Chen, W and Ren, Z and Yao, M and Wang, F}, title = {CRISPR/Cas: An Emerging Toolbox for Engineering Virus Resistance in Plants.}, journal = {Plants (Basel, Switzerland)}, volume = {13}, number = {23}, pages = {}, pmid = {39683106}, issn = {2223-7747}, support = {2023BBB044//Fei Wang/ ; 2022BBA0060//Fei Wang/ ; HBZY2023B004-3//Minghua Yao/ ; 2023HBSTX4-06//Ning Li/ ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas have been recognized as powerful genome-editing tools in diverse eukaryotic species, including plants, and thus hold great promise for engineering virus resistance in plants. Nevertheless, further attention is required regarding various issues associated with applying new powerful technologies in the field. This mini-review focuses on the recent advances in using CRISPR/Cas9 and CRISPR/Cas13 systems to combat DNA and RNA viruses in plants. We explored the utility of CRISPR/Cas for targeting the viral genome and editing host susceptibility genes in plants. We also provide insights into the limitations and challenges of using CRISPR/Cas for plant virus interference and propose individual combinatorial solutions. In conclusion, CRISPR/Cas technology has the potential to offer innovative and highly efficient approaches for controlling viruses in important crops in the near future.}, }
@article {pmid39682777, year = {2024}, author = {Ryu, M and Yurube, T and Takeoka, Y and Kanda, Y and Tsujimoto, T and Miyazaki, K and Ohnishi, H and Matsuo, T and Kumagai, N and Kuroshima, K and Hiranaka, Y and Kuroda, R and Kakutani, K}, title = {Gene-Silencing Therapeutic Approaches Targeting PI3K/Akt/mTOR Signaling in Degenerative Intervertebral Disk Cells: An In Vitro Comparative Study Between RNA Interference and CRISPR-Cas9.}, journal = {Cells}, volume = {13}, number = {23}, pages = {}, pmid = {39682777}, issn = {2073-4409}, support = {JP21K09323//Japan Society for the Promotion of Science/ ; JP24K02565//Japan Society for the Promotion of Science/ ; }, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *TOR Serine-Threonine Kinases/metabolism ; *Proto-Oncogene Proteins c-akt/metabolism ; *Signal Transduction ; *RNA Interference ; *Intervertebral Disc Degeneration/therapy/genetics/metabolism/pathology ; Gene Silencing ; Phosphatidylinositol 3-Kinases/metabolism ; Nucleus Pulposus/metabolism/pathology ; Intervertebral Disc/metabolism/pathology ; Autophagy/genetics ; Regulatory-Associated Protein of mTOR/metabolism/genetics ; RNA, Small Interfering/metabolism/genetics ; Male ; Female ; Middle Aged ; }, abstract = {The mammalian target of rapamycin (mTOR), a serine/threonine kinase, promotes cell growth and inhibits autophagy. The following two complexes contain mTOR: mTORC1 with the regulatory associated protein of mTOR (RAPTOR) and mTORC2 with the rapamycin-insensitive companion of mTOR (RICTOR). The phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling pathway is important in the intervertebral disk, which is the largest avascular, hypoxic, low-nutrient organ in the body. To examine gene-silencing therapeutic approaches targeting PI3K/Akt/mTOR signaling in degenerative disk cells, an in vitro comparative study was designed between small interfering RNA (siRNA)-mediated RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) gene editing. Surgically obtained human disk nucleus pulposus cells were transfected with a siRNA or CRISPR-Cas9 plasmid targeting mTOR, RAPTOR, or RICTOR. Both of the approaches specifically suppressed target protein expression; however, the 24-h transfection efficiency differed by 53.8-60.3% for RNAi and 88.1-89.3% for CRISPR-Cas9 (p < 0.0001). Targeting mTOR, RAPTOR, and RICTOR all induced autophagy and inhibited apoptosis, senescence, pyroptosis, and matrix catabolism, with the most prominent effects observed with RAPTOR CRISPR-Cas9. In the time-course analysis, the 168-h suppression ratio of RAPTOR protein expression was 83.2% by CRISPR-Cas9 but only 8.8% by RNAi. While RNAi facilitates transient gene knockdown, CRISPR-Cas9 provides extensive gene knockout. Our findings suggest that RAPTOR/mTORC1 is a potential therapeutic target for degenerative disk disease.}, }
@article {pmid39681230, year = {2024}, author = {Nafari, NB and Zamani, M and Mosayyebi, B}, title = {Recent advances in lateral flow assays for MicroRNA detection.}, journal = {Clinica chimica acta; international journal of clinical chemistry}, volume = {567}, number = {}, pages = {120096}, doi = {10.1016/j.cca.2024.120096}, pmid = {39681230}, issn = {1873-3492}, abstract = {Lateral flow assays (LFAs) have emerged as pivotal tools for the rapid and reliable detection of microRNAs (miRNAs). It is believed that these biomarkers are crucial for the diagnosis and prognosis of various diseases, particularly cancer. Traditional miRNA detection techniques, such as quantitative PCR, are highly sensitive but have limited efficacy due to their complexity, high cost, and technical requirements. LFAs are valuable due to their simplicity, affordability, and portability, making them ideal for point-of-care testing in low-resource environments. However, challenges remain in developing highly sensitive and accurate LFA devices for miRNA detection. This review explores recent advancements in LFAs to improve miRNA detection sensitivity and specificity. Key innovations include signal amplification using isothermal methods, the application of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas systems for direct targeting of miRNAs, and the incorporation of nanomaterials, such as gold nanoparticles and nanorods, to enhance signal intensity. Using artificial intelligence (AI) algorithms enables precise, automated, and rapid quantification of miRNAs. Moreover, this review examines the ability of LFA-based devices to detect multiple miRNAs simultaneously. One of the most significant advancements is the detection of miR-21 levels as low as 20 pM and let-7a levels as low as 40 pM within ten minutes. This highlights the potential of these devices for clinical diagnostics.}, }
@article {pmid39680881, year = {2025}, author = {Zeng, J and Huang, X and Yang, Y and Wang, J and Shi, Y and Li, H and Hu, N and Yu, B and Mu, J}, title = {Near-Infrared Optogenetic Nanosystem for Spatiotemporal Control of CRISPR-Cas9 Gene Editing and Synergistic Photodynamic Therapy.}, journal = {ACS applied materials &amp; interfaces}, volume = {17}, number = {1}, pages = {701-710}, doi = {10.1021/acsami.4c18656}, pmid = {39680881}, issn = {1944-8252}, mesh = {*Photochemotherapy ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; Humans ; Mice ; *Infrared Rays ; Nanoparticles/chemistry ; Optogenetics/methods ; Cell Line, Tumor ; Neoplasms/therapy/drug therapy/genetics ; Photosensitizing Agents/chemistry/pharmacology ; Mice, Inbred BALB C ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism ; Mice, Nude ; }, abstract = {Controlling CRISPR/Cas9 gene editing at the spatiotemporal resolution level, especially for in vivo applications, remains a great challenge. Here, we developed a near-infrared (NIR) light-activated nanophotonic system (UCPP) for controlled CRISPR-Cas9 gene editing and synergistic photodynamic therapy (PDT). Lanthanide-doped upconversion nanoparticles are not only employed as carriers for intracellular plasmid delivery but also serve as the nanotransducers to convert NIR light (980 nm) into visible light with emission at 460 and 650 nm, which could result in simultaneous activation of gene editing and PDT processes, respectively. Such unique design not only achieves light-controlled precise gene editing of hypoxia-inducible factor 1α with minimal off-target effect, which effectively ameliorates the hypoxic state at tumor sites, but also facilitates the deep-seated PDT process with synergistic antitumor effect. This optogenetically activatable CRISPR-Cas9 nanosystem holds great potential for spatially controlled in vivo gene editing and targeted cancer therapy.}, }
@article {pmid39680738, year = {2024}, author = {Yang, S and Hu, G and Wang, J and Song, J}, title = {CRISPR/Cas-Based Gene Editing Tools for Large DNA Fragment Integration.}, journal = {ACS synthetic biology}, volume = {}, number = {}, pages = {}, doi = {10.1021/acssynbio.4c00632}, pmid = {39680738}, issn = {2161-5063}, abstract = {In recent years, gene editing technologies have rapidly evolved to enable precise and efficient genomic modification. These strategies serve as a crucial instrument in advancing our comprehension of genetics and treating genetic disorders. Of particular interest is the manipulation of large DNA fragments, notably the insertion of large fragments, which has emerged as a focal point of research in recent years. Nevertheless, the techniques employed to integrate larger gene fragments are frequently confronted with inefficiencies, off-target effects, and elevated costs. It is therefore imperative to develop efficient tools capable of precisely inserting kilobase-sized DNA fragments into mammalian genomes to support genetic engineering, gene therapy, and synthetic biology applications. This review provides a comprehensive overview of methods developed in the past five years for integrating large DNA fragments with a particular focus on burgeoning CRISPR-related technologies. We discuss the opportunities associated with homology-directed repair (HDR) and emerging CRISPR-transposase and CRISPR-recombinase strategies, highlighting their potential to revolutionize gene therapies for complex diseases. Additionally, we explore the challenges confronting these methodologies and outline potential future directions for their improvement with the overarching goal of facilitating the utilization and advancement of tools for large fragment gene editing.}, }
@article {pmid39680576, year = {2024}, author = {Guguin, J and Chen, TY and Cuinat, S and Besson, A and Bertiaux, E and Boutaud, L and Ardito, N and Imaz Murguiondo, M and Cabet, S and Hamel, V and Thomas, S and Pain, B and Edery, P and Putoux, A and Tang, TK and Mazoyer, S and Delous, M}, title = {A Taybi-Linder syndrome-related RTTN variant impedes neural rosette formation in human cortical organoids.}, journal = {PLoS genetics}, volume = {20}, number = {12}, pages = {e1011517}, pmid = {39680576}, issn = {1553-7404}, mesh = {Humans ; *Organoids/metabolism ; *Microcephaly/genetics ; *Neural Stem Cells/metabolism ; Induced Pluripotent Stem Cells/metabolism ; Cell Cycle Proteins/genetics/metabolism ; RNA, Small Nuclear/genetics/metabolism ; Centrosome/metabolism ; CRISPR-Cas Systems ; Mutation ; Exome Sequencing ; Cerebral Cortex/metabolism ; }, abstract = {Taybi-Linder syndrome (TALS) is a rare autosomal recessive disorder characterized by severe microcephaly with abnormal gyral pattern, severe growth retardation and bone abnormalities. It is caused by pathogenic variants in the RNU4ATAC gene. Its transcript, the small nuclear RNA U4atac, is involved in the excision of ~850 minor introns. Here, we report a patient presenting with TALS features but no pathogenic variants were found in RNU4ATAC, instead the homozygous RTTN c.2953A>G variant was detected by whole-exome sequencing. After deciphering the impact of the variant on the RTTN protein function at centrosome in engineered RTTN-depleted RPE1 cells and patient fibroblasts, we analysed neural stem cells (NSC) derived from CRISPR/Cas9-edited induced pluripotent stem cells and revealed major cell cycle and mitotic abnormalities, leading to aneuploidy, cell cycle arrest and cell death. In cortical organoids, we discovered an additional function of RTTN in the self-organisation of NSC into neural rosettes, by observing delayed apico-basal polarization of NSC. Altogether, these defects contributed to a marked delay of rosette formation in RTTN-mutated organoids, thus impeding their overall growth and shedding light on mechanisms leading to microcephaly.}, }
@article {pmid39680522, year = {2024}, author = {Zheng, J and Li, B and Jia, L and Zhang, J and Gong, Z and Le, Y and Nian, X and Li, X and Liu, B and Yu, D and Zhang, Z and Li, C}, title = {Tumorigenicity decrease in Bcl-xL deficient MDCK cells ensuring the safety for influenza vaccine production.}, journal = {PloS one}, volume = {19}, number = {12}, pages = {e0311069}, pmid = {39680522}, issn = {1932-6203}, mesh = {Animals ; Dogs ; *bcl-X Protein/genetics/metabolism ; Madin Darby Canine Kidney Cells ; *Influenza Vaccines ; Mice ; *Apoptosis ; Cell Proliferation ; CRISPR-Cas Systems ; Mice, Nude ; Carcinogenesis/genetics ; Cell Movement ; Gene Editing ; Mice, Inbred BALB C ; }, abstract = {Madin-Darby canine kidney (MDCK) cells are the recognized cell strain for influenza vaccine production. However, the tumorigenic potential of MDCK cells raises concerns about their use in biological product manufacturing. To reduce MDCK cells' tumorigenicity and ensure the safety of influenza vaccine production, a B-cell lymphoma extra-large (Bcl-xL) gene, which plays a pivotal role in apoptosis regulation, was knocked-out in original MDCK cells by CRISPR-Cas9 gene editing technology, so that a homozygous MDCK-Bcl-xL-/- cell strain was acquired and named as BY-02. Compared with original MDCK cells, the proliferation and migration ability of BY-02 were significantly reduced, while apoptosis level was significantly increased, the endogenous mitochondrial apoptotic pathway were also modulated after Bcl-xL knock-out in MDCK cells. For tumor formation assays in nude mouse tests, all ten mice injected with original MDCK cells presented tumors growth in the injection site, in contrast to only one mouse injected with BY-02 cells presented tumors growth. These findings suggest that Bcl-xL knock-down is an effective strategy to inhibit tumor formation in MDCK cells, making BY-02 a promising genetically engineered cell strain for influenza vaccine production.}, }
@article {pmid39680038, year = {2025}, author = {Van, R and Pan, X and Rostami, S and Liu, J and Agarwal, PK and Brooks, B and Rajan, R and Shao, Y}, title = {Exploring CRISPR-Cas9 HNH-Domain-Catalyzed DNA Cleavage Using Accelerated Quantum Mechanical Molecular Mechanical Free Energy Simulation.}, journal = {Biochemistry}, volume = {64}, number = {1}, pages = {289-299}, doi = {10.1021/acs.biochem.4c00651}, pmid = {39680038}, issn = {1520-4995}, mesh = {*CRISPR-Cas Systems ; *DNA Cleavage ; *Quantum Theory ; Molecular Dynamics Simulation ; Thermodynamics ; DNA/chemistry/metabolism/genetics ; Catalytic Domain ; CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; }, abstract = {The target DNA (tDNA) cleavage catalyzed by the CRISPR Cas9 enzyme is a critical step in the Cas9-based genome editing technologies. Previously, the tDNA cleavage from an active SpyCas9 enzyme conformation was modeled by Palermo and co-workers (Nierzwicki et al., Nat. Catal. 2022 5, 912) using ab initio quantum mechanical molecular mechanical (ai-QM/MM) free energy simulations, where the free energy barrier was found to be more favorable than that from a pseudoactive enzyme conformation. In this work, we performed ai-QM/MM simulations based on another catalytically active conformation (PDB 7Z4J) of the Cas9 HNH domain from cryo-electron microscopy experiments. For the wildtype enzyme, we acquired a free energy profile for the tDNA cleavage that is largely consistent with the previous report. Furthermore, we explored the role of the active-site K866 residue on the catalytic efficiency by modeling the K866A mutant and found that the K866A mutation increased the reaction free energy barrier, which is consistent with the experimentally observed reduction in the enzyme activity.}, }
@article {pmid39679901, year = {2025}, author = {Zhang, W and Li, Z and Lun, X and Guo, Y}, title = {Telomerase-Responsive CRISPR System-Regulated Nanobomb for Triggering Research on Telomerase "Self-Detonation".}, journal = {ACS applied materials &amp; interfaces}, volume = {17}, number = {1}, pages = {725-738}, doi = {10.1021/acsami.4c18859}, pmid = {39679901}, issn = {1944-8252}, mesh = {*Telomerase/metabolism/genetics ; Humans ; Animals ; Mice ; MCF-7 Cells ; *Aptamers, Nucleotide/chemistry ; Female ; CRISPR-Cas Systems/genetics ; Nanoparticles/chemistry ; Mice, Nude ; Silicon Dioxide/chemistry ; Naphthalenes/chemistry/pharmacology ; Breast Neoplasms/pathology/metabolism ; Mice, Inbred BALB C ; Antineoplastic Agents/pharmacology/chemistry ; Cell Proliferation/drug effects ; Aminobenzoates ; }, abstract = {Targeting tumor markers is one of the most important approaches to tumor therapy, and the "suicide" pattern of tumor marker response is a very challenging study. Telomerase, as one of the key factors associated with human longevity and cancer progression, is considered to be an emerging biomarker for cancer diagnosis. The targeted drug delivery nanobomb─BIBR1532@HSN/FQDNA/MUC1 aptamer (B@HDA) is prepared in this study based on hollow silica nanoparticles (HSN) and CRISPR systems. Amino-modified FQDNA and amino-modified MUC1 aptamer are covalently attached to the surface of carboxyl-functionalized HSN. The modified MUC1 aptamer directs the nanobomb to specifically target breast cancer cells (MCF-7) and FQDNA sequesters the telomerase inhibitor (BIBR1532) within the HSN. Telomerase primers (TPs) is recognized by the highly expressed telomerase in MCF-7 cells and is elongated to form DNA substrates. The substrate pairs with crRNA bases to effectively activate CRISPR-Cas12a. The activated CRISPR-Cas12a precisely cut FQDNA, releasing BIBR1532, which inhibits telomerase activity. This strategy achieves telomerase "suicide". The nanobomb described above has the following advantages. (1) The "closing" effect of FQDNA contributes to reducing the nonspecific release of BIBR1532. (2) B@HDA, combined with CRISPR, regulates mitochondrial dysfunction and cell senescence in MCF-7 cells. (3) In the tumor-bearing mouse model, B@HDA, combined with CRISPR, exhibits good biocompatibility and an obvious tumor ablation effect on MCF-7 tumors, suggesting potential application prospects across a wide range of cancer cell lines. In summary, the proposed nanobomb provides a tunable switch approach for the specific inhibition of telomerase and the reduction of tumor cell growth, representing a promising avenue for promoting senescence and treating cancer.}, }
@article {pmid39678914, year = {2024}, author = {Rostampour, M and Panahi, B and Masoumi Jahandizi, R}, title = {The CRISPR-Cas system in Lactiplantibacillus plantarum strains: identification and characterization using a genome mining approach.}, journal = {Frontiers in microbiology}, volume = {15}, number = {}, pages = {1394756}, pmid = {39678914}, issn = {1664-302X}, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CAS) genes make up bacteria's adaptive immune system against bacteriophages. In this study, 675 sequences of Lactiplantibacillus plantarum isolates deposited in GenBank were analyzed in terms of diversity, occurrence, and evolution of the CRISPR-Cas system. This study investigated the presence, structural variations, phylogenetic relationships, and diversity of CRISPR-Cas systems in 675 L. plantarum strains. The analysis revealed that 143 strains harbor confirmed CRISPR-Cas systems, with subtype II-A being predominant. Moreover, targeting phages and plasmid diversity between the predicted systems were dissected. The results indicated that approximately 22% of the isolates with verified and complete CRISPR systems exhibited the coexistence of both subtypes II-A and I-E within their genomes. The results further showed that in subtype II-A, the length of the repeat sequence was 36 nucleotides, on average. In addition, the number of spacers in subtypes II-A and I-E varied between 1-24 and 3-16 spacers, respectively. The results also indicated that subtype II-A has nine protospacer adjacent motifs, which are 5'-CC-3', 5'-GAA-3', 5'-TGG-3', 5'-CTT-3', 5'-GGG-3', 5'-CAT-3', 5'-CTC-3', 5'-CCT-3', and 5'-CGG-3'. In addition, the identified systems displayed a potential for targeting Lactobacillus phages. The investigation of the relationship between the targeting of Lactobacillus phages by the antiphage system in L. plantarum species showed that subtype II-A had the highest diversity in targeting Lactobacillus phages than subtype I-E. In conclusion, current findings offer a perspective on the prevalence and evolution of the CRISPR-Cas system in L. plantarum, contributing novel insights to the expanding field of CRISPR-Cas systems within lactobacillus strains. This knowledge establishes a foundation for future applied studies focused on enhancing phage resistance in industrial fermentation, reducing contamination risks, and improving product quality. The identified targeting diversity may also foster advancements in phage therapy through the development of CRISPR-based antimicrobials.}, }
@article {pmid39677770, year = {2024}, author = {Hoelting, K and Madlmayr, A and Hoeger, B and Lewitz, D and Weng, M and Haider, T and Duggan, M and Ross, R and Horgen, FD and Sperandio, M and Dietrich, A and Gudermann, T and Zierler, S}, title = {TRPM7 activity drives human CD4 T-cell activation and differentiation in a magnesium dependent manner.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, pmid = {39677770}, issn = {2692-8205}, support = {P20 GM103466/GM/NIGMS NIH HHS/United States ; U54 GM138062/GM/NIGMS NIH HHS/United States ; }, abstract = {T lymphocyte activation is a crucial process in the regulation of innate and adaptive immune responses. The ion channel-kinase TRPM7 has previously been implicated in cellular Mg[2+] homeostasis, proliferation, and immune cell modulation. Here, we show that pharmacological and genetic silencing of TRPM7 leads to diminished human CD4 T-cell activation and proliferation following TCR mediated stimulation. In both primary human CD4 T cells and CRISPR/Cas-9 engineered Jurkat T cells, loss of TRPM7 led to altered Mg[2+] homeostasis, Ca[2+] signaling, reduced NFAT translocation, decreased IL-2 secretion and ultimately diminished proliferation and differentiation. While the activation of primary human CD4 T cells was dependent on TRPM7, polarization of naïve CD4 T cells into regulatory T cells (Treg) was not. Taken together, these results highlight TRPM7 as a key protein of cellular Mg[2+] homeostasis and CD4 T-cell activation. Its role in lymphocyte activation suggests therapeutic potential for TRPM7 in numerous T-cell mediated diseases.}, }
@article {pmid39677748, year = {2024}, author = {Shamloo, S and Schloßhauer, JL and Tiwari, S and Fischer, KD and Ghebrechristos, Y and Kratzenberg, L and Bejoy, AM and Aifantis, I and Wang, E and Imig, J}, title = {RNA Binding of GAPDH Controls Transcript Stability and Protein Translation in Acute Myeloid Leukemia.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, doi = {10.1101/2024.12.02.626357}, pmid = {39677748}, issn = {2692-8205}, abstract = {Dysregulation of RNA binding proteins (RBPs) is a hallmark in cancerous cells. In acute myeloid leukemia (AML) RBPs are key regulators of tumor proliferation. While classical RBPs have defined RNA binding domains, RNA recognition and function in AML by non-canonical RBPs (ncRBPs) remain unclear. Given the inherent complexity of targeting AML broadly, our goal was to uncover potential ncRBP candidates critical for AML survival using a CRISPR/Cas-based screening. We identified the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a pro-proliferative factor in AML cells. Based on cross-linking and immunoprecipitation (CLIP), we are defining the global targetome, detecting novel RNA targets mainly located within 5'UTRs, including GAPDH, RPL13a, and PKM. The knockdown of GAPDH unveiled genetic pathways related to ribosome biogenesis, translation initiation, and regulation. Moreover, we demonstrated a stabilizing effect through GAPDH binding to target transcripts including its own mRNA. The present findings provide new insights on the RNA functions and characteristics of GAPDH in AML.}, }
@article {pmid39676682, year = {2024}, author = {Chen, J and Lin, X and Xiang, W and Chen, Y and Zhao, Y and Huang, L and Liu, L}, title = {DNA target binding-induced pre-crRNA processing in type II and V CRISPR-Cas systems.}, journal = {Nucleic acids research}, volume = {}, number = {}, pages = {}, doi = {10.1093/nar/gkae1241}, pmid = {39676682}, issn = {1362-4962}, support = {32022047//National Natural Science Foundation of China/ ; 3502Z20227020//Natural Science Foundation of Xiamen, China/ ; 2023J01023//Natural Science Foundation of Fujian Province/ ; }, abstract = {Precursor (pre)-CRISPR RNA (crRNA) processing can occur in both the repeat and spacer regions, leading to the removal of specific segments from the repeat and spacer sequences, thereby facilitating crRNA maturation. The processing of pre-crRNA repeat by Cas effector and ribonuclease has been observed in CRISPR-Cas9 and CRISPR-Cas12a systems. However, no evidence of pre-crRNA spacer cleavage by any enzyme has been reported in these systems. In this study, we demonstrate that DNA target binding triggers efficient cleavage of pre-crRNA spacers by type II and V Cas effectors such as Cas12a, Cas12b, Cas12i, Cas12j and Cas9. We show that the pre-crRNA spacer cleavage catalyzed by Cas12a and Cas9 has distinct characteristics. Activation of the cleavage activity in Cas12a is induced by both single-stranded DNA (ssDNA) and double-stranded DNA target binding, whereas only ssDNA target binding triggers cleavage in Cas9 toward the pre-crRNA spacer. We present a series of structures elucidating the underlying mechanisms governing conformational activation in both Cas12a and Cas9. Furthermore, leveraging the trans-cutting activity of the pre-crRNA spacer, we develop a one-step DNA detection method characterized by its simplicity, high sensitivity, and excellent specificity.}, }
@article {pmid39676076, year = {2024}, author = {Gao, Z and Bak, RO}, title = {Integration of large genetic payloads using prime editing and site-specific integrases.}, journal = {Nature protocols}, volume = {}, number = {}, pages = {}, pmid = {39676076}, issn = {1750-2799}, support = {101041231//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; R238-2016-3349//Lundbeckfonden (Lundbeck Foundation)/ ; 8056-00010B//Innovationsfonden (Innovation Fund Denmark)/ ; NNF21OC0071259//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; }, }
@article {pmid39675005, year = {2024}, author = {Rubio, A and Garzón, A and Moreno-Rodríguez, A and Pérez-Pulido, AJ}, title = {Biological warfare between two bacterial viruses in a defense archipelago sheds light on the spread of CRISPR-Cas systems.}, journal = {Cell reports}, volume = {43}, number = {12}, pages = {115085}, doi = {10.1016/j.celrep.2024.115085}, pmid = {39675005}, issn = {2211-1247}, mesh = {*CRISPR-Cas Systems/genetics ; Acinetobacter baumannii/genetics ; Biological Warfare ; Genome, Bacterial ; }, abstract = {CRISPR-Cas systems are adaptive immunity systems of bacteria and archaea that prevent infection by viruses and other external mobile genetic elements. It is currently known that these defense systems can be co-opted by the same viruses. We have found one of these viruses in the opportunistic pathogen Acinetobacter baumannii, and the same system has been also found in an integration hotspot of the bacterial genome that harbors other multiple defense systems. The CRISPR-Cas system appears to especially target another virus that could compete with the system itself for the same integration site. This virus is prevalent in strains of the species belonging to the so-called Global Clone 2, which causes the most frequent outbreaks worldwide. Knowledge of this viral warfare involving antiviral systems could be useful in the fight against infections caused by bacteria, and it would also shed light on how CRISPR-Cas systems expand in bacteria.}, }
@article {pmid39673958, year = {2025}, author = {Yang, R and Guan, X and Zhang, J and Moon, J and Guo, C and Jia, Z and Hou, C and Ganbaatar, U and Tricarico, S and Ma, AWK and Liu, C}, title = {Quencher-free CRISPR-based molecular detection using an amphiphilic DNA fluorescence probe.}, journal = {Biosensors &amp; bioelectronics}, volume = {271}, number = {}, pages = {117054}, pmid = {39673958}, issn = {1873-4235}, support = {U01 CA269147/CA/NCI NIH HHS/United States ; }, mesh = {Humans ; *Biosensing Techniques/methods ; *Fluorescent Dyes/chemistry ; *Human papillomavirus 16/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *DNA, Viral/analysis/genetics ; DNA, Single-Stranded/chemistry ; DNA Probes/chemistry/genetics ; Carbocyanines/chemistry ; Lab-On-A-Chip Devices ; Papillomavirus Infections/diagnosis/virology ; CRISPR-Associated Proteins/chemistry ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {Rapid, sensitive, and specific nucleic acid detection methods play crucial roles in clinical diagnostics and healthcare. Here, we report a novel amphiphilic DNA fluorescence probe for CRISPR-based nucleic acid detection. Unlike conventional fluorophore-quencher probe detection system, our amphiphilic DNA fluorescence probe features a hydrophobic Cy5 fluorophore head and a hydrophilic single-stranded DNA (ssDNA) tail. By combining the amphiphilic DNA fluorescence probe with a paper-based microfluidic device, we developed a quencher-free, CRISPR-based detection system for target nucleic acid quantification. In the presence of the target nucleic acid, the activated CRISPR-Cas12a enzyme cleaves the hydrophilic ssDNA tail of the amphiphilic DNA fluorescence probe, releasing the hydrophobic Cy5 head and altering the wettability of the CRISPR reaction solution. When the CRISPR reaction product is applied to the paper-based microfluidic device, the migration of the cleaved Cy5 head along the hydrophilic microfluidic channel is limited. The higher the target nucleic acid concentration, the shorter the fluorescence diffusion distance, enabling visual quantification of the nucleic acid target. We used human papillomavirus-16 (HPV-16) DNA as a model to evaluate the analytical performance of the system. Furthermore, we validated its clinical feasibility by testing clinical swab samples, achieving results comparable to the traditional PCR method. Our quencher-free CRISPR-based detection system shows potential for simple, affordable, and sensitive clinical diagnostics of HPV-associated cancer and other infectious diseases.}, }
@article {pmid39656986, year = {2024}, author = {Jordan, B}, title = {[New-generation editing].}, journal = {Medecine sciences : M/S}, volume = {40}, number = {11}, pages = {869-871}, doi = {10.1051/medsci/2024149}, pmid = {39656986}, issn = {1958-5381}, mesh = {*Gene Editing/methods/trends ; Humans ; CRISPR-Cas Systems ; Animals ; Bacteria/genetics ; }, abstract = {Recent work on bacterial insertion sequences reveals that some of them use an RNA sequence (called Bridge RNA or Seek RNA) to define both donor and target DNA specificity. This opens the way to easy insertion of kilobase DNA sequences at pre-defined sites in the genome, announcing a host of new possibilities. The system still needs a lot of tweaking, as it has only been demonstrated in bacteria, but it holds great promise for genome editing and engineering.}, }
@article {pmid39673266, year = {2024}, author = {Molina-Sánchez, MD and Martínez-Abarca, F and Millán, V and Mestre, MR and Stehantsev, P and Stetsenko, A and Guskov, A and Toro, N}, title = {Adaptive immunity of type VI CRISPR-Cas systems associated with reverse transcriptase-Cas1 fusion proteins.}, journal = {Nucleic acids research}, volume = {52}, number = {22}, pages = {14229-14243}, pmid = {39673266}, issn = {1362-4962}, support = {P20_00047//Consejeria de Transformacion Economica, industria, Conocimiento y Universidades/ ; //ERDF A way of making Europe/ ; }, mesh = {*CRISPR-Cas Systems ; *RNA-Directed DNA Polymerase/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; *Adaptive Immunity/genetics ; Bacterial Proteins/genetics/metabolism ; Gastrointestinal Microbiome/genetics/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Bacteria/genetics/immunology ; RNA Interference ; }, abstract = {Cas13-containing type VI CRISPR-Cas systems specifically target RNA; however, the mechanism of spacer acquisition remains unclear. We have previously reported the association of reverse transcriptase-Cas1 (RT-Cas1) fusion proteins with certain types of VI-A systems. Here, we show that RT-Cas1 fusion proteins are also recruited by type VI-B systems in bacteria from gut microbiomes, constituting a VI-B1 variant system that includes a CorA-encoding locus in addition to the CRISPR array and the RT-Cas1/Cas2 adaptation module. We found that type VI RT-CRISPR systems were functional for spacer acquisition, CRISPR array processing and interference activity, demonstrating that adaptive immunity mediated by these systems can function independently of other in trans systems. We provide evidence that the RT associated with these systems enables spacer acquisition from RNA molecules. We also found that CorA encoded by type VI-B1 RT-associated systems can transport divalent metal ions and downregulate Cas13b-mediated RNA interference. These findings highlight the importance of RTs in RNA-targeting CRISPR-Cas systems, potentially enabling the integration of RNA-derived spacers into CRISPR arrays as a mechanism against RNA-based invaders in specific environments.}, }
@article {pmid39672387, year = {2025}, author = {He, X and Li, P and Cao, H and Zhang, X and Zhang, M and Yu, X and Sun, Y and Ghonaim, AH and Ma, H and Li, Y and Shi, K and Zhu, H and He, Q and Li, W}, title = {Construction of a recombinant African swine fever virus with firefly luciferase and eGFP reporter genes and its application in high-throughput antiviral drug screening.}, journal = {Antiviral research}, volume = {233}, number = {}, pages = {106058}, doi = {10.1016/j.antiviral.2024.106058}, pmid = {39672387}, issn = {1872-9096}, mesh = {Animals ; *African Swine Fever Virus/genetics/drug effects ; Chlorocebus aethiops ; *Green Fluorescent Proteins/genetics ; *Antiviral Agents/pharmacology ; Vero Cells ; *Genes, Reporter ; Swine ; *Luciferases, Firefly/genetics ; *African Swine Fever/virology ; *Drug Evaluation, Preclinical/methods ; High-Throughput Screening Assays/methods ; Virus Replication/drug effects ; CRISPR-Cas Systems ; Gene Editing/methods ; Macrophages, Alveolar/virology/drug effects ; }, abstract = {African Swine Fever (ASF) is a highly lethal and contagious disease in pigs caused by African Swine Fever Virus (ASFV), which primarily infects domestic pigs and wild boars, with a mortality rate of up to 100%. Currently, there are no commercially available vaccines or drugs that are both safe and effective against ASFV. The ASFV 0428C strain was continuously passaged in Vero cells, and the adapted ASFV demonstrated efficient replication in Vero cells. The adapted ASFV was used as the parental virus, and an expression cassette encoding a dual reporter gene for firefly luciferase (Fluc) and enhanced green fluorescent protein (eGFP) was inserted into the ASFV genome using CRISPR/Cas9 gene editing technology to construct a recombinant ASFV variant (rASFV-FLuc-eGFP). rASFV-Fluc-eGFP was genetically stable, effectively infected porcine alveolar macrophages (PAM) and Vero cells, and expressed Fluc and eGFP concurrently. This study provides a tool for investigating the infection and pathogenic mechanisms of ASFV, as well as for screening essential host genes and antiviral drugs. Additionally, a high-throughput screening model of antiviral drugs was established based on rASFV-FLuc-eGFP in passaged cells, 218 compounds from the FDA-approved compound library were screened, and 5 candidate compounds with significant inhibitory effects in Vero cells were identified. The inhibitory effects on ASFV were further validated in both Vero and PAM cells, resulting in the identification of Salvianolic acid C (SAC), which demonstrated inhibitory effects and safety in both cell types. SAC is a candidate drug for the prevention and control of ASFV and shows promising application prospects.}, }
@article {pmid39671433, year = {2024}, author = {Taylor, T and Zhu, HV and Moorthy, SD and Khader, N and Mitchell, JA}, title = {The cells are all-right: Regulation of the Lefty genes by separate enhancers in mouse embryonic stem cells.}, journal = {PLoS genetics}, volume = {20}, number = {12}, pages = {e1011513}, pmid = {39671433}, issn = {1553-7404}, support = {R01 HG010045/HG/NHGRI NIH HHS/United States ; }, mesh = {Animals ; Mice ; *Enhancer Elements, Genetic ; *Mouse Embryonic Stem Cells/metabolism ; *Left-Right Determination Factors/genetics/metabolism ; *Chromatin/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Gene Expression Regulation, Developmental ; }, abstract = {Enhancers play a critical role in regulating precise gene expression patterns essential for development and cellular identity; however, how gene-enhancer specificity is encoded within the genome is not clearly defined. To investigate how this specificity arises within topologically associated domains (TAD), we performed allele-specific genome editing of sequences surrounding the Lefty1 and Lefty2 paralogs in mouse embryonic stem cells. The Lefty genes arose from a tandem duplication event and these genes interact with each other in chromosome conformation capture assays which place these genes within the same TAD. Despite their physical proximity, we demonstrate that these genes are primarily regulated by separate enhancer elements. Through CRISPR-Cas9 mediated deletions to remove the intervening chromatin between the Lefty genes, we reveal a distance-dependent dosage effect of the Lefty2 enhancer on Lefty1 expression. These findings indicate a role for chromatin distance in insulating gene expression domains in the Lefty locus in the absence of architectural insulation.}, }
@article {pmid39671335, year = {2024}, author = {Kolodziejczyk, A and Hoffmann, J and Cubillos, P and Albert, M}, title = {Electroporation of Sliced Human Cortical Organoids for Studies of Gene Function.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {213}, pages = {}, doi = {10.3791/67598}, pmid = {39671335}, issn = {1940-087X}, mesh = {Humans ; *Electroporation/methods ; *Organoids/cytology/metabolism ; *CRISPR-Cas Systems ; Cerebral Cortex/cytology ; Gene Knockout Techniques/methods ; Neocortex/cytology ; }, abstract = {Human cortical organoids have become important tools for studying human brain development, neurodevelopmental disorders, and human brain evolution. Studies analyzing gene function by overexpression or knockout have been instrumental in animal models to provide mechanistic insights into the regulation of neocortex development. Here, we present a detailed protocol for CRISPR/Cas9-mediated acute gene knockout by electroporation of sliced human cortical organoids. The slicing of cortical organoids aids the identification of ventricle-like structures for injection and subsequent electroporation, making this a particularly well-suited model for acute genetic manipulation during human cortical development. We describe the design of guide RNAs and the validation of targeting efficiency in vitro and in cortical organoids. Electroporation of cortical organoids is performed at mid-neurogenic stages, enabling the targeting of most major cell classes in the developing neocortex, including apical radial glia, basal progenitor cells, and neurons. Taken together, the electroporation of sliced human cortical organoids represents a powerful technique to investigate gene function, gene regulation, and cell morphology during cortical development.}, }
@article {pmid39671009, year = {2024}, author = {Yu, Y and Li, Q and Yu, H and Li, Q}, title = {Comparative Analysis of Promoter Activity in Crassostrea gigas Embryos: Implications for Bivalve Gene Editing.}, journal = {Marine biotechnology (New York, N.Y.)}, volume = {27}, number = {1}, pages = {20}, pmid = {39671009}, issn = {1436-2236}, support = {42276111//National Natural Science Foundation of China/ ; ZR2022MC171//Natural Science Foundation of Shandong Province/ ; }, mesh = {Animals ; *Crassostrea/genetics/embryology ; *Promoter Regions, Genetic ; *Green Fluorescent Proteins/genetics/metabolism ; *Gene Editing/methods ; *Histones/metabolism/genetics ; Embryo, Nonmammalian/metabolism ; CRISPR-Cas Systems ; Peptide Elongation Factor 1/genetics/metabolism ; Aquaculture ; }, abstract = {In recent years, CRISPR/Cas9 gene editing technology has emerged as a powerful genetic tool with potential application in aquaculture. Crassostrea gigas, as a valuable species in aquaculture, holds promising potential for genetic enhancement and breeding through gene editing. However, the lack of efficient promoters for driving exogenous gene expression poses a major obstacle in bivalve gene editing. In this study, we isolated the promoter sequences of the β-tub and histone H3.3A genes from C. gigas. DNA expression constructs were generated by linking the promoters with the enhanced green fluorescent protein (EGFP) reporter and compared with the promoter activity of the endogenous EF-1α gene and an exogenous OsHV-1 promoter in C. gigas embryos. All four promoters effectively drive the expression of EGFP during early embryonic development in C. gigas. Among these four promoters, the β-tub promoter is the most potent promoter in driving EGFP expression in C. gigas embryos as early as 4.5 h after fertilization. The OsHV-1 promoter showed similar activity as β-tub promoter and appeared to be more active than the EF-1α and histone H3.3A promoters in C. gigas embryos. Furthermore, we assessed their performance in other three C. gigas relatives (Crassostrea ariakensis, Crassostrea nippona, and Crassostrea sikamea) and similar results were found. Collectively, these data suggest that the β-tub promoter is an effective promoter in directing gene expression in directing gene expression in oyster embryos, thus offering a potential application for gene editing in bivalves.}, }
@article {pmid39670656, year = {2024}, author = {Jung, JK and Dreyer, KS and Dray, KE and Muldoon, JJ and George, J and Shirman, S and Cabezas, MD and d'Aquino, AE and Verosloff, MS and Seki, K and Rybnicky, GA and Alam, KK and Bagheri, N and Jewett, MC and Leonard, JN and Mangan, NM and Lucks, JB}, title = {Developing, Characterizing, and Modeling CRISPR-Based Point-of-Use Pathogen Diagnostics.}, journal = {ACS synthetic biology}, volume = {}, number = {}, pages = {}, doi = {10.1021/acssynbio.4c00469}, pmid = {39670656}, issn = {2161-5063}, support = {R01 EB026510/EB/NIBIB NIH HHS/United States ; }, abstract = {Recent years have seen intense interest in the development of point-of-care nucleic acid diagnostic technologies to address the scaling limitations of laboratory-based approaches. Chief among these are combinations of isothermal amplification approaches with CRISPR-based detection and readouts of target products. Here, we contribute to the growing body of rapid, programmable point-of-care pathogen tests by developing and optimizing a one-pot NASBA-Cas13a nucleic acid detection assay. This test uses the isothermal amplification technique NASBA to amplify target viral nucleic acids, followed by the Cas13a-based detection of amplified sequences. We first demonstrate an in-house formulation of NASBA that enables the optimization of individual NASBA components. We then present design rules for NASBA primer sets and LbuCas13a guide RNAs for the fast and sensitive detection of SARS-CoV-2 viral RNA fragments, resulting in 20-200 aM sensitivity. Finally, we explore the combination of high-throughput assay condition screening with mechanistic ordinary differential equation modeling of the reaction scheme to gain a deeper understanding of the NASBA-Cas13a system. This work presents a framework for developing a mechanistic understanding of reaction performance and optimization that uses both experiments and modeling, which we anticipate will be useful in developing future nucleic acid detection technologies.}, }
@article {pmid39670632, year = {2024}, author = {Chen, W and Liu, L and Cheng, L}, title = {Conditionally Activated Cross-Linked crRNAs for CRISPR/Cas12a Based Nucleic Acid Detection.}, journal = {ACS synthetic biology}, volume = {}, number = {}, pages = {}, doi = {10.1021/acssynbio.4c00695}, pmid = {39670632}, issn = {2161-5063}, abstract = {CRISPR/Cas systems, particularly CRISPR/Cas12a, have revolutionized nucleic acid detection due to their exceptional specificity and sensitivity. However, CRISPR/Cas12a's cleavage activity can interfere with amplification processes, such as reverse transcription (RT) and isothermal amplification (e.g., RPA), potentially compromising detection sensitivity and accuracy. While modified CRISPR/Cas12a systems employing caging and decaging strategies have been developed to address this, these approaches typically require extensive optimization of photolabile groups and complex assay configurations. Here, we present a universal, photochemically controlled strategy for CRISPR/Cas12a-based detection that overcomes these challenges. Our approach involves cross-linking a polymeric crRNA with a photoresponsive cross-linker, effectively inactivating it during amplification and enabling rapid activation through brief light exposure to cleave the cross-linker and release active crRNA. This method obviates the need for labor-intensive optimizations and modifications, making it highly versatile and suitable for rapid, on-site detection applications. Our strategy demonstrates enhanced versatility and applicability, particularly for the immediate detection of newly emerging or unexpected nucleic acid sequences, supporting applications in pathogen detection, genetic screening, and point-of-care diagnostics.}, }
@article {pmid39670384, year = {2025}, author = {Blennemann, M and Verma, A and Bachl, S and Carnevale, J and Engelhardt, BE}, title = {Understanding TCR T cell knockout behavior using interpretable machine learning.}, journal = {Pacific Symposium on Biocomputing. Pacific Symposium on Biocomputing}, volume = {30}, number = {}, pages = {382-393}, pmid = {39670384}, issn = {2335-6936}, mesh = {Humans ; *Receptors, Antigen, T-Cell/immunology/genetics ; *Machine Learning ; *Computational Biology ; *T-Lymphocytes/immunology ; *Neoplasms/immunology/genetics ; Gene Knockout Techniques ; Neural Networks, Computer ; CRISPR-Cas Systems ; }, abstract = {Genetic perturbation of T cell receptor (TCR) T cells is a promising method to unlock better TCR T cell performance to create more powerful cancer immunotherapies, but understanding the changes to T cell behavior induced by genetic perturbations remains a challenge. Prior studies have evaluated the effect of different genetic modifications with cytokine production and metabolic activity assays. Live-cell imaging is an inexpensive and robust approach to capture TCR T cell responses to cancer. Most methods to quantify T cell responses in live-cell imaging data use simple approaches to count T cells and cancer cells across time, effectively quantifying how much space in the 2D well each cell type covers, leaving actionable information unexplored. In this study, we characterize changes in TCR T cell's interactions with cancer cells from live-cell imaging data using explainable artificial intelligence (AI). We train convolutional neural networks to distinguish behaviors in TCR T cell with CRISPR knock outs of CUL5, RASA2, and a safe harbor control knockout. We use explainable AI to identify specific interaction types that define different knock-out conditions. We find that T cell and cancer cell coverage is a strong marker of TCR T cell modification when comparing similar experimental time points, but differences in cell aggregation characterize CUL5KO and RASA2KO behavior across all time points. Our pipeline for discovery in live-cell imaging data can be used for characterizing complex behaviors in arbitrary live-cell imaging datasets, and we describe best practices for this goal.}, }
@article {pmid39667940, year = {2024}, author = {Yang, B and Wu, C and Teng, Y and Chou, KJ and Guarnieri, MT and Xiong, W}, title = {Tailoring microbial fitness through computational steering and CRISPRi-driven robustness regulation.}, journal = {Cell systems}, volume = {15}, number = {12}, pages = {1133-1147.e4}, doi = {10.1016/j.cels.2024.11.012}, pmid = {39667940}, issn = {2405-4720}, mesh = {*CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Computer Simulation ; Genetic Fitness/genetics ; Escherichia coli/genetics/metabolism ; Organisms, Genetically Modified ; }, abstract = {The widespread application of genetically modified microorganisms (GMMs) across diverse sectors underscores the pressing need for robust strategies to mitigate the risks associated with their potential uncontrolled escape. This study merges computational modeling with CRISPR interference (CRISPRi) to refine GMM metabolic robustness. Utilizing ensemble modeling, we achieved high-throughput in silico screening for enzymatic targets susceptible to expression alterations. Translating these insights, we developed functional CRISPRi, boosting fitness control via multiplexed gene knockdown. Our method, enhanced by an insulator-improved gRNA structure and an off-switch circuit controlling a compact Cas12m, resulted in rationally engineered strains with escape frequencies below National Institutes of Health standards. The effectiveness of this approach was confirmed under various conditions, showcasing its ability for secure GMM management. This research underscores the resilience of microbial metabolism, strategically modifying key nodes to halt growth without provoking significant resistance, thereby enabling more reliable and precise GMM control. A record of this paper's transparent peer review process is included in the supplemental information.}, }
@article {pmid39667861, year = {2025}, author = {Wu, P and Zhang, Y and Shan, Q and Wang, Z and Cheng, S and Wang, L and Liu, B and Li, W and Chen, Z and Luo, J and Liang, Y}, title = {The investigation of the mechanism underlying variations in oxidative stress tolerance of Lacticaseibacillus paracasei resulting from fermentation methods through endogenous CRISPR-Cas9 editing methodology.}, journal = {Food microbiology}, volume = {127}, number = {}, pages = {104697}, doi = {10.1016/j.fm.2024.104697}, pmid = {39667861}, issn = {1095-9998}, mesh = {*Oxidative Stress ; *Fermentation ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Lacticaseibacillus paracasei/genetics/metabolism/physiology/growth &amp; development ; Probiotics ; Bacterial Proteins/genetics/metabolism ; }, abstract = {The probiotic effects of lactic acid bacteria make them widely used in human and animal breeding industry. However, the presence of oxidative stress during the production and application process can cause bacterial damage or even death, significantly compromising the functionality of probiotics. Despite its potential for broader application scenarios that could provide a more comprehensive understanding of bacteria's internal adaptation strategies, there is a lack of research investigating oxidative stress from the perspective of culture methods. In this study, the tolerance to oxidative stress was compared between bacteria cultivated through solid-state fermentation (SSF) and liquid-state fermentation (LSF), and the physiological and transcriptional disparities between these two bacterial strains were investigated. Additionally, a novel and efficient gene editing method was developed to elucidate the genetic basis underlying these differences in tolerance. The results demonstrated a significantly higher tolerance to oxidative stress in SSF bacteria compared to LSF bacteria, along with a stronger capacity for maintaining intracellular microenvironment stability and the activity of key metabolic enzymes. It is noteworthy that the bacteria from SSF significantly enhance the transport of carbohydrate substances and facilitate intracellular metabolic flow. Gene editing experiments have confirmed the crucial role of genes glpF and glpO in regulating the glycerol metabolism pathway, which is essential for enhancing the tolerance of bacteria from SSF to oxidative stress. Based on these findings, the mechanism underlying the disparity in oxidative stress tolerance resulting from different culture methods has been summarized. Furthermore, investigation into different culture modes has revealed that moderate oxygen levels during cultivation significantly influence variation in bacterial tolerance to oxidative stress. Importantly, these variations are species-specific and depend on the ecological niche distribution of Lactobacilli. These findings elucidate a novel mechanism by which Lacticaseibacillus paracasei Zhang tolerates oxidative stress, and also suggest that distinct cultivation and processing methods should be tailored based on the specific Lactobacilli groups to achieve optimal application effects in production.}, }
@article {pmid39667860, year = {2025}, author = {Zhang, X and Chen, W and Wang, C}, title = {Regulation of citrinin biosynthesis in Monascus purpureus: Impacts on growth, morphology, and pigments production.}, journal = {Food microbiology}, volume = {127}, number = {}, pages = {104698}, doi = {10.1016/j.fm.2024.104698}, pmid = {39667860}, issn = {1095-9998}, mesh = {*Monascus/metabolism/growth &amp; development/genetics ; *Citrinin/biosynthesis/metabolism ; *Fungal Proteins/genetics/metabolism ; *Pigments, Biological/biosynthesis/metabolism ; Gene Expression Regulation, Fungal ; Mycelium/growth &amp; development/metabolism/genetics ; CRISPR-Cas Systems ; Hyphae/growth &amp; development/metabolism ; Spores, Fungal/growth &amp; development/metabolism/genetics ; Gene Knockout Techniques ; }, abstract = {Fungal hyphae self-assemble a variety of cellular macrostates, ranging from suspended mycelium to dense pellets, all inextricably linked to their productivity. In this study, using CRISPR/Cas technology, we constructed a ctnA knockout strain (ΔctnA) and an overexpression strain (A2) so as to investigate the effects of interfering with citrinin biosynthesis on the growth, morphology and pigmentation of M.purpureus. Results indicated that deletion of ctnA in M. purpureus RP2 led to increased mycelium length, delayed conidium formation, and a citrinin content of 22% of the wild-type strain. Conversely, ctnA overexpression in strain A2 resulted in delayed mycelial growth, normal conidium formation, and a citrinin content of 120% compared to the wild-type strain, with minimal effects on pigments content. Notably, the ΔctnA strain formed small, tightly structured pellets (mean diameter 1.2 ± 0.06 mm) and exhibited low citrinin content, promoting pigments production. Our findings suggest a complex interplay between citrinin biosynthesis and morphological development, providing insights for optimizing metabolite production in industrial applications.}, }
@article {pmid39667702, year = {2024}, author = {Chaudhary, N and Sharma, K and Harpreet Kaur,  and Prajapati, S and Mohan, B and Taneja, N}, title = {CRISPR-Cas-assisted phage engineering for personalized antibacterial treatments.}, journal = {Indian journal of medical microbiology}, volume = {53}, number = {}, pages = {100771}, doi = {10.1016/j.ijmmb.2024.100771}, pmid = {39667702}, issn = {1998-3646}, abstract = {BACKGROUND: In the age of modern medicine, CRISPR-Cas system-aided phage engineering has emerged as a major game changer for developing personalized antibacterial treatments. Modifying genomic DNA at a specific location leads to the inactivation of target genes, the acquisition of novel genetic features, and the correction of lethal gene mutations. Phages can be modified to precisely detect and control bacteria because of the vast possibilities of CRISPR-Cas-based genetic engineering.<br><br>OBJECTIVES: The primary objective of this review is to explore the basic principles, mechanisms, limitations, and perspectives of CRISPR-Cas system-aided phage engineering in producing tailored antibacterial therapeutics. Furthermore, this study will address how editing phage genomes using CRISPR-Cas technology allows for precise bacteria targeting, broadening phage host range, and improving infection control tactics.<br><br>CONTENT: The arrival of the CRISPR-Cas system has transformed the field of phage engineering and aided in the precise modification of phag&#x435; genomes to broaden the phage host range. This novel strategy uses the accuracy of the CRISPR-Cas system to design engineered bacteriophages, giving targeted options for infection control. These recent advancements have the potential to alter the era of modern medicine.}, }
@article {pmid39666857, year = {2024}, author = {Xie, F and Zhao, H and Liu, J and Yang, X and Neuber, M and Agrawal, AA and Kaur, A and Herrmann, J and Kalinina, OV and Wei, X and Müller, R and Fu, C}, title = {Autologous DNA mobilization and multiplication expedite natural products discovery from bacteria.}, journal = {Science (New York, N.Y.)}, volume = {386}, number = {6727}, pages = {eabq7333}, doi = {10.1126/science.abq7333}, pmid = {39666857}, issn = {1095-9203}, mesh = {*Biological Products/metabolism ; *Streptomyces/genetics/metabolism ; *CRISPR-Cas Systems ; *Multigene Family ; *Plasmids/genetics ; DNA, Bacterial/genetics ; Genome, Bacterial ; Drug Discovery ; Biosynthetic Pathways ; DNA Replication ; Drug Resistance, Bacterial/genetics ; }, abstract = {The transmission of antibiotic-resistance genes, comprising mobilization and relocation events, orchestrates the dissemination of antimicrobial resistance. Inspired by this evolutionarily successful paradigm, we developed ACTIMOT, a CRISPR-Cas9-based approach to unlock the vast chemical diversity concealed within bacterial genomes. ACTIMOT enables the efficient mobilization and relocation of large DNA fragments from the chromosome to replicative plasmids within the same bacterial cell. ACTIMOT circumvents the limitations of traditional molecular cloning methods involving handling and replicating large pieces of genomic DNA. Using ACTIMOT, we mobilized and activated four cryptic biosynthetic gene clusters from Streptomyces, leading to the discovery of 39 compounds across four distinct classes. This work highlights the potential of ACTIMOT for accelerating the exploration of biosynthetic pathways and the discovery of natural products.}, }
@article {pmid39666823, year = {2024}, author = {Li, Y and Li, Z and Yan, P and Hua, C and Kong, J and Wu, W and Cui, Y and Duan, Y and Li, S and Li, G and Ji, S and Chen, Y and Zhao, Y and Yang, P and Hu, C and Lu, M and Chen, M and Xiao, Y}, title = {Antiviral signaling of a type III CRISPR-associated deaminase.}, journal = {Science (New York, N.Y.)}, volume = {}, number = {}, pages = {eadr0393}, doi = {10.1126/science.adr0393}, pmid = {39666823}, issn = {1095-9203}, abstract = {Prokaryotes have evolved diverse defense strategies against viral infection, such as foreign nucleic acid degradation by CRISPR-Cas systems and DNA/RNA synthesis inhibition via nucleotide pool depletion. Here, we report an antiviral mechanism of type III CRISPR-Cas-regulated ATP depletion, where ATP is converted into ITP by CRISPR-Cas-associated adenosine deaminase (CAAD) upon activation by either cA4 or cA6, followed by hydrolysis into IMP by Nudix hydrolase, ultimately resulting in cell growth arrest. The cryo-electron microscopy structures of CAAD in its apo and activated forms, together with biochemical evidence, revealed how cA4/cA6 binds to the CARF domain and abrogates CAAD autoinhibition, inducing substantial conformational changes that reshape the structure of CAAD and induce its deaminase activity. Our results reveal the mechanism of a CRISPR-Cas-regulated ATP depletion antiviral strategy.}, }
@article {pmid39666765, year = {2024}, author = {Shao, G and Zhu, X and Hua, R and Chen, Y and Yang, G}, title = {Development of a Copro-RPA-CRISPR/Cas12a assay to detect Echinococcus granulosus nucleic acids isolated from canine feces using NaOH-based DNA extraction method.}, journal = {PLoS neglected tropical diseases}, volume = {18}, number = {12}, pages = {e0012753}, pmid = {39666765}, issn = {1935-2735}, mesh = {Animals ; Dogs ; *Echinococcus granulosus/genetics/isolation &amp; purification ; *Feces/parasitology ; *Echinococcosis/diagnosis/veterinary/parasitology ; *Dog Diseases/diagnosis/parasitology ; *CRISPR-Cas Systems ; *DNA, Helminth/genetics/isolation &amp; purification ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/genetics/isolation &amp; purification ; Bacterial Proteins ; Endodeoxyribonucleases ; }, abstract = {BACKGROUND: Cystic echinococcosis (CE), caused by Echinococcus granulosus sensu lato (E. granulosus s.l.), remains a significant zoonotic parasitic disease affecting both livestock and humans. It arises from the ingestion of food and water contaminated with canine feces containing E. granulosus eggs. The detection of these eggs in canine feces is essential for guiding effective preventative measures against the disease. Therefore, the development of a novel accurate, rapid, and visually interpretable point-of-care test is crucial for controlling CE.<br><br>METHODS: We combined recombinase polymerase amplification (RPA) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) with a CRISPR-associated protein 12a (Cas12a) system, forming the RPA-CRISPR/Cas12a assay. This assay targeted the E. granulosus mitochondrial nad2 gene and utilized a lateral flow strip for visual readout. To improve field applicability, we integrated a simple and cost-effective NaOH-Based DNA extraction method. Clinical validation included testing DNA extracted from eighteen canine fecal samples, followed by comparison with quantitative PCR (qPCR) and two commercial enzyme-linked immunosorbent assay (ELISA) kits.<br><br>RESULTS: The RPA-CRISPR/Cas12a assay showed a detection limit of 1 fg/&#x3bc;L DNA, without any cross-reactivity with related tapeworms such as Echinococcus multilocularis, Dipylidium caninum, Taenia hydatigera, Taenia multiceps, and Taenia pisiformis. When applied to 62 clinical fecal samples from dogs, the RPA-CRISPR/Cas12a assay demonstrated 68% sensitivity, while the developed RPA-CRISPR/Cas12a-NaOH assay exhibited 45% sensitivity. In the field performance comparison of the RPA-CRISPR/Cas12a and the RPA-CRISPR/Cas12a-NaOH assay with qPCR and two ELISA kits, the sensitivity, consistency rate, and Youden's index suggested good or fair agreement with the currently employed detection methods.<br><br>CONCLUSION: This study describes the development and validation of the RPA-CRISPR/Cas12a and RPA-CRISPR/Cas12a-NaOH assays for detecting E. granulosus in canine feces. The developed assays surpassed previous detection methods in providing enhanced diagnostic sensitivity and enabling point-of-care testing. Moreover, these assays hold potential for surveilling E. granulosus in low-income countries.}, }
@article {pmid39666677, year = {2024}, author = {Blokhina, Y and Buchwalter, A}, title = {Modeling the consequences of age-linked rDNA hypermethylation with dCas9-directed DNA methylation in human cells.}, journal = {PloS one}, volume = {19}, number = {12}, pages = {e0310626}, pmid = {39666677}, issn = {1932-6203}, support = {T32 HL007731/HL/NHLBI NIH HHS/United States ; }, mesh = {Humans ; *DNA Methylation ; *DNA, Ribosomal/genetics/metabolism ; *Promoter Regions, Genetic ; *Aging/genetics ; Pol1 Transcription Initiation Complex Proteins/metabolism/genetics ; RNA Polymerase I/metabolism/genetics ; CRISPR-Cas Systems ; Transcription, Genetic ; CRISPR-Associated Protein 9/metabolism/genetics ; Animals ; }, abstract = {Ribosomal DNA (rDNA) genes encode the structural RNAs of the ribosome and are present in hundreds of copies in mammalian genomes. Age-linked DNA hypermethylation throughout the rDNA constitutes a robust "methylation clock" that accurately reports age, yet the consequences of hypermethylation on rDNA function are unknown. We confirmed that pervasive hypermethylation of rDNA occurs during mammalian aging and senescence while rDNA copy number remains stable. We found that DNA methylation is exclusively found on the promoters and gene bodies of inactive rDNA. To model the effects of age-linked methylation on rDNA function, we directed de novo DNA methylation to the rDNA promoter or gene body with a nuclease-dead Cas9 (dCas9)-DNA methyltransferase fusion enzyme in human cells. Hypermethylation at each target site had no detectable effect on rRNA transcription, nucleolar morphology, or cellular growth rate. Instead, human UBF and Pol I remain bound to rDNA promoters in the presence of increased DNA methylation. These data suggest that promoter methylation is not sufficient to impair transcription of the human rDNA and imply that the human rDNA transcription machinery may be resilient to age-linked rDNA hypermethylation.}, }
@article {pmid39666461, year = {2024}, author = {Yan, B and Liu, Y and Cai, Y and Liu, Y and Chen, Y}, title = {Protocol for establishing CRISPR-Cas12a for efficient genome editing of Pseudomonas aeruginosa phages.}, journal = {STAR protocols}, volume = {5}, number = {4}, pages = {103488}, pmid = {39666461}, issn = {2666-1667}, mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Pseudomonas aeruginosa/genetics/virology ; Genome, Viral/genetics ; Pseudomonas Phages/genetics ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {We developed an efficient type V CRISPR-Cas12a system tailored specifically for Pseudomonas aeruginosa phages, showcasing its remarkable cleavage activity and the ability to precisely introduce genetic modifications, including point mutations, deletions, and insertions, into phage genomes. Here, we present a protocol for establishing CRISPR-Cas12a for genome editing of Pseudomonas aeruginosa phages. We describe steps for the construction of pCRISPR-12a plasmid and guide RNA and the utilization of the type V CRISPR-Cas12a system for precise genetic editing of phages. For complete details on the use and execution of this protocol, please refer to Chen et al.[1].}, }
@article {pmid39665756, year = {2024}, author = {Kent, K and Nozawa, K and Parkes, R and Dean, L and Daniel, F and Leng, M and Jain, A and Malovannaya, A and Matzuk, MM and X Garcia, T}, title = {Large-scale CRISPR/Cas9 deletions within the WFDC gene cluster uncover gene functionality and critical roles in mammalian reproduction.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {51}, pages = {e2413195121}, pmid = {39665756}, issn = {1091-6490}, support = {T32 GM139534/GM/NIGMS NIH HHS/United States ; RP210227//Cancer Prevention and Research Institute of Texas (CPRIT)/ ; S10OD026804//HHS | NIH | NIH Office of the Director (OD)/ ; Fellowship//Lalor Foundation (The Lalor Foundation)/ ; R01HD095341//HHS | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; R01HD106056//HHS | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; R01 HD088412/HD/NICHD NIH HHS/United States ; T32HD098068//HHS | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; P30 CA125123/CA/NCI NIH HHS/United States ; Fellowship//Japan Society for the Promotion of Science Overseas Research/ ; RP170005//Cancer Prevention and Research Institute of Texas (CPRIT)/ ; R01 HD106056/HD/NICHD NIH HHS/United States ; T32 HD098068/HD/NICHD NIH HHS/United States ; 2021-302//Male Contraceptive Initiative/ ; R01 HD095341/HD/NICHD NIH HHS/United States ; S10 OD026804/OD/NIH HHS/United States ; P30CA125123//HHS | NIH | National Cancer Institute (NCI)/ ; P50HD103555//HHS | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; Fellowship//A.I. &amp; Manet Schepps Discovery Foundation/ ; P50 HD103555/HD/NICHD NIH HHS/United States ; R01HD088412//HHS | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; }, mesh = {Animals ; Male ; Mice ; *Multigene Family ; *CRISPR-Cas Systems ; *Infertility, Male/genetics ; Reproduction/genetics ; Mice, Knockout ; Fertility/genetics ; Humans ; Spermatogenesis/genetics ; Female ; Proteinase Inhibitory Proteins, Secretory/genetics/metabolism ; Gene Deletion ; Spermatozoa/metabolism ; Sperm Motility/genetics ; }, abstract = {Despite 96 million years of evolution separating humans and rodents, 11 closely related reproductive tract-specific genes in humans-SPINT3, WFDC6, EPPIN, WFDC8, WFDC9, WFDC10A, WFDC11, WFDC10B, WFDC13, SPINT4, and WFDC3-and the 13 reproductive tract-specific orthologous genes in mice, form highly conserved syntenic gene clusters indicative of conserved, combined critical functions. Further, despite significant progress toward a nonhormonal male contraceptive targeting the protein encoded by one of these genes, epididymal peptidase inhibitor (EPPIN), and associations found between mutations in EPPIN and an increased risk of male infertility, neither EPPIN nor any closely related whey acidic protein four-disulfide core (WFDC) gene have been explored functionally. To clarify the involvement of WFDC genes in male fertility, we strategically used CRISPR/Cas9 to generate mice lacking 13, 10, 5, or 4 genes within the cluster and demonstrated that males with deletions of 13, 10, or 4 genes (Wfdc6a, Eppin, Wfdc8, and Wfdc6a) were sterile due to an arrest in spermatogenesis, preventing formation beyond round spermatids. In contrast, the five gene knockout (KO) males (lacking Wfdc16, Wfdc9, Wfdc10, Wfdc11, and Wfdc13), despite normal spermatogenesis and sperm counts, were infertile due to defects in sperm motility and increased sperm death. Similarly to our previously reported Spint3 single gene KO, Wfdc3 single KO mice were fertile with no obvious reproductive phenotype. Our KO mouse studies to explore the entire WFDC locus of closely related genes have clarified the functional requirements of WFDC locus genes in different aspects of male fertility. Our research has implications for improving clinical diagnoses of male infertility and identifying additional targets for nonhormonal male contraception.}, }
@article {pmid39663809, year = {2024}, author = {Tian, Q and Zhou, H and Zhao, Z and Zhang, Y and Zhao, W and Cai, L and Guo, T}, title = {Single and dual RPA-CRISPR/Cas assays for point-of-need detection of Stewart's wilt pathogen (Pantoea stewartii subsp. stewartii) of corn and Maize dwarf mosaic virus.}, journal = {Pest management science}, volume = {}, number = {}, pages = {}, doi = {10.1002/ps.8597}, pmid = {39663809}, issn = {1526-4998}, support = {//Hainan Province Key Research and Development Program grant (ZDYF2024XDNY227)/ ; //the Chinese Academy of Inspection and Quarantine Basic Research program grant (2023JK020)/ ; }, abstract = {BACKGROUND: Pantoea stewartii subsp. stewartii and Maize dwarf mosaic virus (MDMV) infections severely affect corn productivity worldwide. Rapid point-of-need diagnoses of quarantine pathogens P. stewartii subsp. stewartii and MDMV are required for early detection, timely disease management and ensuring phytosanitary regulations. Recombinase polymerase amplification (RPA) is an isothermal technique suitable for rapid diagnostics using minimally processed samples. Integrating CRISPR/Cas collateral activities with the RPA assays further enhances the specificity and sensitivity of molecular toolkits for diagnostic assays.<br><br>RESULTS: RPA-CRISPR/Cas12a assay targeting the intergenic spacer region between capsular polysaccharide genes cpsA and cpsB of P. stewartii subsp. stewartii detected 1&#x2009;&#xd7;&#x2009;10[-6]&#x2009;ng DNA &#x3bc;L[-1] using real-time fluorescence and blue light observation methods, and 1&#x2009;&#xd7;&#x2009;10[-4]&#x2009;ng DNA &#x3bc;L[-1] using the lateral flow dipstick (LFD). Likewise, RPA-CRISPR/Cas13a assay detected MDMV coat protein (CP) gene with an ultrasensitive detection limit of 3.69&#x2009;&#xd7;&#x2009;10[-7]&#x2009;ng&#x2009;&#x3bc;L[-1] using the real-time fluorescence and blue light observation methods, and 3.69&#x2009;&#xd7;&#x2009;10[-5]&#x2009;ng&#x2009;&#x3bc;L[-1] using the LFD. The dual RPA-CRISPR/Cas assays detected both pathogens without compromising the speed and/or detection sensitivity of the single assays.<br><br>CONCLUSION: The validated assays provide a useful and sensitive molecular tool for detecting two quarantine pathogens of maize within a minimal resource framework suitable for fast-tracking the containment strategies. &#xa9; 2024 The Author(s). Pest Management Science published by John Wiley &amp; Sons Ltd on behalf of Society of Chemical Industry.}, }
@article {pmid39662170, year = {2025}, author = {Wang, M and Chen, W and Li, M and Lin, F and Zhong, J and Ouyang, W and Cai, C and Zeng, G and Liu, H}, title = {TE-RPA: One-tube telomerase extension recombinase polymerase amplification-based electrochemical biosensor for precise diagnosis of urothelial carcinoma.}, journal = {Biosensors &amp; bioelectronics}, volume = {271}, number = {}, pages = {117042}, doi = {10.1016/j.bios.2024.117042}, pmid = {39662170}, issn = {1873-4235}, mesh = {*Telomerase/genetics/urine ; *Biosensing Techniques/methods ; Humans ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; *Electrochemical Techniques/methods ; *Recombinases/chemistry/metabolism ; Limit of Detection ; CRISPR-Cas Systems ; Urinary Bladder Neoplasms/urine/diagnosis/genetics ; Biomarkers, Tumor/urine/genetics ; Urologic Neoplasms/diagnosis/genetics/urine ; }, abstract = {Telomerase demonstrates potential as a non-invasive urinary biomarker for urothelial carcinoma (UC); however, current detection methods are either labor-intensive or exhibit suboptimal performance. There is a need for alternative approaches to enable rapid and early diagnosis of UC. In this study, we propose TE-RPA, which combines telomerase extension (TE) with recombinase polymerase amplification (RPA) for one-tube isothermal amplification. The GC content and length of the telomerase substrate were first considered during the screening process. TE-RPA exponential amplification was initiated by the addition of MgOAc along with a forward primer derived from the products of telomerase-mediated extension and a corresponding reverse primer. The amplification product from TE-RPA was subsequently detected using CRISPR-Cas12a system for trans-cleavage of signal probes on the surface of screen-printed electrode in an electrochemical biosensor, resulting in a current change that reflects the corresponding concentration of telomerase. The TE-RPA/CRISPR-Cas12a/electrochemical sensing platform achieves a limit of detection (LOD) for telomerase activity as low as a single-cell level. In addition, the platform attained an area under the curve (AUC) value of 0.9589 in a clinical evaluation involving urine samples from 43 suspected UC patients. Overall, our proposed platform not only offers an efficient method for telomerase isothermal amplification but also provides a portable and precise diagnostic tool for UC.}, }
@article {pmid39661519, year = {2024}, author = {Pan, Q and Zhang, Z and Xiong, Y and Bao, Y and Chen, T and Xu, P and Liu, Z and Ma, H and Yu, Y and Zhou, Z and Wei, W}, title = {Mapping functional elements of the DNA damage response through base editor screens.}, journal = {Cell reports}, volume = {43}, number = {12}, pages = {115047}, doi = {10.1016/j.celrep.2024.115047}, pmid = {39661519}, issn = {2211-1247}, mesh = {*DNA Damage ; Humans ; *DNA Repair ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Protein Serine-Threonine Kinases/metabolism/genetics ; Cisplatin/pharmacology ; Mutation/genetics ; Replication Protein A/metabolism/genetics ; HEK293 Cells ; }, abstract = {Maintaining genomic stability is vital for cellular equilibrium. In this study, we combined CRISPR-mediated base editing with pooled screening technologies to identify numerous mutations in lysine residues and protein-coding genes. The loss of these lysine residues and genes resulted in either sensitivity or resistance to DNA-damaging agents. Among the identified variants, we characterized both loss-of-function and gain-of-function mutations in response to DNA damage. Notably, we discovered that the K494 mutation of C17orf53 disrupts its interaction with RPA proteins, leading to increased sensitivity to cisplatin. Additionally, our analysis identified STK35 as a previously unrecognized gene involved in DNA damage response (DDR) pathways, suggesting that it may play a critical role in DNA repair. We believe that this resource will offer valuable insights into the broader functions of DNA damage response genes and accelerate research on variants relevant to cancer therapy.}, }
@article {pmid39661507, year = {2024}, author = {Ordóñez, A and Ron, D and Harding, HP}, title = {Protocol for iterative enrichment of integrated sgRNAs via derivative CRISPR-Cas9 libraries from genomic DNA of sorted fixed cells.}, journal = {STAR protocols}, volume = {5}, number = {4}, pages = {103493}, pmid = {39661507}, issn = {2666-1667}, mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; DNA/genetics ; Gene Library ; Lentivirus/genetics ; Gene Editing/methods ; }, abstract = {Here, we present a protocol for iterative enrichment of integrated single guide RNA (sgRNA) via derivative CRISPR-Cas9 from genomic DNA (gDNA) of phenotypically sorted fixed cells. We describe steps for high-scale lentiviral production, genome-wide screening, extracting gDNA from fixed cells, cloning of integrated sgRNAs, and high-scale transformation. This protocol introduces three key advantages: (1) applicability to fixed cells, (2) bypassing epigenetic drift, and (3) pause points lowering the contamination risk. We believe this approach will benefit researchers applying somatic cell genetics in cell biology. For complete details on the use and execution of this protocol, please refer to Ordoñez et al.[1].}, }
@article {pmid39660762, year = {2024}, author = {Zeng, T and Wu, Q and Liu, Y and Qi, Q and Shen, W and Gu, W and Zhang, Y and Xiong, W and Xie, Z and Qi, X and Tian, T and Zhou, X}, title = {Unraveling the Cleavage Reaction of Hydroxylamines with Cyclopropenones Considering Biocompatibility.}, journal = {Journal of the American Chemical Society}, volume = {146}, number = {51}, pages = {35077-35089}, doi = {10.1021/jacs.4c09757}, pmid = {39660762}, issn = {1520-5126}, mesh = {*Cyclopropanes/chemistry/pharmacology ; Humans ; HeLa Cells ; *Hydroxylamines/chemistry ; Biocompatible Materials/chemistry/pharmacology/chemical synthesis ; Density Functional Theory ; Molecular Structure ; CRISPR-Cas Systems ; Prodrugs/chemistry/pharmacology/chemical synthesis ; }, abstract = {We develop a latent biocompatible cleavage reaction involving the hitherto unexplored interaction between hydroxylamines and cyclopropenones. Our study addresses the regioselectivity challenges commonly observed in asymmetric cyclopropenone transformations, substantiated by variations in substrate, Density Functional Theory calculations, and in situ NMR analysis. This reaction is characterized by high efficiency, broad substrate scope, stability, latent biocompatibility, and mild reaction conditions. Significantly, it facilitates fluorescence activation and functions as a controlled release mechanism for prodrugs, showing great promise in biological assays. Our success in achieving the controlled release of nitrogen mustard in HeLa cells underscores its potential application in cellular contexts. Additionally, we introduce a simple and highly efficient method for synthesizing α, β-substituted pentenolides, applicable to a variety of substrates. Moreover, we extend this cleavage reaction to the CRISPR-Cas9 system, achieving precise, on-demand regulation of guide RNA activity. The introduction of this cleavage reaction offers a promising tool for biochemical research and biotechnological applications.}, }
@article {pmid39658572, year = {2024}, author = {Adachi, Y and Terakura, S and Osaki, M and Okuno, Y and Sato, Y and Sagou, K and Takeuchi, Y and Yokota, H and Imai, K and Steinberger, P and Leitner, J and Hanajiri, R and Murata, M and Kiyoi, H}, title = {Cullin-5 deficiency promotes chimeric antigen receptor T cell effector functions potentially via the modulation of JAK/STAT signaling pathway.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10376}, pmid = {39658572}, issn = {2041-1723}, mesh = {*Cullin Proteins/metabolism/genetics ; Humans ; Animals ; *Receptors, Chimeric Antigen/metabolism/immunology ; *Signal Transduction ; Mice ; *T-Lymphocytes/immunology/metabolism ; Antigens, CD19/metabolism/immunology ; Immunotherapy, Adoptive/methods ; Phosphorylation ; Cell Line, Tumor ; Cell Proliferation ; STAT3 Transcription Factor/metabolism ; CRISPR-Cas Systems ; Janus Kinases/metabolism ; STAT5 Transcription Factor/metabolism/genetics ; Xenograft Model Antitumor Assays ; STAT Transcription Factors/metabolism ; }, abstract = {Chimeric antigen receptor (CAR) T cell is a promising therapy for cancer, but factors that enhance the efficacy of CAR T cell remain elusive. Here we perform a genome-wide CRISPR screening to probe genes that regulate the proliferation and survival of CAR T cells following repetitive antigen stimulations. We find that genetic ablation of CUL5, encoding a core element of the multi-protein E3 ubiquitin-protein ligase complex, cullin-RING ligase 5, enhances human CD19 CAR T cell expansion potential and effector functions, potentially via the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway. In this regard, CUL5 knockout CD19 CAR T cells show sustained STAT3 and STAT5 phosphorylation, as well as delayed phosphorylation and degradation of JAK1 and JAK3. In vivo, shRNA-mediated knockdown of CUL5 enhances CD19 CAR T treatment outcomes in tumor-bearing mice. Our findings thus imply that targeting CUL5 in the ubiquitin system may enhance CAR T cell effector functions to enhance immunotherapy efficacy.}, }
@article {pmid39658506, year = {2024}, author = {Shen, H and Weng, Z and Zhao, H and Song, H and Wang, F and Fan, C and Song, P}, title = {Random Sanitization in DNA Information Storage Using CRISPR-Cas12a.}, journal = {Journal of the American Chemical Society}, volume = {146}, number = {51}, pages = {35155-35164}, doi = {10.1021/jacs.4c11380}, pmid = {39658506}, issn = {1520-5126}, mesh = {*CRISPR-Cas Systems/genetics ; DNA/chemistry ; Information Storage and Retrieval ; DNA, Single-Stranded/chemistry ; Thermodynamics ; }, abstract = {DNA information storage provides an excellent solution for metadata storage due to its high density, programmability, and long-term stability. However, current research primarily focuses on the processes of storing and reading data, lacking comprehensive solutions for secure metadata wiping. Herein, we present a method of random sanitization in DNA information storage using CRISPR-Cas12a (RSDISC) based on precise control of the thermodynamic energy of primer-template hybridization. We utilize the collateral cleavage (trans-activity) of single-stranded DNA (ssDNA) by CRISPR-Cas12a to achieve selective sanitization of files in metadata. This method enables ssDNA degradation with different GC contents, lengths, and secondary structures to achieve a sanitization efficiency up to 99.9% for 28,258 oligonucleotides in DNA storage within one round. We demonstrate that the number of erasable files could reach 10[12] based on a model of primer-template hybridization efficiency. Overall, RSDISC provides a random sanitization approach to set the foundation of information encryption, file classification, memory deallocation, and accurate reading in DNA storage.}, }
@article {pmid39658085, year = {2024}, author = {Hak, H and Ostendorp, S and Reza, A and Ishgur Greenberg, S and Pines, G and Kehr, J and Spiegelman, Z}, title = {Rapid on-site detection of crop RNA viruses using CRISPR/Cas13a.}, journal = {Journal of experimental botany}, volume = {}, number = {}, pages = {}, doi = {10.1093/jxb/erae495}, pmid = {39658085}, issn = {1460-2431}, abstract = {Plant viruses are destructive pathogens for various crop species. Rapid, sensitive, and specific detection is crucial for the effective containment of emerging and resistance-breaking viruses. CRISPR/Cas has been established as a new tool for plant virus identification. However, its application for direct detection of viruses in the field is still limited. In this study, we present a CRISPR/Cas13a-based method for rapid detection of different viruses directly from RNA of several crop species, including tomato, cucumber and rapeseed. This method was used to identify the emerging tomato brown rugose fruit virus (ToBRFV), a prominent pathogen in tomato cultivation, and distinguish it from closely related viruses in infected tomato plants. ToBRFV could be identified in a 100-fold dilution and early during infection, prior to the onset of viral symptoms. Finally, we developed a user-friendly, extraction-free, 15-minute protocol for on-site virus detection using a portable fluorescent viewer and a mobile phone camera. This protocol was successfully applied for ToBRFV identification in several commercial greenhouses. These results demonstrate that CRISPR/Cas13a is a robust technology for on-site detection of multiple viruses in different crop plants. This method could be swiftly adapted to identify newly emerging pests, which threaten global food security.}, }
@article {pmid39657918, year = {2024}, author = {Li, X and Zhang, S and Wang, C and Ren, B and Yan, F and Li, S and Spetz, C and Huang, J and Zhou, X and Zhou, H}, title = {Efficient in situ epitope tagging of rice genes by nuclease-mediated prime editing.}, journal = {The Plant cell}, volume = {}, number = {}, pages = {}, doi = {10.1093/plcell/koae316}, pmid = {39657918}, issn = {1532-298X}, abstract = {In situ epitope tagging is crucial for probing gene expression, protein localization, and the dynamics of protein interactions within their natural cellular context. However, the practical application of this technique in plants presents considerable hurdles. Here, we comprehensively explored the potential of the CRISPR/Cas nuclease-mediated prime editing and different DNA repair pathways in epitope tagging of endogenous rice (Oryza sativa) genes. We found that a SpCas9 nuclease/microhomology-mediated end joining (MMEJ)-based prime editing (PE) strategy (termed NM-PE) facilitates more straightforward and efficient gene tagging compared to the conventional and other derivative PE methods. Furthermore, the PAM-flexible SpRY and ScCas9 nucleases-based prime editors have been engineered and implemented for the tagging of endogenous genes with diverse epitopes, significantly broadening the applicability of NM-PE in rice. Moreover, NM-PE has been successfully adopted in simultaneous tagging of the MAP kinase (MPK) genes OsMPK1 and OsMPK13 in rice plants with c-Myc and HA tags, respectively. Taken together, our results indicate great potential of the NM-PE toolkit in the targeted gene tagging for Rice Protein Tagging Project, gene function study and genetic improvement.}, }
@article {pmid39657724, year = {2024}, author = {Rasoul, A and Johnston, C and LaChance, J and Sedbrook, JC and Alonso, AP}, title = {Propelling sustainable energy: Multi-omics analysis of pennycress FATTY ACID ELONGATION1 knockout for biofuel production.}, journal = {Plant physiology}, volume = {}, number = {}, pages = {}, doi = {10.1093/plphys/kiae650}, pmid = {39657724}, issn = {1532-2548}, abstract = {The aviation industry's growing interest in renewable jet fuel has encouraged the exploration of alternative oilseed crops. Replacing traditional fossil fuels with a sustainable, domestically sourced crop can substantially reduce carbon emissions, thus mitigating global climate instability. Pennycress (Thlaspi arvense L.) is an emerging oilseed intermediate crop that can be grown during the offseason between maize (Zea mays) and soybean (Glycine max) to produce renewable biofuel. Pennycress is being domesticated through breeding and mutagenesis, providing opportunities for trait enhancement. Here, we employed metabolic engineering strategies to improve seed oil composition and bolster the plant's economic competitiveness. FATTY ACID ELONGATION1 (FAE1) was targeted using CRISPR-Cas 9 gene editing to eliminate very long chain fatty acids (VLCFAs) from pennycress seed oil, thereby enhancing its cold flow properties. Through an integrated multi-omics approach, we investigated the impact of eliminating VLCFAs in developing and mature plant embryos. Our findings revealed improved cold-germination efficiency in fae1, with seedling emergence occurring up to three days earlier at 10 °C. However, these alterations led to a trade-off between storage oil content and composition. Additionally, these shifts in lipid biosynthesis were accompanied by broad metabolic changes, such as the accumulation of glucose and ADP-glucose quantities consistent with increased starch production. Furthermore, shifts to shorter FA chains triggered the upregulation of heat shock proteins, underscoring the importance of VLCFAs in stress signaling pathways. Overall, this research provides crucial insights for optimizing pennycress seed oil while preserving essential traits for biofuel applications.}, }
@article {pmid39656015, year = {2025}, author = {Hernandez-Rodriguez, Y and Bullard, AM and Busch, RJ and Marshall, A and Vargas-Muñiz, JM}, title = {Strategies for genetic manipulation of the halotolerant black yeast Hortaea werneckii: ectopic DNA integration and marker-free CRISPR/Cas9 transformation.}, journal = {Microbiology spectrum}, volume = {13}, number = {1}, pages = {e0243024}, pmid = {39656015}, issn = {2165-0497}, mesh = {*CRISPR-Cas Systems ; *Transformation, Genetic ; *Gene Editing/methods ; Ascomycota/genetics/metabolism ; DNA, Fungal/genetics ; }, abstract = {Hortaea werneckii is a halotolerant black yeast commonly found in hypersaline environments. This yeast is also the causative agent of tinea nigra, a superficial mycosis of the palm of the hand and soles of the feet of humans. In addition to their remarkable halotolerance, this black yeast exhibits an unconventional cell division cycle, alternating between fission and budding cell division. Cell density and the salt concentration in their environment regulate which cell division cycle H. werneckii uses. Although H. werneckii have been extensively studied due to their unique physiology and cell biology, deciphering the underlying mechanisms behind these remarkable phenotypes has been limited due to the lack of genetic tools available. Here, we report a new ectopic integration protocol for H. werneckii using polyethylene glycol-CaCl2 mediated protoplast transformation. This approach relies on a drug (hygromycin B) resistance gene to select for successful integration of the genetic construct. The same construct was used to express cytosolic green fluorescent protein. Finally, we developed a marker-free CRISPR/Cas9 protocol for targeted gene deletion using the melanin synthesis pathway as a visual reporter of successful transformation. These transformation strategies will allow testing hypotheses related to H. werneckii cell biology and physiology.IMPORTANCEHortaea werneckii is a remarkable yeast capable of growing in high salt concentration, and its cell division cycle alternates between fission-like and budding. For these unique attributes, H. werneckii has gathered interest in research programs studying extremophile fungi and cell division. Most of our understanding of H. werneckii biology comes from genomic analyses, the usage of drugs to target a particular pathway, or the heterologous expression of its genes in S. cerevisiae. Nonetheless, H. werneckii has remained genetically intractable. Here, we report on two strategies to transform H. werneckii: ectopic integration of a plasmid and gene deletion using CRISPR/Cas9. These approaches will be fundamental to expanding the experimental techniques available to study H. werneckii, including live-cell imaging of cellular processes and reverse genetic approaches.}, }
@article {pmid39655997, year = {2024}, author = {Li, M and Zheng, T and Zhu, J and Zhang, H and Fan, L}, title = {Cas12a/crRNA recognition initiated self-priming mediated chain extension for colorimetric cell-free DNA (cfDNA) analysis.}, journal = {The Analyst}, volume = {}, number = {}, pages = {}, doi = {10.1039/d4an01432d}, pmid = {39655997}, issn = {1364-5528}, abstract = {Cell-free DNA (cfDNA) has attracted increasing attention as a promising biomarker in liquid biopsy due to its crucial role in disease diagnosis. However, previous cfDNA detection methods are commonly based on the development of target-specific primers and integrated signal amplification strategies, which may induce false-positive results. This paper presents a sensitive yet accurate method for cfDNA detection that combines phosphorothioated-terminal hairpin creation with a self-priming extension process. This approach initiates a self-priming mediated chain extension-based signal cycle following the trans-cleavage of H0@MBs when the CRISPR-Cas12a complex is activated by target cfDNA, resulting in the production of a substantial quantity of pyrophosphate. A pyrophosphate sensing probe (pp probe) was utilized, facilitating both high-efficiency and stable colorimetric signaling. This innovative technique for colorimetric detection of target cfDNA demonstrated exceptional sensitivity with a low limit of detection of 1.04 fM and greatly enhanced selectivity, with the complete detection process taking around 60 min. In addition, this technique is capable of detecting cfDNA from the culture medium of HEK293 cells, indicating its clinical application potential. Compared with the previous CRISPR-Cas system-based cfDNA method that necessitates an amplification step before detection, Cas12a was directly used to identify a target sequence that can avoid false target amplification. This technique is simple, accurate, and rapid, engineered to identify cancer-associated cfDNA via a highly sensitive colorimetric change, which is expected to be beneficial for applications requiring point-of-care cancer detection.}, }
@article {pmid39655603, year = {2024}, author = {Wu, S and Yang, Y and Lian, X and Zhang, F and Hu, C and Tsien, J and Chen, Z and Sun, Y and Vaidya, A and Kim, M and Sung, YC and Xiao, Y and Bian, X and Wang, X and Tian, Z and Guerrero, E and Robinson, J and Basak, P and Qin, T and Siegwart, DJ}, title = {Isosteric 3D Bicyclo[1.1.1]Pentane (BCP) Core-Based Lipids for mRNA Delivery and CRISPR/Cas Gene Editing.}, journal = {Journal of the American Chemical Society}, volume = {146}, number = {50}, pages = {34733-34742}, pmid = {39655603}, issn = {1520-5126}, support = {P30 CA142543/CA/NCI NIH HHS/United States ; R01 CA269787/CA/NCI NIH HHS/United States ; R01 EB025192/EB/NIBIB NIH HHS/United States ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *RNA, Messenger/chemistry/genetics ; *Lipids/chemistry ; Animals ; Bridged Bicyclo Compounds/chemistry ; Mice ; Nanoparticles/chemistry ; Humans ; }, abstract = {Lipid nanoparticles (LNPs) are an essential component of messenger RNA (mRNA) vaccines and genome editing therapeutics. Ionizable amino lipids, which play the most crucial role in enabling mRNA to overcome delivery barriers, have, to date, been restricted to two-dimensional (2D) architectures. Inspired by improved physicochemical properties resulting from the incorporation of three-dimensionality (3D) into small-molecule drugs, we report the creation of 3D ionizable lipid designs through the introduction of bicyclo[1.1.1]pentane (BCP) core motifs. BCP-based lipids enabled efficient in vivo mRNA delivery to the liver and spleen with significantly greater performance over 2D benzene- and cyclohexane-based analogues. Notably, lead BCP-NC2-C12 LNPs mediated ∼90% reduction in the PCSK9 serum protein level via CRISPR/Cas9 gene knockout, outperforming 2D controls and clinically used DLin-MC3-DMA LNPs at the same dose. Here, we introduce BCP-based designs with superior in vivo activity, thereby expanding the chemical scope of ionizable amino lipids from 2D to 3D and offering a promising avenue to improve mRNA and gene editing efficiency for the continued development of genetic medicines.}, }
@article {pmid39655408, year = {2024}, author = {Butterfield, SP and Sizer, RE and Saunders, FL and White, RJ}, title = {Selective Recruitment of a Synthetic Histone Acetyltransferase Can Boost CHO Cell Productivity.}, journal = {Biotechnology journal}, volume = {19}, number = {12}, pages = {e202400474}, pmid = {39655408}, issn = {1860-7314}, support = {//Engineering and Physical Sciences Research Council/ ; }, mesh = {CHO Cells ; *Cricetulus ; Animals ; *Histone Acetyltransferases/metabolism/genetics ; *Promoter Regions, Genetic/genetics ; Histones/metabolism/genetics ; Antibodies, Monoclonal/genetics/metabolism ; Acetylation ; CRISPR-Cas Systems/genetics ; Epigenesis, Genetic/genetics ; Transgenes ; Cytomegalovirus/genetics ; Cricetinae ; }, abstract = {Industrial production of biologics typically involves the integration of transgenes into host cell genomes, most often Chinese hamster ovary (CHO) cells. Epigenetic control of transgene expression is a major determinant of production titers. Although the cytomegalovirus (CMV) promoter has long been used to drive industrial transgene expression, we found that its associated histones are suboptimally acetylated in CHO cells, providing an opportunity to enhance productivity through epigenetic manipulation. Expression of monoclonal antibody mRNAs increased up to 12-fold when a CRISPR-dCas9 system delivered the catalytic domain of a histone acetyltransferase to the CMV promoter. This effect was far stronger than when promoter DNA was selectively demethylated using dCas9 fused to a 5-methylcytosine dioxygenase. Mechanistically, acetylation-mediated transcriptional activation involved heightened phosphorylation and activity of RNA polymerase II, enabling it to escape promoter-proximal pausing at the transgene. This approach almost doubled the titer and specific productivity of antibody-producing CHO cells, demonstrating the potential for biomanufacturing.}, }
@article {pmid39655309, year = {2024}, author = {Singh, K and Fronza, R and Evens, H and Chuah, MK and VandenDriessche, T}, title = {Comprehensive analysis of off-target and on-target effects resulting from liver-directed CRISPR-Cas9-mediated gene targeting with AAV vectors.}, journal = {Molecular therapy. Methods &amp; clinical development}, volume = {32}, number = {4}, pages = {101365}, pmid = {39655309}, issn = {2329-0501}, abstract = {Comprehensive genome-wide studies are needed to assess the consequences of adeno-associated virus (AAV) vector-mediated gene editing. We evaluated CRISPR-Cas-mediated on-target and off-target effects and examined the integration of the AAV vectors employed to deliver the CRISPR-Cas components to neonatal mice livers. The guide RNA (gRNA) was specifically designed to target the factor IX gene (F9). On-target and off-target insertions/deletions were examined by whole-genome sequencing (WGS). Efficient F9-targeting (36.45% ± 18.29%) was apparent, whereas off-target events were rare or below the WGS detection limit since only one single putative insertion was detected out of 118 reads, based on >100 computationally predicted off-target sites. AAV integrations were identified by WGS and shearing extension primer tag selection ligation-mediated PCR (S-EPTS/LM-PCR) and occurred preferentially in CRISPR-Cas9-induced double-strand DNA breaks in the F9 locus. In contrast, AAV integrations outside F9 were not in proximity to any of ∼5,000 putative computationally predicted off-target sites (median distance of 70 kb). Moreover, without relying on such off-target prediction algorithms, analysis of DNA sequences close to AAV integrations outside the F9 locus revealed no homology to the F9-specific gRNA. This study supports the use of S-EPTS/LM-PCR for direct in vivo comprehensive, sensitive, and unbiased off-target analysis.}, }
@article {pmid39654402, year = {2024}, author = {Torelli, F and da Fonseca, DM and Butterworth, SW and Young, JC and Treeck, M}, title = {Paracrine rescue of MYR1-deficient Toxoplasma gondii mutants reveals limitations of pooled in vivo CRISPR screens.}, journal = {eLife}, volume = {13}, number = {}, pages = {}, pmid = {39654402}, issn = {2050-084X}, support = {MR/V03314X/1/MRC_/Medical Research Council/United Kingdom ; CC2132/ARC_/Arthritis Research UK/United Kingdom ; 10.35802/223192/WT_/Wellcome Trust/United Kingdom ; TO 1349/1-1//Deutsche Forschungsgemeinschaft/ ; CC2132/WT_/Wellcome Trust/United Kingdom ; CC0199/ARC_/Arthritis Research UK/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; }, mesh = {*Toxoplasma/genetics ; Animals ; Mice ; *Protozoan Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Toxoplasmosis/parasitology/genetics ; Paracrine Communication ; Macrophages/parasitology ; Mice, Inbred C57BL ; Female ; Host-Parasite Interactions/genetics ; Mutation ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Toxoplasma gondii is an intracellular parasite that subverts host cell functions via secreted virulence factors. Up to 70% of parasite-controlled changes in the host transcriptome rely on the MYR1 protein, which is required for the translocation of secreted proteins into the host cell. Mice infected with MYR1 knock-out (KO) strains survive infection, supporting a paramount function of MYR1-dependent secreted proteins in Toxoplasma virulence and proliferation. However, we have previously shown that MYR1 mutants have no growth defect in pooled in vivo CRISPR-Cas9 screens in mice, suggesting that the presence of parasites that are wild-type at the myr1 locus in pooled screens can rescue the phenotype. Here, we demonstrate that MYR1 is not required for the survival in IFN-γ-activated murine macrophages, and that parasites lacking MYR1 are able to expand during the onset of infection. While ΔMYR1 parasites have restricted growth in single-strain murine infections, we show that the phenotype is rescued by co-infection with wild-type (WT) parasites in vivo, independent of host functional adaptive immunity or key pro-inflammatory cytokines. These data show that the major function of MYR1-dependent secreted proteins is not to protect the parasite from clearance within infected cells. Instead, MYR-dependent proteins generate a permissive niche in a paracrine manner, which rescues ΔMYR1 parasites within a pool of CRISPR mutants in mice. Our results highlight an important limitation of otherwise powerful in vivo CRISPR screens and point towards key functions for MYR1-dependent Toxoplasma-host interactions beyond the infected cell.}, }
@article {pmid39654027, year = {2024}, author = {Liang, Y and Xie, SC and Lv, YH and He, YH and Zheng, XN and Cong, W and Elsheikha, HM and Zhu, XQ}, title = {A novel single-tube LAMP-CRISPR/Cas12b method for rapid and visual detection of zoonotic Toxoplasma gondii in the environment.}, journal = {Infectious diseases of poverty}, volume = {13}, number = {1}, pages = {94}, pmid = {39654027}, issn = {2049-9957}, support = {2021YFC2300800//National Key Research and Development Program of China/ ; 2021YFC2300802//National Key Research and Development Program of China/ ; 2021YFC2300804//National Key Research and Development Program of China/ ; LXXMsxnd202101//Shanxi Provincial Agricultural and Rural Research Program/ ; RFSXIHLT202101//the Research Fund of Shanxi Province for Introduced High-level Leading Talents/ ; 2021XG001//the Special Research Fund of Shanxi Agricultural University for High-level Talents/ ; }, mesh = {*Toxoplasma/genetics/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Animals ; Molecular Diagnostic Techniques/methods ; Humans ; Sensitivity and Specificity ; Cats ; Zoonoses/parasitology/diagnosis ; Feces/parasitology ; Toxoplasmosis/diagnosis/parasitology ; }, abstract = {BACKGROUND: Toxoplasma gondii oocysts, excreted in cat feces, pose a significant health risk to humans through contaminated soil and water. Rapid and accurate detection of T. gondii in environmental samples is essential for public health protection.<br><br>METHODS: We developed a novel, single-tube detection method that integrates loop-mediated isothermal amplification (LAMP), the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12b system, and lateral flow immunoassay strips for rapid, visual identification of T. gondii. This method targets the T. gondii B1 gene, initially amplifies it with LAMP, directed by a single-guide RNA (sgRNA). It then recognizes the amplified target gene and activates trans-cleavage, cutting nearby single-stranded DNA (ssDNA) reporters. Fluorescence detection was performed using a 6-Carboxyfluorescein (FAM)-12N-Black Hole Quencher-1 (BHQ1) reporter, while Fluorescein Isothiocyanate (FITC)-12N-Biotin enabled visual detection on lateral flow strips. The method was tested for its ability to detect various T. gondii genotypes and related parasites, assessing its specificity and broad-spectrum applicability. It was further applied to real-world environmental samples to evaluate its practicality.<br><br>RESULTS: The LAMP-CRISPR/Cas12b method exhibited high specificity and broad-spectrum detection capability, successfully identifying nine T. gondii genotypes and distinguishing them from 11 other parasitic species. Sensitivity testing at both molecular (plasmid) and practical (oocyst) levels showed detection limits of 10 &#xa0;copies/&#x3bc;L and 0.1 oocyst, respectively. When applied to 112 environmental samples (soil, water, and cat feces), the method demonstrated 100% sensitivity, accurately reflecting known infection rates.<br><br>CONCLUSIONS: This LAMP-CRISPR/Cas12b single-tube method offers a robust, innovative approach for monitoring zoonotic T. gondii in environmental samples, with significant implications for public health surveillance.}, }
@article {pmid39652422, year = {2025}, author = {Ashkin, EL and Tang, YJ and Xu, H and Hung, KL and Belk, JA and Cai, H and Lopez, SS and Dolcen, DN and Hebert, JD and Li, R and Ruiz, PA and Keal, T and Andrejka, L and Chang, HY and Petrov, DA and Dixon, JR and Xu, Z and Winslow, MM}, title = {A STAG2-PAXIP1/PAGR1 axis suppresses lung tumorigenesis.}, journal = {The Journal of experimental medicine}, volume = {222}, number = {1}, pages = {}, pmid = {39652422}, issn = {1540-9538}, support = {F99CA284289/CA/NCI NIH HHS/United States ; F99 CA274692/CA/NCI NIH HHS/United States ; 28FT-0019//Tobacco-Related Disease Research Program/ ; //Stanford University/ ; K00 CA245784/CA/NCI NIH HHS/United States ; DGE-2146755//National Science Foundation Graduate Research Fellowship Program/ ; MFE-176568//Canadian Institute of Health Research/ ; R01 CA234349/CA/NCI NIH HHS/United States ; F99 CA284289/CA/NCI NIH HHS/United States ; P30 CA124435/CA/NCI NIH HHS/United States ; GT14928/HHMI/Howard Hughes Medical Institute/United States ; R01 CA231253/CA/NCI NIH HHS/United States ; R01-CA231253/NH/NIH HHS/United States ; PF-21-112-01-MM//American Cancer Society/ ; }, mesh = {*Lung Neoplasms/pathology/metabolism/genetics ; Humans ; Animals ; *Cell Cycle Proteins/metabolism/genetics ; Cell Line, Tumor ; Mice ; *Carcinogenesis/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Antigens, Nuclear/metabolism/genetics ; Tumor Suppressor Proteins/metabolism/genetics ; Cohesins ; }, abstract = {The cohesin complex is a critical regulator of gene expression. STAG2 is the most frequently mutated cohesin subunit across several cancer types and is a key tumor suppressor in lung cancer. Here, we coupled somatic CRISPR-Cas9 genome editing and tumor barcoding with an autochthonous oncogenic KRAS-driven lung cancer model and showed that STAG2 is uniquely tumor-suppressive among all core and auxiliary cohesin components. The heterodimeric complex components PAXIP1 and PAGR1 have highly correlated effects with STAG2 in human lung cancer cell lines, are tumor suppressors in vivo, and are epistatic to STAG2 in oncogenic KRAS-driven lung tumorigenesis in vivo. STAG2 inactivation elicits changes in gene expression, chromatin accessibility, and 3D genome conformation that impact the cancer cell state. Gene expression and chromatin accessibility similarities between STAG2- and PAXIP1-deficient neoplastic cells further relate STAG2-cohesin to PAXIP1/PAGR1. These findings reveal a STAG2-PAXIP1/PAGR1 tumor-suppressive axis and uncover novel PAXIP1-dependent and PAXIP1-independent STAG2-cohesin-mediated mechanisms of lung tumor suppression.}, }
@article {pmid39651221, year = {2024}, author = {Yun, S and Chekuri, A and Art, J and Kondabolu, K and Slaugenhaupt, SA and Zeltner, N and Kleinstiver, BP and Morini, E and Alves, CRR}, title = {Engineered CRISPR-Base Editors as a Permanent Treatment for Familial Dysautonomia.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, pmid = {39651221}, issn = {2692-8205}, support = {P01 HL142494/HL/NHLBI NIH HHS/United States ; DP2 CA281401/CA/NCI NIH HHS/United States ; R01 NS095640/NS/NINDS NIH HHS/United States ; K01 NS134784/NS/NINDS NIH HHS/United States ; R01 NS124561/NS/NINDS NIH HHS/United States ; R01 NS125353/NS/NINDS NIH HHS/United States ; }, abstract = {Familial dysautonomia (FD) is a fatal autosomal recessive congenital neuropathy caused by a T-to-C mutation in intron 20 of the Elongator acetyltransferase complex subunit 1 (ELP1) gene, which causes tissue-specific skipping of exon 20 and reduction of ELP1 protein. Here, we developed a base editor (BE) approach to precisely correct this mutation. By optimizing Cas9 variants and screening multiple gRNAs, we identified a combination that was able to promote up to 70% on-target editing in HEK293T cells harboring the ELP1 T-to-C mutation. These editing levels were sufficient to restore exon 20 inclusion in the ELP1 transcript. Moreover, we optimized an engineered dual intein-split system to deliver these constructs in vivo. Mediated by adeno-associated virus (AAV) delivery, this BE strategy effectively corrected the liver and brain ELP1 splicing defects in a humanized FD mouse model carrying the ELP1 T-to-C mutation and rescued the FD phenotype in iPSC-derived sympathetic neurons. Importantly, we observed minimal off-target editing demonstrating high levels of specificity with these optimized base editors. These findings establish a novel and highly precise BE-based therapeutic approach to correct the FD mutation and associated splicing defects and provide the foundation for the development of a transformative, permanent treatment for this devastating disease.}, }
@article {pmid39651213, year = {2024}, author = {Singh, MA and Chang, MM and Wang, Q and Rodgers, C and Lutz, BR and Olanrewaju, AO}, title = {Rapid enzymatic assay for antiretroviral drug monitoring using CRISPR-Cas12a enabled readout.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, doi = {10.1101/2024.11.25.625292}, pmid = {39651213}, issn = {2692-8205}, abstract = {Maintaining efficacy of human immunodeficiency virus (HIV) medications is challenging among children because of dosing difficulties, the limited number of approved drugs, and low rates of medication adherence. Drug level feedback (DLF) can support dose optimization and timely interventions to prevent treatment failure, but current tests are heavily instrumented and centralized. We developed the REverse-transcriptase ACTivity-crispR (REACTR) assay for rapid measurement of HIV drugs based on the extent of DNA synthesis by HIV reverse transcriptase. CRISPR-Cas enzymes bind to synthesized DNA, triggering collateral cleavage of quenched reporters and generating fluorescence. We measured azidothymidine triphosphate (AZT-TP), a key drug in pediatric HIV treatment, and investigated the impact of assay time and DNA template length on REACTR's sensitivity. REACTR selectively measured clinically relevant AZT-TP concentrations in the presence of genomic DNA and peripheral blood mononuclear cell lysate. REACTR has the potential to enable rapid point-of-care HIV DLF to improve pediatric HIV care.}, }
@article {pmid39651158, year = {2024}, author = {Kiernan, KA and Taylor, DW}, title = {Visualization of a multi-turnover Cas9 after product release.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, doi = {10.1101/2024.11.25.625307}, pmid = {39651158}, issn = {2692-8205}, abstract = {While the most widely used CRISPR-Cas enzyme is the S. pyogenes Cas9 endonuclease (Cas9), it exhibits single-turnover enzyme kinetics which leads to long residence times on product DNA. This blocks access to DNA repair machinery and acts as a major bottleneck during CRISPR-Cas9 gene editing. Although Cas9 can eventually be forcibly removed by extrinsic factors (translocating polymerases, helicases, chromatin modifying complexes, etc), the mechanisms contributing to Cas9 dissociation following cleavage remain poorly understood. Interestingly, it's been shown that Cas9 can be more easily dislodged when complexes collide with the PAM-distal region of the Cas9 complex or when the strength of Cas9 interactions in this region are weakened. Here, we employ truncated guide RNAs as a strategy to weaken PAM-distal nucleic acid interactions and still support Cas9 activity. We find that guide truncation promotes much faster Cas9 turnover and used this to capture previously uncharacterized Cas9 reaction states. Kinetics-guided cryo-EM enabled us to enrich for rare, transient states that are often difficult to capture in standard workflows. From a single dataset, we examine the entire conformational landscape of a multi-turnover Cas9, including the first detailed snapshots of Cas9 dissociating from product DNA. We discovered that while the PAM-distal product dissociates from Cas9 following cleavage, tight binding of the PAM-proximal product directly inhibits re-binding of new targets. Our work provides direct evidence as to why Cas9 acts as a single-turnover enzyme and will guide future Cas9 engineering efforts.}, }
@article {pmid39650142, year = {2024}, author = {Xiang, H and Chen, B and Wang, S and Zeng, W and Jiang, J and Kong, W and Huang, H and Mi, Q and Ni, S and Gao, Q and Li, Z}, title = {Development of an RNA virus vector for non-transgenic genome editing in tobacco and generation of berberine bridge enzyme-like mutants with reduced nicotine content.}, journal = {aBIOTECH}, volume = {5}, number = {4}, pages = {449-464}, pmid = {39650142}, issn = {2662-1738}, abstract = {UNLABELLED: Tobacco (Nicotiana tabacum) plants synthesize the psychoactive pyridine alkaloid nicotine, which has sparked growing interest in reducing nicotine levels through genome editing aiming at inactivating key biosynthetic genes. Although stable transformation-mediated genome editing is effective in tobacco, its polyploid nature complicates the complete knockout of genes and the segregation of transgenes from edited plants. In this study, we developed a non-transgenic genome editing method in tobacco by delivering the CRISPR/Cas machinery via an engineered negative-strand RNA rhabdovirus vector, followed by the regeneration of mutant plants through tissue culture. Using this method, we targeted six berberine bridge enzyme-like protein (BBL) family genes for mutagenesis, which are implicated in the last steps of pyridine alkaloid biosynthesis, in the commercial tobacco cultivar Hongda. We generated a panel of 16 mutant lines that were homozygous for mutations in various combinations of BBL genes. Alkaloid profiling revealed that lines homozygous for BBLa and BBLb mutations exhibited drastically reduced nicotine levels, while other BBL members played a minor role in nicotine synthesis. The decline of nicotine content in these lines was accompanied by reductions in anatabine and cotinine levels but increases in nornicotine and its derivative myosmine. Preliminary agronomic evaluation identified two low-nicotine lines with growth phenotypes comparable to those of wild-type plants under greenhouse and field conditions. Our work provides potentially valuable genetic materials for breeding low-nicotine tobacco and enhances our understanding of alkaloid biosynthesis.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42994-024-00188-y.}, }
@article {pmid39650138, year = {2024}, author = {Zou, J and Li, Y and Wang, K and Wang, C and Zhuo, R}, title = {Prime editing enables precise genome modification of a Populus hybrid.}, journal = {aBIOTECH}, volume = {5}, number = {4}, pages = {497-501}, pmid = {39650138}, issn = {2662-1738}, abstract = {UNLABELLED: CRISPR/Cas-based genome editing has been extensively employed in the breeding and genetic improvement of trees, yet precise editing remains challenging in these species. Prime editing (PE), a revolutionary technology for precise editing, allows for arbitrary base substitutions and the insertion/deletion of small fragments. In this study, we focused on the model tree poplar 84K (Populus alba × P. glandulosa). We used the 2 × 35S promoter to express a fusion protein of spCas9 nickase (nCas9) and engineered Moloney murine leukemia virus (MMLV), and the Arabidopsis thaliana AtU6 promoter to express an engineered PE guide RNA (epegRNA) and Nick gRNA, pioneering the establishment of the Prime Editor 3 (PE3) system in dicot poplar. Single-base substitutions, multiple-base substitutions, and small-fragment insertions/deletions were edited into three endogenous target genes. The desired edits were identified in hygromycin-resistant (transformed) calli at seven out of nine target sites, with an average editing efficiency ranging from 0.1 to 3.6%. Furthermore, stable T0 plants contained the desired edits at four out of nine targets, with editing efficiencies ranging from 3.6 to 22.2%. Establishment of the PE3 system provides a powerful tool for the precise modification of the poplar genome.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42994-024-00177-1.}, }
@article {pmid39648412, year = {2025}, author = {Wang, J and Yang, R and Wang, F and Zhang, J and Dong, Y and Wang, J and Yu, M and Xu, Y and Liu, L and Cheng, Y and Zhang, C and Yang, Y and Yang, W and Wang, J and Chen, G and Huang, Y and Tian, Y and Jian, R and Ni, B and Wu, W and Ruan, Y}, title = {CRISPR-Cas9 screening identifies the role of FER as a tumor suppressor.}, journal = {The Journal of pathology}, volume = {265}, number = {2}, pages = {158-171}, doi = {10.1002/path.6374}, pmid = {39648412}, issn = {1096-9896}, support = {2023NSCQZDX0035//Natural Science Foundation of Chongqing Municipality/ ; 82273118//National Natural Science Foundation of China/ ; CX2019JS202//New Technology Cultivation Project of Military Medicine/ ; }, mesh = {*CRISPR-Cas Systems ; Humans ; Animals ; *Genes, Tumor Suppressor ; Mice ; Cell Transformation, Neoplastic/genetics/pathology/metabolism ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; }, abstract = {It is important to systematically identify tumor suppressor genes (TSGs) to improve our understanding of tumorigenesis and develop strategies for early diagnosis and mitigating disease progression. In the present study, we used an in vivo genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) screen and identified FPS/FES-related (FER) as a TSG. Single-cell RNA sequencing (scRNA-seq) revealed that normal cells with low FER expression exhibited elevated malignant transformation potential and stemness properties. FER knockout promoted the tumorigenic transformation, characterized by high colony-forming efficiency and suspension growth ability, acquired tumorigenicity in vivo, increased metabolic activity, dedifferentiation properties, and immune evasion. Moreover, analysis revealed that low FER expression tumors share molecular phenotypes with FER knockout cells, suggesting the consistent role of FER in tumor initiation and progression. Taken together, our findings not only provide insights into the essential role of FER as a tumor suppressor in tumor initiation and progression but also highlight its potential as a target for future clinical diagnosis. © 2024 The Pathological Society of Great Britain and Ireland.}, }
@article {pmid39647408, year = {2025}, author = {Zheng, L and Fan, B and Fu, Y and Wei, J and Ye, Y and Gui, Y and Zhang, S and Wei, Y and Yin, J and Li, J and Jin, M and Pang, B}, title = {Single-tube detection of a foodborne bacterial pathogen using user-friendly portable device.}, journal = {Biosensors &amp; bioelectronics}, volume = {271}, number = {}, pages = {117035}, doi = {10.1016/j.bios.2024.117035}, pmid = {39647408}, issn = {1873-4235}, mesh = {*Biosensing Techniques/instrumentation ; *Salmonella enteritidis/isolation &amp; purification/pathogenicity/genetics ; *Food Microbiology/instrumentation ; Humans ; Equipment Design ; Nucleic Acid Amplification Techniques/instrumentation ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Foodborne Diseases/microbiology ; }, abstract = {Timely and reliable detection of foodborne bacterial pathogen is crucial for reducing disease burden in low- and middle-income countries. However, laboratory-based methods are often inaccessibility in resource-limited settings. Here, we developed a single-tube assay and a low-cost palm-sized device for on-site detection of the representative foodborne bacterial pathogen, Salmonella Enteritidis. Our assay incorporates the advantages of protein-nucleic acid signal transduction, EXPonential Amplification Reaction (EXPAR), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 12a (Cas12a). After systematically investigating the compatibility of these components, we developed a "three-in-one" integration reaction, termed ST-EXPAR-CRISPR assay. This assay requires only one tube, one controlled temperature (39 °C) and simple operation, eliminating the need for bacterial isolation, nucleic acid extraction, or washing steps. ST-EXPAR-CRISPR assay is capable of detecting as few as 37 CFU/mL of target bacterium. Using our kit and portable device, untrained volunteers successfully detected contamination in food samples outdoors. The simplicity of the detection process and minimal hardware requirements make our assay highly promising for application in point-of-care and on-site scenarios. Moreover, the ST-EXPAR-CRISPR assay can be easily modified to detect other targets by changing the nucleic acid sequence with low research and development cost, potentially reducing the global disease burden.}, }
@article {pmid39644903, year = {2024}, author = {Kohabir, KAV and Linthorst, J and Nooi, LO and Brouwer, R and Wolthuis, RMF and Sistermans, EA}, title = {Synthetic mismatches enable specific CRISPR-Cas12a-based detection of genome-wide SNVs tracked by ARTEMIS.}, journal = {Cell reports methods}, volume = {4}, number = {12}, pages = {100912}, pmid = {39644903}, issn = {2667-2375}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Proto-Oncogene Proteins B-raf/genetics ; Aldehyde Dehydrogenase, Mitochondrial/genetics ; Genome, Human/genetics ; Polymorphism, Single Nucleotide/genetics ; Endodeoxyribonucleases/genetics/metabolism ; CRISPR-Associated Proteins/genetics/metabolism ; BRCA2 Protein/genetics ; Neoplasms/genetics/diagnosis ; Tumor Suppressor Protein p53/genetics ; Base Pair Mismatch ; Bacterial Proteins ; }, abstract = {Detection of pathogenic DNA variants is vital in cancer diagnostics and treatment monitoring. While CRISPR-based diagnostics (CRISPRdx) offer promising avenues for cost-effective, rapid, and point-of-care testing, achieving single-nucleotide detection fidelity remains challenging. We present an in silico pipeline that scans the human genome for targeting pathogenic mutations in the seed region (ARTEMIS), the most stringent crRNA domain. ARTEMIS identified 12% of pathogenic SNVs as Cas12a recognizable, including 928 cancer-associated variants such as BRAF[V600E], BRCA2[E1953∗], TP53[V272M], and ALDH2[E504K]. Cas12a exhibited remarkable tolerance to single mismatches within the seed region. Introducing deliberate synthetic mismatches within the seed region yielded on-target activity with single-nucleotide fidelity. Both positioning and nucleobase types of mismatches influenced detection accuracy. With improved specificity, Cas12a could accurately detect and semi-quantify BRAF[V600E] in cfDNA from cell lines and patient liquid biopsies. These results provide insights toward rationalized crRNA design for high-fidelity CRISPRdx, supporting personalized and cost-efficient healthcare solutions in oncologic diagnostics.}, }
@article {pmid39644528, year = {2025}, author = {Guo, H and Ma, P and Dong, X and Qin, M and Zhang, Y and Wang, Z}, title = {CRISPR/Cas12a-mediated fluorescent aptasensor based on DNA walker amplification for oxytetracycline detection.}, journal = {Biosensors &amp; bioelectronics}, volume = {271}, number = {}, pages = {117031}, doi = {10.1016/j.bios.2024.117031}, pmid = {39644528}, issn = {1873-4235}, mesh = {*Oxytetracycline/analysis ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry ; *Milk/chemistry ; Animals ; *CRISPR-Cas Systems ; *Honey/analysis ; Limit of Detection ; Food Contamination/analysis ; Spectrometry, Fluorescence/methods ; Anti-Bacterial Agents/analysis ; Nucleic Acid Amplification Techniques/methods ; Fluorescent Dyes/chemistry ; DNA/chemistry/genetics ; }, abstract = {Nowadays, although most oxytetracycline (OTC) are used only in animals, their leaching into the environment and residues in food lead to noticeable health problems. Fortunately, by studying the molecular dynamics of OTC aptamers and the mutations of key bases, aptamer's base fragments involved in identifying OTC was confirmed for the first time, which provided theoretical guidance for the ingenious design of aptamer detection. Thereafter, benefiting from the amplification impact by DNA walker, the disadvantage of low efficiency caused by CRISPR/Cas12a was effectively avoided. In the established delicate aptasensing platform, small quantity of OTC promoted DNA walker to produce a massive of activators, which stimulated the trans-cleavage activity of CRISPR/Cas12a. By implying nanoclusters (NCs) with covalent organic framework (COF), the sensitive fluorescence "turn-on" method for OTC detection was realized. Under the optimum conditions, the linear range of aptasensor was 0.05-50 μM, and the detection limit was 0.041 μM. In addition, the aptasensing method can be successfully used for the quantitative detection of OTC in milk and honey samples with a recovery rate of 95.67%-104.10% and 95.33%-109.70%, implying excellent analytical performance and practical application prospect.}, }
@article {pmid39644349, year = {2024}, author = {Wang, YX and Ding, ZJ and Wang, QL and Zhao, CC and Liu, SQ and Du, SL and Zhou, S and Zheng, LY and Gao, M and Shen, CC and Chen, XD}, title = {CRISPR-Cas9 screening identified novel subtypes of cutaneous melanoma based on essential cancer genes.}, journal = {Archives of dermatological research}, volume = {317}, number = {1}, pages = {86}, pmid = {39644349}, issn = {1432-069X}, support = {MS2023007//Scientific Research Project of Nantong Municipal Health Commission/ ; ZD2022006//Key Medical Research Projects of Jiangsu Provincial Health Commission/ ; }, mesh = {Humans ; *Melanoma/genetics/immunology/pathology ; *Skin Neoplasms/genetics/immunology/pathology/diagnosis/therapy ; *CRISPR-Cas Systems/genetics ; Prognosis ; Biomarkers, Tumor/genetics ; Gene Expression Regulation, Neoplastic ; Male ; Female ; Melanoma, Cutaneous Malignant ; Cell Line, Tumor ; Databases, Genetic ; Middle Aged ; Gene Expression Profiling ; }, abstract = {Our primary objective was to identify genes critical for cutaneous melanoma (CM) and related typing, based on essential genes, to generate novel insights for clinical management and immunotherapy of patients with CM. We analyzed RNA sequencing (RNA-seq) data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx), and sequencing data of 29 CM cell line from Cancer Cell Line Encyclopedia (CCLE) databases. Combined with DepMap database, 406 CM essential cancer genes were finally obtained. Based on the expression of essential genes in cancer, the patients included in TCGA and Gene Expression Omnibus (GEO) databases were divided into three different molecular subtypes (C1, C2, and C3) by the NMF algorithm. Data analysis from TCGA and GEO datasets revealed that subtype C3 had the poorest prognosis, while subtype C1 exhibited the best prognosis. Combined with the CIBERSORT, ESTIMATE and ssGSEA algorithm, patients with different molecular subtypes can be divided into two immune subtypes (hot and cold). We found that subtype C1 was characterized by hot tumors, in contrast to subtypes C2 and C3, which were characterized by cold tumors. Then, we used univariate Cox regression, LASSO, and multifactor Cox regression analysis to select risk genes and constructed a prognostic model based on eight genes: RABIF, CDCA8, FOXM1, SPRR2E, AIP, CAP1, CTSW, and IFITM3. All patients were divided into two risk subtypes (high and low) according to the median of risk scores. We found that most hot tumor subtypes were found in the low-risk subtypes and most patients with this subtype survived for longer. Ultimately, we selected RABIF, which exhibits the highest risk coefficient, for histological and cytological verification. The results showed that RABIF was overexpressed in melanoma. Inhibition of RABIF expression could suppress the proliferation and invasion of melanoma cells and promote the apoptosis of melanoma cells. In conclusion, we used CRISPR-Cas9 screening to verify the association between molecular subtypes (C1, C2, and C3), immune subtypes (hot and cold), and risk subtypes (high and low) in patients with CM, particularly in distinguishing survival and prognosis. These findings can be used to guide clinical management and immunotherapy of patients with CM.}, }
@article {pmid39643619, year = {2024}, author = {Khan, NM and Wilderman, A and Kaiser, JM and Kamalakar, A and Goudy, SL and Cotney, J and Drissi, H}, title = {Enhanced osteogenic potential of iPSC-derived mesenchymal progenitor cells following genome editing of GWAS variants in the RUNX1 gene.}, journal = {Bone research}, volume = {12}, number = {1}, pages = {70}, pmid = {39643619}, issn = {2095-4700}, support = {R21 AR071536/AR/NIAMS NIH HHS/United States ; AR071536//U.S. Department of Health &amp; Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/ ; }, mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; *Osteogenesis/genetics ; Humans ; *Core Binding Factor Alpha 2 Subunit/genetics/metabolism ; *Gene Editing/methods ; *Mesenchymal Stem Cells/metabolism/cytology ; *Genome-Wide Association Study ; Animals ; Cell Differentiation/genetics ; Mice ; Osteoblasts/metabolism/cytology ; Bone Density/genetics ; CRISPR-Cas Systems/genetics ; }, abstract = {Recent genome-wide association studies (GWAS) identified 518 significant loci associated with bone mineral density (BMD), including variants at the RUNX1 locus (rs13046645, rs2834676, and rs2834694). However, their regulatory impact on RUNX1 expression and bone formation remained unclear. This study utilized human induced pluripotent stem cells (iPSCs) differentiated into osteoblasts to investigate these variants' regulatory roles. CRISPR/Cas9 was employed to generate mutant (Δ) iPSC lines lacking these loci at the RUNX1 locus. Deletion lines (Δ1 and Δ2) were created in iPSCs to assess the effects of removing regions containing these loci. Deletion lines exhibited enhanced osteogenic potential, with increased expression of osteogenic marker genes and Alizarin Red staining. Circularized chromosome conformation capture (4C-Seq) was utilized to analyze interactions between BMD-associated loci and the RUNX1 promoter during osteogenesis. Analysis revealed altered chromatin interactions with multiple gene promoters including RUNX1 isoform, as well as SETD4, a histone methyltransferase, indicating their regulatory influence. Interestingly, both deletion lines notably stimulated the expression of the long isoform of RUNX1, with more modest effects on the shorter isoform. Consistent upregulation of SETD4 and other predicted targets within the Δ2 deletion suggested its removal removed a regulatory hub constraining expression of multiple genes at this locus. In vivo experiments using a bone defect model in mice demonstrated increased bone regeneration with homozygous deletion of the Δ2 region. These findings indicate that BMD-associated variants within the RUNX1 locus regulate multiple effector genes involved in osteoblast commitment, providing valuable insights into genetic regulation of bone density and potential therapeutic targets.}, }
@article {pmid39643320, year = {2025}, author = {Gao, J and Huang, S and Jiang, J and Miao, Q and Zheng, R and Kang, Y and Tang, W and Zuo, H and He, J and Xie, J}, title = {Dual-CRISPR/Cas12a-assisted RT-RAA visualization system for rapid on-site detection of nervous necrosis virus (NNV).}, journal = {Analytica chimica acta}, volume = {1335}, number = {}, pages = {343469}, doi = {10.1016/j.aca.2024.343469}, pmid = {39643320}, issn = {1873-4324}, mesh = {*CRISPR-Cas Systems/genetics ; *Nodaviridae/genetics/isolation &amp; purification ; Animals ; Fish Diseases/virology/diagnosis ; CRISPR-Associated Proteins/genetics ; Endodeoxyribonucleases/genetics ; Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins ; }, abstract = {BACKGROUND: Nervous necrosis virus (NNV) poses a severe threat to the aquaculture industry, particularly infecting fish fry with devastating mortality rates and inflicting heavy economic losses. Traditional detection methods, such as cell culture and conventional RT-PCR, are not only time-consuming and require specialized laboratory facilities but also hard to eliminate contamination. Rapid and accurate on-site detection methods in aquaculture settings are crucial for effective control of NNV outbreaks in fish farms.<br><br>RESULTS: This study developed a one-tube visualization system for rapid and precise identification of NNV in a pond-side setting. This system utilizes the dual-clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-assisted reverse transcription-recombinase aided amplification (RT-RAA) detection method, employing fluorescence intensity to indicate positive results for easy interpretation by field operators. The key to this system involved the meticulous selection of RT-RAA primer sets and CRISPR RNA (crRNA) primer sets targeting two genes of NNV, the capsid protein (CP) and RNA-dependent RNA polymerase (RdRp), distributing on two particles of genomic sequences. The assay demonstrated a speed and efficiency process within 30&#xa0;min and a detection limit of 0.5 copies/&#x3bc;L, achieving 100&#xa0;% accuracy when compared to qRT-PCR. The practical utility and effectiveness were validated by using 32 field samples. The results underscored the simplicity, rapidity, and reliability of the system, confirming its potential as a robust tool for NNV diagnosis in fish farms.<br><br>SIGNIFICANCE: This study introduces the first application of a dual-CRISPR/Cas12a-assisted RT-RAA visualization system for diagnosing NNV infections. The novel approach substantially enhances on-site diagnostic capabilities, offering a rapid, reliable, and cost-effective solution for fish farm operators. This innovation not only streamlines the detection process but also ensures timely intervention, thereby mitigating the impact of NNV on aquaculture.}, }
@article {pmid39643311, year = {2025}, author = {He, H and Zhang, X and Deng, M and Zhou, Y and Pang, H and Yang, H and Lyu, J and Feng, Y and Geng, X and Guo, X and Luo, G and Guo, B}, title = {In-situ nucleic acid amplification induced by DNA self-assembly for rapid and ultrasensitive detection of miRNA.}, journal = {Analytica chimica acta}, volume = {1335}, number = {}, pages = {343457}, doi = {10.1016/j.aca.2024.343457}, pmid = {39643311}, issn = {1873-4324}, mesh = {*Nucleic Acid Amplification Techniques/methods ; *MicroRNAs/analysis/genetics ; Humans ; *DNA/chemistry/genetics ; *Limit of Detection ; Biosensing Techniques/methods ; }, abstract = {BACKGROUND: To improve the sensitivity and specificity of nucleic acid detection, coupling two or more signal amplification systems is a feasible pattern, such as nucleic acid isothermal amplification coupling genome-editing technology, and cascaded DNA self-assembly circuits. And representative signal amplification strategies include loop-mediated isothermal amplification (LAMP), clustered regularly interspaced short palindromic repeats/associated proteins (CRISPR/Cas) systems, and catalyzed hairpin assembly (CHA). However, these detection strategies often require the enrichment of intermediate products, the replacement of reaction conditions and the design of multiple probes, which may seriously affect the reliability of detection results.<br><br>RESULTS: Herein, we propose a novel nucleic acid detection system which is named as catalyzed hairpin assembly (CHA) coupled with embedded primer triggered isothermal amplification (CEA for short). DNA self-assembly probes in CEA contain a specially designed primer. When target nucleic acid (e.g., miRNA) initiates CHA reaction (the first signal amplification), the self-assembly product of CHA will expose a primer (named as embedded primer). The embedded primer will trigger a special nucleic acid isothermal amplification in situ, then generate plenty of double-stranded DNA products in 30&#xa0;min with varying lengths (the second signal amplification). Compared to that of a typical CHA reaction, the sensitivity of CEA has increased by three orders of magnitude and the detection limit is as low as 0.228&#xa0;fM. Besides, it has excellent detection performance in cancer and stem cell samples.<br><br>SIGNIFICANCE: By coupling embedded primer with DNA self-assembly system, a new nucleic acid detection system (CEA) with one-step operation and dual signal amplification has been successfully established. Compared with traditional dual signal amplification systems, CEA can not only significantly improve the reaction efficiency, but also greatly reduce the difficulty of detection system design and experimental operation.}, }
@article {pmid39643309, year = {2025}, author = {Li, X and Wang, C and Chai, J and Liu, H and Jiang, X and Li, Y and Li, Z and Li, Y}, title = {A novel all-in-one target-powered entropy-driven dynamic DNA networks to regulate the activity of CRISPR/AsCas12a for enhanced DNA detection.}, journal = {Analytica chimica acta}, volume = {1335}, number = {}, pages = {343455}, doi = {10.1016/j.aca.2024.343455}, pmid = {39643309}, issn = {1873-4324}, mesh = {*Entropy ; *CRISPR-Cas Systems/genetics ; *DNA/chemistry ; *Biosensing Techniques/methods ; Nucleic Acid Hybridization ; CRISPR-Associated Proteins/metabolism/chemistry ; Nucleic Acid Amplification Techniques ; Bacterial Proteins/chemistry/metabolism/genetics ; Endodeoxyribonucleases/chemistry/metabolism ; }, abstract = {BACKGROUND: The non-enzyme autonomous DNA nanodevices have been developed to detect various analytes through the programmability of Watson-Crick base pairing. Nevertheless, by comparison with enzymatic biosensors, the usage of enzyme-free DNA networks to create biosensors for testing low amounts of targets is still subject to the finite number of cycles. Besides, these biosensors still require the incorporation of other amplification strategies to improve the sensitivity, which complicates the detection workflow and lacks of a uniform compatible system to respond to the target in one pot.<br><br>RESULTS: Here, we put forward a novel way for rapid and sensitive DNA diagnostic via EDN (entropy-driven dynamic network) coupling with CRISPR/AsCas12a-powered amplification. In the absence of the target, the autonomous hybridization among the substrate and crRNA is kinetically hindered by enclosing complementary regions, which leads to the loss of the activation function of Cas12a. On the contrary, the target initiates the EDN, reconfiguring the activator strand from a duplex to branch construction, which provides a valid means to adjust the hybridization with crRNA, thereby controlling the indiscriminate collateral cleavage activities of the CRISPR/AsCas12a. Compared with the traditional EDN, synergistic activation between the EDN and the CRISPR catalyst could dramatically enhance the detection signal of the target in one pot. Furthermore, the proposed approach provides universal platforms through the rational functional and structural design of DNA assembly modules.<br><br>SIGNIFICANCE AND NOVELTY: Overall, this target-triggered EDN switches the activator strand to regulate the activity of AsCas12a (called TERA), which showed nearly one orders of magnitude sensitivity than the conventional Cas12a alone assay, resulting in a devisable universal CRISPR sensing platform that favours the fast, robust and one-pot detection of nucleic molecules.}, }
@article {pmid39642411, year = {2025}, author = {Li, H and Li, Y and Zhang, T and Liu, S and Song, C and Wang, K and Yan, W and Wang, Z and Yang, Q and Yang, X and Wang, H}, title = {Genome-wide CRISPR screen reveals specific role of type I interferon signaling pathway in Newcastle disease virus establishment of persistent infection.}, journal = {Veterinary microbiology}, volume = {300}, number = {}, pages = {110288}, doi = {10.1016/j.vetmic.2024.110288}, pmid = {39642411}, issn = {1873-2542}, mesh = {*Newcastle disease virus/immunology/genetics/physiology ; *Interferon Type I/genetics/metabolism/immunology ; Humans ; *Signal Transduction ; A549 Cells ; Animals ; *Newcastle Disease/virology/immunology ; Virus Replication ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Chickens/virology ; }, abstract = {Newcastle disease virus (NDV) is a potent oncolytic agent that exhibits sensitivity to a wide range of cancer cells. Unfortunately, some cancer cells are able to resist NDV-mediated oncolysis, by developing a persistent infection. The mechanism of persistency of infection remains poorly understood. In this study, a genome-wide CRISPR screen was conducted on non-small cell lung cancer cells (A549) to identify key host factors for NDV infection. Interestingly, a persistent infection was established in the surviving cells. CRISPR high-throughput screening results showed that members of the type I interferon signaling pathway (JAK1, STAT1, STAT2 and IRF9) were identified as top hits in the surviving cells. Further studies found that the type I IFN signaling pathway is intact in A549 cells, and a violent cytokine storm was induced after NDV infection. Both NDV infection and cytokine storm can induce cell death in A549 cells. We further blocked the type I interferon signaling pathway, and impaired type I interferon signaling pathway promoted NDV replication, but it did attenuate cell death induced by cytokine storm. Furthermore, persistent infection is more easily established in type I interferon signaling pathway-impaired A549 cells than in wild-type A549 cells. These findings suggest that the type I interferon signaling pathway plays a decisive role in persistent infection by regulating the antiviral immunity and cytokine storm inducing cell death.}, }
@article {pmid39641617, year = {2024}, author = {Jiang, H and Qian, C and Deng, Y and Lv, X and Liu, Y and Li, A and Li, X}, title = {Novel Multimode Assay Based on Asymmetrically Competitive CRISPR and Raman Barcode Spectra for Multiple Hepatocellular Carcinoma Biomarkers Detection.}, journal = {Analytical chemistry}, volume = {96}, number = {50}, pages = {20004-20014}, doi = {10.1021/acs.analchem.4c04593}, pmid = {39641617}, issn = {1520-6882}, mesh = {*Spectrum Analysis, Raman/methods ; *Carcinoma, Hepatocellular/diagnosis/genetics ; Humans ; *Liver Neoplasms/diagnosis/genetics ; *Biomarkers, Tumor/genetics ; *MicroRNAs/analysis ; Gold/chemistry ; Metal Nanoparticles/chemistry ; CRISPR-Cas Systems/genetics ; }, abstract = {Commercial pregnancy test strips (PTS) possess the advantages of lower price, higher stability, and better repeatability and have been popularized to integrate with novel sensing strategies to detect other disease biomarkers, which accelerates the commercialization process of those novel sensing strategies. However, the current integration of novel sensing strategies into commercial PTS still faced the problems of insufficient quantification, low sensitivity, and lack of multiple detection capabilities. Hence, we proposed the concept of "visual classification recognition, spectral signal subdivision" for multiple hepatocellular carcinoma biomarkers (miRNA122 and miRNA233) detection with dual signals based on asymmetric competitive CRISPR (acCRISPR) and surface-enhanced Raman spectroscopy coupling with PTS, named the acCRISPR-PTS-SERS assay. In this assay, acCRISPR was used as a nonamplified cascaded signal amplification method to improve the sensitivity of detection. Two AuNPs-based core-shell Raman tags, each corresponding to different miRNA biomarkers, were used to achieve both visual recognition and spectral segmentation to enhance the quantification of PTS detection and the capability for multiple detection. Under the optimal conditions, the LOD for miRNA122 and miRNA223 were 10.36 and 4.65 fM, respectively. The sensitivity was enhanced by nearly 2 orders of magnitude. In the future, simultaneous hand-held detection for fingerprint barcodes of different cancers can be achieved with the assistance of a microfluidic chip and smartphone.}, }
@article {pmid39640855, year = {2024}, author = {Hu, S and Gan, M and Wei, Z and Shang, P and Song, L and Feng, J and Chen, L and Niu, L and Wang, Y and Zhang, S and Shen, L and Zhu, L and Zhao, Y}, title = {Identification of host factors for livestock and poultry viruses: genome-wide screening technology based on the CRISPR system.}, journal = {Frontiers in microbiology}, volume = {15}, number = {}, pages = {1498641}, pmid = {39640855}, issn = {1664-302X}, abstract = {Genome-wide CRISPR library screening technology is a gene function research tool developed based on the CRISPR/Cas9 gene-editing system. The clustered regularly interspaced short palindromic repeats/CRISPR-associated genes (CRISPR/Cas) system, considered the third generation of gene editing after zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), is widely used for screening various viral host factors. CRISPR libraries are classified into three main categories based on the different functions of Cas9 enzymes: CRISPR knockout (CRISPR KO) library screening, CRISPR transcriptional activation (CRISPRa) library screening, and CRISPR transcriptional interference (CRISPRi) library screening. Recently, genome-wide CRISPR library screening technology has been used to identify host factors that interact with viruses at various stages, including adsorption, endocytosis, and replication. By specifically modulating the expression of these host factors, it becomes possible to cultivate disease-resistant varieties, establish disease models, and design and develop vaccines, among other applications. This review provides an overview of the development and technical processes of genome-wide CRISPR library screening, as well as its applications in identifying viral host factors in livestock and poultry.}, }
@article {pmid39639565, year = {2024}, author = {Yuan, A and Sun, T and Chen, L and Zhang, D and Xie, W and Peng, H}, title = {CRISPR/Cas12a Corona Nanomachine for Detecting Circulating Tumor Nucleic Acids in Serum.}, journal = {Analytical chemistry}, volume = {96}, number = {50}, pages = {20074-20081}, doi = {10.1021/acs.analchem.4c04993}, pmid = {39639565}, issn = {1520-6882}, mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gold/chemistry ; Circulating Tumor DNA/blood/genetics ; Metal Nanoparticles/chemistry ; Endodeoxyribonucleases/chemistry/metabolism ; CRISPR-Associated Proteins/chemistry/metabolism ; Biosensing Techniques ; Limit of Detection ; Bacterial Proteins ; }, abstract = {Circulating tumor nucleic acids (CTNAs), which consist of cell-free DNA or RNA released from tumor cells, are utilized as potential biomarkers for diagnosing and managing tumor prognosis. There is a significant demand for developing a highly sensitive and reliable assay for CTNAs detection. In this study, we engineered a CRISPR/Cas12a corona nanomachine capable of detecting circulating tumor DNA and RNA in serum. This nanomachine consists of a protein shell incorporating Cas12a/crRNA ribonucleoprotein complexes and a scaffold AuNP core decorated with substrate ssDNA strands. The protective CRISPR corona shields the nucleic acid core from degradation by nuclease DNase/RNase, thereby enhancing the stability of the CRISPR/Cas12a corona nanomachine in biological fluids, even tolerating up to undiluted human serum and FBS. Upon encountering target CTNAs, the CRISPR/Cas12a is activated through the sequence-specific hybridization between crRNA and CTNAs. Subsequently, the activated CRISPR/Cas12a autonomously cleaves the collateral ssDNA substrates on AuNPs, releasing the fluorophore-labeled fragment and generating an increasing fluorescent signal. The CRISPR corona nanomachine was successfully employed to detect various CTNAs, including circulating tumor (ct)DNA/RNA (EGFR L858R) and microRNA-21, achieving a limit of detection of 0.14 pM for ctDNA and 1.0 pM for RNA. This CRISPR corona nanomachine enables simultaneous detection of both DNA and RNA in complex biological samples, offering a promising tool for early diagnosis.}, }
@article {pmid39639368, year = {2024}, author = {Yu, L and Zou, J and Hussain, A and Jia, R and Fan, Y and Liu, J and Nie, X and Zhang, X and Jin, S}, title = {Systemic evaluation of various CRISPR/Cas13 orthologs for knockdown of targeted transcripts in plants.}, journal = {Genome biology}, volume = {25}, number = {1}, pages = {307}, pmid = {39639368}, issn = {1474-760X}, support = {2023ZD04074//Science and Technology Innovation 2025 Major Project of Ningbo/ ; 2022ZD0402001-04//Collaborative Innovation Center for Modern Science and Technology and Industrial Development of Jiangxi Traditional Medicine/ ; 32325039//Science Fund for Distinguished Young Scholars of Guangdong Province/ ; 32272128//Innovative Research Group Project of the National Natural Science Foundation of China/ ; 2021hszd013//Hubei Hongshan Laboratory/ ; }, mesh = {*CRISPR-Cas Systems ; *Nicotiana/genetics ; *Gene Editing ; Gene Knockdown Techniques ; Gossypium/genetics ; Plants, Genetically Modified ; RNA, Plant/genetics/metabolism ; }, abstract = {BACKGROUND: CRISPR/Cas13 system, recognized for its compact size and specificity in targeting RNA, is currently employed for RNA degradation. However, the potential of various CRISPR/Cas13 subtypes, particularly concerning the knockdown of endogenous transcripts, remains to be comprehensively characterized in plants.<br><br>RESULTS: Here we present a full spectrum of editing profiles for seven Cas13 orthologs from five distinct subtypes: VI-A (LwaCas13a), VI-B (PbuCas13b), VI-D (RfxCas13d), VI-X (Cas13x.1 and Cas13x.2), and VI-Y (Cas13y.1 and Cas13y.2). A systematic evaluation of the knockdown effects on two endogenous transcripts (GhCLA and GhPGF in cotton) as well as an RNA virus (TMV in tobacco) reveals that RfxCas13d, Cas13x.1, and Cas13x.2 exhibit enhanced stability with editing efficiencies ranging from 58 to 80%, closely followed by Cas13y.1 and Cas13y.2. Notably, both Cas13x.1 and Cas13y.1 can simultaneously degrade two endogenous transcripts through a tRNA-crRNA cassette approach, achieving editing efficiencies of up to 50%. Furthermore, different Cas13 orthologs enable varying degrees of endogenous transcript knockdown with minimal off-target effects, generating germplasms that exhibit a diverse spectrum of mutant phenotypes. Transgenic tobacco plants show significant reductions in damage, along with mild oxidative stress and minimal accumulation of viral particles after TMV infection.<br><br>CONCLUSIONS: In conclusion, our study presents an efficient and reliable platform for transcriptome editing that holds promise for plant functional research and future crop improvement.}, }
@article {pmid39639057, year = {2024}, author = {Sakurai, T and Takei, N and Wei, Y and Hayashi, M and Kamiyoshi, A and Kawate, H and Watanabe, S and Sato, M and Shindo, T}, title = {Efficient genome editing of two-cell mouse embryos via modified CRISPR/Cas electroporation.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {30347}, pmid = {39639057}, issn = {2045-2322}, mesh = {Animals ; *Gene Editing/methods ; *Electroporation/methods ; *CRISPR-Cas Systems ; Mice ; *Embryo, Mammalian/metabolism ; Female ; Gene Knock-In Techniques/methods ; Zygote/metabolism ; }, abstract = {Creating genetically modified (GM) animals using CRISPR/Cas mediated through the electroporation of two-cell stage embryos, rather than fertilized eggs, holds considerable potential. The full potential of genome editing using two-cell stage embryos is only beginning to be explored. We developed an improved electroporation method to prevent blastomere fusion in two-cell-stage embryos, enabling efficient genome editing. Using this method, we demonstrated that the indel mutation rates and ssODN knock-in (KI) efficiencies in two-cell-stage embryos are comparable to those in fertilized eggs, with a tendency for higher efficiency in long DNA KI. This study highlights the potential value of two-cell-stage embryos and provides enhanced animal model production opportunities. Furthermore, realizing genome editing in two-cell-stage embryos extends the editing timeframe from fertilized egg to two-cell-stage embryo, offering promising avenues for future research in embryo genome editing techniques.}, }
@article {pmid39638158, year = {2024}, author = {Garg, P and Singhal, G and Pareek, S and Kulkarni, P and Horne, D and Nath, A and Salgia, R and Singhal, SS}, title = {Unveiling the potential of gene editing techniques in revolutionizing Cancer treatment: A comprehensive overview.}, journal = {Biochimica et biophysica acta. Reviews on cancer}, volume = {1880}, number = {1}, pages = {189233}, doi = {10.1016/j.bbcan.2024.189233}, pmid = {39638158}, issn = {1879-2561}, abstract = {Gene editing techniques have emerged as powerful tools in biomedical research, offering precise manipulation of genetic material with the potential to revolutionize cancer treatment strategies. This review provides a comprehensive overview of the current landscape of gene editing technologies, including CRISPR-Cas systems, base editing, prime editing, and synthetic gene circuits, highlighting their applications and potential in cancer therapy. It discusses the mechanisms, advantages, and limitations of each gene editing approach, emphasizing their transformative impact on targeting oncogenes, tumor suppressor genes, and drug resistance mechanisms in various cancer types. The review delves into population-level interventions and precision prevention strategies enabled by gene editing technologies, including gene drives, synthetic gene circuits, and precision prevention tools, for controlling cancer-causing genes, targeting pre-cancerous lesions, and implementing personalized preventive measures. Ethical considerations, regulatory challenges, and future directions in gene editing research for cancer treatment are also addressed. This review highlights how gene editing could revolutionize precision medicine by enhancing patient care and advancing cancer treatments with targeted, personalized methods. For these benefits to be fully realized, collaboration among researchers, doctors, regulators, and patient advocates is crucial in fighting cancer and meeting clinical needs.}, }
@article {pmid39638020, year = {2024}, author = {Qiu, Y and Chen, S and Li, J and Liu, DA and Hu, R and Xu, Y and Chen, K and Yuan, J and Zhang, X and Li, X}, title = {Crispr-cas biosensing for rapid detection of viral infection.}, journal = {Clinica chimica acta; international journal of clinical chemistry}, volume = {567}, number = {}, pages = {120071}, doi = {10.1016/j.cca.2024.120071}, pmid = {39638020}, issn = {1873-3492}, abstract = {With the frequent outbreaks of viral diseases globally, accurate and rapid diagnosis of viral infections is of significant importance for disease prevention and control. The CRISPR-Cas combined biosensing strategy, as an emergent nucleic acid detection technology, exhibits notable advantages including high specificity, elevated sensitivity, operational simplicity, and cost-effectiveness, thereby demonstrating significant potential in the domain of rapid viral diagnostics. This paper summarizes the principles of the CRISPR-Cas system, the novel biotechnologies, and the latest research progress in virus detection using the combined biosensing strategy. Additionally, this paper discusses the challenges faced by CRISPR-Cas biosensing strategies and outlines future development directions, which provides a reference for further research and clinical applications in the rapid diagnosis of viral infections.}, }
@article {pmid39637860, year = {2024}, author = {Ursch, LT and Müschen, JS and Ritter, J and Klermund, J and Bernard, BE and Kolb, S and Warmuth, L and Andrieux, G and Miller, G and Jiménez-Muñoz, M and Theis, FJ and Boerries, M and Busch, DH and Cathomen, T and Schumann, K}, title = {Modulation of TCR stimulation and pifithrin-α improve the genomic safety profile of CRISPR-engineered human T cells.}, journal = {Cell reports. Medicine}, volume = {5}, number = {12}, pages = {101846}, doi = {10.1016/j.xcrm.2024.101846}, pmid = {39637860}, issn = {2666-3791}, mesh = {Humans ; *T-Lymphocytes/immunology/drug effects ; *Receptors, Antigen, T-Cell/genetics/immunology/metabolism ; *CRISPR-Cas Systems/genetics ; *Benzothiazoles/pharmacology ; *Lymphocyte Activation/drug effects/immunology ; *Toluene/analogs &amp; derivatives/pharmacology ; *Gene Editing/methods ; Cell Proliferation/drug effects ; Receptors, Chimeric Antigen/genetics/immunology ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {CRISPR-engineered chimeric antigen receptor (CAR) T cells are at the forefront of novel cancer treatments. However, several reports describe the occurrence of CRISPR-induced chromosomal aberrations. So far, measures to increase the genomic safety of T cell products focused mainly on the components of the CRISPR-Cas9 system and less on T cell-intrinsic features, such as their massive expansion after T cell receptor (TCR) stimulation. Here, we describe driving forces of indel formation in primary human T cells. Increased T cell activation and proliferation speed correlate with larger deletions. Editing of non-activated T cells reduces the risk of large deletions with the downside of reduced knockout efficiencies. Alternatively, the addition of the small-molecule pifithrin-α limits large deletions, chromosomal translocations, and aneuploidy in a p53-independent manner while maintaining the functionality of CRISPR-engineered T cells, including CAR T cells. Controlling T cell activation and pifithrin-α treatment are easily implementable strategies to improve the genomic integrity of CRISPR-engineered T cells.}, }
@article {pmid39637703, year = {2025}, author = {Thangaraj, A and Kaul, R and Sharda, S and Kaul, T}, title = {Revolutionizing cotton cultivation: A comprehensive review of genome editing technologies and their impact on breeding and production.}, journal = {Biochemical and biophysical research communications}, volume = {742}, number = {}, pages = {151084}, doi = {10.1016/j.bbrc.2024.151084}, pmid = {39637703}, issn = {1090-2104}, mesh = {*CRISPR-Cas Systems ; Crops, Agricultural/genetics/growth &amp; development ; *Gene Editing/methods ; Genome, Plant ; *Gossypium/genetics/growth &amp; development ; *Plant Breeding/methods ; Plants, Genetically Modified/genetics/growth &amp; development ; }, abstract = {Cotton (Gossypium hirsutum L.), a vital global cash crop, significantly impacts both the agricultural and industrial sectors, providing essential fiber for textiles and valuable byproducts such as cottonseed oil and animal feed. The cultivation of cotton supports millions of livelihoods worldwide, particularly in developing regions, making it a cornerstone of rural economies. Despite its importance, cotton production faces numerous challenges, including biotic stresses from pests and diseases, and abiotic stresses like drought, salinity, and extreme temperatures. These challenges necessitate innovative solutions to ensure sustainable production. Genome editing technologies, particularly CRISPR/Cas9, have revolutionized cotton breeding by enabling precise genetic modifications. These advancements hold promise for developing cotton varieties with enhanced resistance to pests, diseases, and environmental stresses. Early genome editing tools like ZFNs and TALENs paved the way for more precise modifications but were limited by complexity and cost. The introduction of CRISPR/Cas-based technology with its simplicity and efficiency, has dramatically transformed the field, making it the preferred tool for genome editing in crops. Improved version of the technology like CRISPR/Cas12a, CRISPR/Cas13, base and prime editing, developed from CRISPR/Cas systems, provide additional tools with distinct mechanisms, further expanding their potential applications in crop improvement. This comprehensive review explores the impact of genome editing on cotton breeding and production. It discusses the technical challenges, including off-target effects and delivery methods for genome editing components, and highlights ongoing research efforts to overcome these hurdles. The review underscores the potential of genome editing technologies to revolutionize cotton cultivation, enhancing yield, quality, and resilience, ultimately contributing to a sustainable future for the cotton industry.}, }
@article {pmid39636137, year = {2024}, author = {Nakata, M and Ueno, M and Kikuchi, Y and Iwami, M and Takayanagi-Kiya, S and Kiya, T}, title = {CRISPR/Cas9- and Single-Stranded ODN-Mediated Knock-In in Silkworm Bombyx mori.}, journal = {Zoological science}, volume = {41}, number = {6}, pages = {540-547}, doi = {10.2108/zs240019}, pmid = {39636137}, issn = {0289-0003}, mesh = {Animals ; *Bombyx/genetics ; *CRISPR-Cas Systems ; *Gene Knock-In Techniques ; Oligodeoxyribonucleotides/genetics ; Gene Editing ; }, abstract = {Although genome editing techniques have made significant progress, introducing exogenous genes into the genome through knock-in remains a challenge in many organisms. In silkworm Bombyx mori, TALEN-mediated knock-in methods have been established. However, difficulties in construction and limitations of the target sequence have hindered the application of these methods. In the present study, we verified several CRISPR/Cas9-mediated knock-in methods to expand the application of gene knock-in techniques and found that the short single-stranded oligodeoxynucleotide (ssODN)-mediated method is the most effective in silkworms. Using ssODN-mediated methods, we established knock-in silkworm strains that harbor an attP sequence, a 50 bp phiC31 integrase recognition site, at either the BmHr38 (Hormone receptor 38) or Bmdsx (doublesex) locus. Additionally, we found that the long ssODN (lsODN)-mediated method successfully introduced the GAL4 gene at the doublesex locus in embryos. The present study provides valuable information on CRISPR/Cas9-mediated knock-in methods in silkworms, expanding the utility of genome editing techniques in insects and paving the way for analyzing gene and genome function in silkworms.}, }
@article {pmid39633273, year = {2024}, author = {Peng, Y and Li, J and Niu, K and Wang, M and Chen, Y and Tong, C and Feng, Q}, title = {Bmlark is essential for embryonic development.}, journal = {BMC genomics}, volume = {25}, number = {1}, pages = {1179}, pmid = {39633273}, issn = {1471-2164}, support = {2021M691096//China&#xa0;Postdoctoral Science Foundation/ ; 32100383//Chinese National Natural Science Foundation/ ; }, mesh = {Animals ; *Bombyx/genetics/embryology/metabolism ; *Embryonic Development/genetics ; Gene Expression Regulation, Developmental ; Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Mutation ; Transcription Factors/genetics/metabolism ; Transcriptome ; }, abstract = {BACKGROUND: Transcription factor lark has been demonstrated to play multiple functions in Drosophila, but the function of this gene in embryonic development remains to be elucidated.<br><br>RESULTS: In this study, the CRISPR/Cas9 gene-editing method was used to construct a Bmlark mutant strain of Bombyx mori to investigate the roles of this gene. The results showed that the homozygous mutant Bmlark[-/-] was lethal. The Bmlark[-/-] embryos showed obvious developmental defects, such as defective sclerotization and melanization of the exoskeleton. A transcriptomic comparison of Bmlark[-/-] and wild-type embryos showed that the differentially expressed genes were mainly enriched in the structure and metabolic processes of chitin and cuticles. While the expression levels of chitin metabolism-related enzyme genes did not significantly change, in the mutant embryos compared to the wild-type embryos, the expression levels of 63 putative cuticle protein genes showed significant differences. Among which, 35 genes were downregulated and 28 genes were upregulated. The expression levels of the transcription factor BmPOUM2 and eight wing disc cuticle protein genes (WCP) also changed. BmPOUM2, WCP5, WCP9, WCP10, WCP11 were downregulated and WCP1, WCP2, WCP3, WCP6 were upregulated in Bmlark[-/-] embryos. While the expression level of TH in the tyrosine-mediated pigmentation pathway was upregulated in the mutant embryos, the expression levels of the four key pigment synthesis genes DDC, aaNAT, Laccase2A, and yellow-f2 were significantly downregulated.<br><br>CONCLUSIONS: The expression levels of 63 putative cuticle protein genes, eight WCP genes, and five pigment synthesis genes significantly changed in Bmlark mutant B. mori compared to those of the wildtype. These results suggest that Bmlark is essential for normal development of cuticle and tyrosine-mediated melanization in silkworm embryos.}, }
@article {pmid39633151, year = {2024}, author = {Wu, WY and Adiego-Pérez, B and van der Oost, J}, title = {Biology and applications of CRISPR-Cas12 and transposon-associated homologs.}, journal = {Nature biotechnology}, volume = {42}, number = {12}, pages = {1807-1821}, pmid = {39633151}, issn = {1546-1696}, mesh = {*CRISPR-Cas Systems/genetics ; *DNA Transposable Elements/genetics ; *Gene Editing/methods ; CRISPR-Associated Proteins/genetics/metabolism ; Humans ; }, abstract = {CRISPR-associated Cas12 proteins are a highly variable collection of nucleic acid-targeting proteins. All Cas12 variants use RNA guides and a single nuclease domain to target complementary DNA or, in rare cases, RNA. The high variability of Cas12 effectors can be explained by a series of independent evolution events from different transposon-associated TnpB-like ancestors. Despite basic structural and functional similarities, this has resulted in unprecedented variation of the Cas12 effector proteins in terms of size, domain composition, guide structure, target identity and interference strategy. In this Review, we compare the unique molecular features of natural and engineered Cas12 and TnpB variants. Furthermore, we provide an overview of established genome editing and diagnostic applications and discuss potential future directions.}, }
@article {pmid39633035, year = {2024}, author = {Rocha, BMO and Sabino, YNV and de Almeida, TC and Palacio, FB and Rotta, IS and Dias, VC and da Silva, VL and Diniz, CG and Azevedo, VAC and Brenig, B and Soares, SC and Paiva, AD and Medeiros, JD and Machado, ABF}, title = {Unlocking Probiotic Potential: Genomic Insights into Weissella paramesenteroides UFTM 2.6.1.}, journal = {Probiotics and antimicrobial proteins}, volume = {}, number = {}, pages = {}, pmid = {39633035}, issn = {1867-1314}, abstract = {Weissella, a genus of lactic acid bacteria, has diverse beneficial attributes including probiotic activity and biotechnological applications. Therefore, the investigation of the Weissella genus has garnered growing interest. In this study, we sequenced the complete genome of Weissella paramesenteroides UFTM 2.6.1 isolated from unpasteurized cow's milk from the Triângulo Mineiro region and performed probiogenomic analyses. Taxonomic characterization confirmed the identity of W. paramesenteroides. The genome comprises 1926 protein-coding genes, mainly related to cell metabolism, information storage and processing, and cellular processes and signaling. Ninety-nine unique genes associated with probiotic functions were identified in the genome of W. paramesenteroides UFTM 2.6.1, including genes involved in stress response, bacterial persistence in the gastrointestinal tract, and biosynthesis of vitamins. In silico analysis of bacteriocin-related genes identified Pediocin, and subsequent in vitro testing confirmed that W. paramesenteroides UFTM 2.6.1 exhibits antimicrobial activity against Listeria spp. Genomic characterization revealed the presence of the replicon pLCK4 and four prophage regions, one of which was intact. Moreover, no CRISPR-Cas array or associated Cas proteins were found, along with an absence of resistance and virulence genes, suggesting a safety aspect of the evaluated strain. Pan-genome analysis unveiled 204 exclusive genes in the genome of W. paramesenteroides UFTM 2.6.1, which includes metabolism and stress-associated genes. In general, the results indicate probiotic potential of W. paramesenteroides UFTM 2.6.1. Further studies are required to ensure the safety and beneficial effects of this bacterium in vivo, aiming for future applications in the food industry and animal and human medicine.}, }
@article {pmid39631395, year = {2025}, author = {Kimble, MT and Sane, A and Reid, RJD and Johnson, MJ and Rothstein, R and Symington, LS}, title = {Repair of replication-dependent double-strand breaks differs between the leading and lagging strands.}, journal = {Molecular cell}, volume = {85}, number = {1}, pages = {61-77.e6}, pmid = {39631395}, issn = {1097-4164}, support = {R35 GM118180/GM/NIGMS NIH HHS/United States ; R35 GM126997/GM/NIGMS NIH HHS/United States ; T32 CA265828/CA/NCI NIH HHS/United States ; }, mesh = {*DNA Breaks, Double-Stranded ; *DNA Replication ; *Saccharomyces cerevisiae/genetics/metabolism ; Saccharomyces cerevisiae Proteins/metabolism/genetics ; DNA End-Joining Repair ; DNA Repair ; Nucleosomes/metabolism/genetics ; DNA, Fungal/genetics/metabolism ; S Phase ; Homologous Recombination ; Histones/metabolism/genetics ; Recombinational DNA Repair ; CRISPR-Associated Protein 9/metabolism/genetics ; CRISPR-Cas Systems ; }, abstract = {Single-strand breaks (SSBs) are one of the most commonly occurring endogenous lesions with the potential to give rise to cytotoxic double-strand breaks (DSBs) during DNA replication. To investigate how replication-dependent DSBs are repaired, we employed Cas9 nickase (nCas9) to generate site- and strand-specific nicks in the budding yeast genome. We found that nCas9-induced nicks are converted to mostly double-ended DSBs during S phase. Repair of replication-associated DSBs requires homologous recombination (HR) and is independent of classical non-homologous end joining. Consistent with a strong bias to repair these lesions using a sister-chromatid template, we observed minimal induction of inter-chromosomal HR by nCas9. In a genome-wide screen to identify factors necessary for the repair of replication-dependent DSBs, we recovered components of the replication-coupled nucleosome assembly (RCNA) pathway. Our findings suggest that the RCNA pathway is especially important to repair DSBs arising from nicks in the leading-strand template through acetylation of histone H3K56.}, }
@article {pmid39631148, year = {2024}, author = {Banta, AB and Cuellar, RA and Nadig, N and Davis, BC and Peters, JM}, title = {The promise of CRISPR-associated transposons for bacterial functional genomics.}, journal = {Current opinion in microbiology}, volume = {83}, number = {}, pages = {102563}, doi = {10.1016/j.mib.2024.102563}, pmid = {39631148}, issn = {1879-0364}, abstract = {CRISPR-associated transposons (CASTs) are naturally occurring amalgamations of CRISPR-Cas machinery and Tn7-like transposons that direct site-specific integration of transposon DNA via programmable guide RNAs. Although the mechanisms of CAST-based transposition have been well studied at the molecular and structural level, CASTs have yet to be broadly applied to bacterial genome engineering and systematic gene phenotyping (i.e. functional genomics) - likely due to their relatively recent discovery. Here, we describe the function and applications of CASTs, focusing on well-characterized systems, including the type I-F CAST from Vibrio cholerae (VcCAST) and type V-K CAST from Scytonema hofmanni (ShCAST). Further, we discuss the potentially transformative impact of targeted transposition on bacterial functional genomics by proposing genome-scale extensions of existing CAST tools.}, }
@article {pmid39629326, year = {2024}, author = {Ishino, Y}, title = {A novel strategy to protect prokaryotic cells from virus infection.}, journal = {Engineering microbiology}, volume = {4}, number = {2}, pages = {100153}, pmid = {39629326}, issn = {2667-3703}, abstract = {The recent discovery of the CRISPR-Cas-mediated acquired immunity system highlights the fact that our knowledge of phage/virus defense mechanisms encoded in bacterial and archaeal genomes is far from complete. Indeed, new prokaryotic immune systems are now continually being discovered. A recent report described a novel glycosylase that recognizes α-glycosyl-hydroxymethyl cytosin (α-Glu-hmC), a modified base observed in the T4 phage genome, where it produces an abasic site, thereby inhibiting the phage propagation.}, }
@article {pmid39628591, year = {2024}, author = {Le, Y and Zhang, M and Wu, P and Wang, H and Ni, J}, title = {Biofuel production from lignocellulose via thermophile-based consolidated bioprocessing.}, journal = {Engineering microbiology}, volume = {4}, number = {4}, pages = {100174}, pmid = {39628591}, issn = {2667-3703}, abstract = {The depletion of fossil fuels and their impact on the environment have led to efforts to develop alternative sustainable fuels. While biofuel derived from lignocellulose is considered a sustainable, renewable, and green energy source, enhancing biofuel production and achieving a cost-effective bioconversion of lignocellulose at existing bio-refineries remains a challenge. Consolidated bioprocessing (CBP) using thermophiles can simplify this operation by integrating multiple processes, such as hydrolytic enzyme production, lignocellulose degradation, biofuel fermentation, and product distillation. This paper reviews recent developments in the conversion of lignocellulose to biofuel using thermophile-based CBP. First, advances in thermostable enzyme and thermophilic lignocellulolytic microorganism discovery and development for lignocellulosic biorefinery use are outlined. Then, several thermophilic CBP candidates and thermophilic microbes engineered to drive CBP of lignocellulose are reviewed. CRISPR/Cas-based genome editing tools developed for thermophiles are also highlighted. The potential applications of the Design-Build-Test-Learn (DBTL) synthetic biology strategy for designing and constructing thermophilic CBP hosts are also discussed in detail. Overall, this review illustrates how to develop highly sophisticated thermophilic CBP hosts for use in lignocellulosic biorefinery applications.}, }
@article {pmid39628447, year = {2024}, author = {Carlsen, FM and Westberg, I and Johansen, IE and Andreasson, E and Petersen, BL}, title = {Strategies and Protocols for Optimized Genome Editing in Potato.}, journal = {The CRISPR journal}, volume = {}, number = {}, pages = {}, doi = {10.1089/crispr.2024.0068}, pmid = {39628447}, issn = {2573-1602}, abstract = {The potato family includes a highly diverse cultivar repertoire and has a high potential for nutritional yield improvement and refinement but must in line with other crops be adapted to biotic and abiotic stresses, for example, accelerated by climate change and environmental demands. The combination of pluripotency, high ploidy, and relative ease of protoplast isolation, transformation, and regeneration together with clonal propagation through tubers makes potato highly suitable for precise genetic engineering. Most potato varieties are tetraploid having a very high prevalence of length polymorphisms and small nucleotide polymorphisms between alleles, often complicating CRISPR-Cas editing designs and strategies. CRISPR-Cas editing in potato can be divided into (i) characterization of target area and in silico-aided editing design, (ii) isolation and editing of protoplast cells, and (iii) the subsequent explant regeneration from single protoplast cells. Implementation of efficient CRISPR-Cas editing relies on efficient editing at the protoplast (cell pool) level and on robust high-throughput editing scoring methods at the cell pool and explant level. Gene and chromatin structure are additional features to optionally consider. Strategies and solutions for addressing key steps in genome editing of potato, including light conditions and schemes for reduced exposure to hormones during explant regeneration, which is often linked to somaclonal variation, are highlighted.}, }
@article {pmid39628369, year = {2024}, author = {Kanitchinda, S and Sritunyalucksana, K and Chaijarasphong, T}, title = {Multiplex CRISPR-Cas Assay for Rapid, Isothermal and Visual Detection of White Spot Syndrome Virus (WSSV) and Enterocytozoon hepatopenaei (EHP) in Penaeid Shrimp.}, journal = {Journal of fish diseases}, volume = {}, number = {}, pages = {e14059}, doi = {10.1111/jfd.14059}, pmid = {39628369}, issn = {1365-2761}, support = {//Mahidol University (Fundamental Fund: fiscal year 2023 by National Science Research and Innovation Fund (NSRF)) (Grant no. FF-056/2566)/ ; }, abstract = {White spot syndrome virus (WSSV) and Enterocytozoon hepatopenaei (EHP) represent the most economically destructive pathogens in the current shrimp industry. WSSV causes white spot disease (WSD) responsible for rapid shrimp mortality, while EHP stunts growth and therefore reduces overall productivity. Despite the importance of timely disease detection, current diagnostic methods for WSSV and EHP are typically singleplex, and those offering multiplex detection face issues such as complexity, low field compatibility and/or low sensitivity. Here, we introduce an orthogonal, multiplex CRISPR-Cas assay for concomitant detection of WSSV and EHP. This method combines recombinase polymerase amplification (RPA) for target DNA enrichment with Cas12a and Cas13a enzymes for fluorescent detection. This assay produces distinct fluorescent colours for different diagnostic outcomes, allowing naked eye visualisation without ambiguity. Further validation reveals that the assay detects as few as 20 and 200 copies of target DNA from EHP and WSSV, respectively, while producing no false positives with DNA from other shrimp pathogens. Moreover, the assay excellently agrees with established PCR methods in evaluation of clinical samples. Requiring only 37°C and less than an hour to complete, multiplex CRISPR-Cas assay presents a promising tool for onsite diagnostics, offering high accuracy while saving time and resources.}, }
@article {pmid39627841, year = {2024}, author = {Sledzinski, P and Nowaczyk, M and Smielowska, MI and Olejniczak, M}, title = {CRISPR/Cas9-induced double-strand breaks in the huntingtin locus lead to CAG repeat contraction through DNA end resection and homology-mediated repair.}, journal = {BMC biology}, volume = {22}, number = {1}, pages = {282}, pmid = {39627841}, issn = {1741-7007}, mesh = {Humans ; *DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems ; *Huntingtin Protein/genetics/metabolism ; Recombinational DNA Repair ; Trinucleotide Repeats/genetics ; Trinucleotide Repeat Expansion/genetics ; DNA Repair ; }, abstract = {BACKGROUND: The expansion of CAG/CTG repeats in functionally unrelated genes is a causative factor in many inherited neurodegenerative disorders, including Huntington's disease (HD), spinocerebellar ataxias (SCAs), and myotonic dystrophy type 1 (DM1). Despite many years of research, the mechanism responsible for repeat instability is unknown, and recent findings indicate the key role of DNA repair in this process. The repair of DSBs induced by genome editing tools results in the shortening of long CAG/CTG repeats in yeast models. Understanding this mechanism is the first step in developing a therapeutic strategy based on the controlled shortening of repeats. The aim of this study was to characterize Cas9-induced DSB repair products at the endogenous HTT locus in human cells and to identify factors affecting the formation of specific types of sequences.<br><br>RESULTS: The location of the cleavage site and the surrounding sequence influence the outcome of DNA repair. DSBs within CAG repeats result in shortening of the repeats in frame in&#x2009;~&#x2009;90% of products. The mechanism of this contraction involves MRE11-CTIP and RAD51 activity and DNA end resection. We demonstrated that a DSB located upstream of CAG repeats induces polymerase theta-mediated end joining, resulting in deletion of the entire CAG tract. Furthermore, using proteomic analysis, we identified novel factors that may be involved in CAG sequence repair.<br><br>CONCLUSIONS: Our study provides new insights into the complex mechanisms of CRISPR/Cas9-induced shortening of CAG repeats in human cells.}, }
@article {pmid39627502, year = {2025}, author = {Ngoi, NYL and Gallo, D and Torrado, C and Nardo, M and Durocher, D and Yap, TA}, title = {Synthetic lethal strategies for the development of cancer therapeutics.}, journal = {Nature reviews. Clinical oncology}, volume = {22}, number = {1}, pages = {46-64}, pmid = {39627502}, issn = {1759-4782}, mesh = {Humans ; *Neoplasms/drug therapy/genetics ; *Synthetic Lethal Mutations ; Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use ; CRISPR-Cas Systems ; Antineoplastic Agents/therapeutic use ; Drug Discovery/methods ; DNA Damage/drug effects ; }, abstract = {Synthetic lethality is a genetic phenomenon whereby the simultaneous presence of two different genetic alterations impairs cellular viability. Importantly, targeting synthetic lethal interactions offers potential therapeutic strategies for cancers with alterations in pathways that might otherwise be considered undruggable. High-throughput screening methods based on modern CRISPR-Cas9 technologies have emerged and become crucial for identifying novel synthetic lethal interactions with the potential for translation into biologically rational cancer therapeutic strategies as well as associated predictive biomarkers of response capable of guiding patient selection. Spurred by the clinical success of PARP inhibitors in patients with BRCA-mutant cancers, novel agents targeting multiple synthetic lethal interactions within DNA damage response pathways are in clinical development, and rational strategies targeting synthetic lethal interactions spanning alterations in epigenetic, metabolic and proliferative pathways have also emerged and are in late preclinical and/or early clinical testing. In this Review, we provide a comprehensive overview of established and emerging technologies for synthetic lethal drug discovery and development and discuss promising therapeutic strategies targeting such interactions.}, }
@article {pmid39626969, year = {2024}, author = {Haussmann, IU and Dix, TC and McQuarrie, DWJ and Dezi, V and Hans, AI and Arnold, R and Soller, M}, title = {Structure-optimized sgRNA selection with PlatinumCRISPr for efficient Cas9 generation of knockouts.}, journal = {Genome research}, volume = {34}, number = {12}, pages = {2279-2292}, pmid = {39626969}, issn = {1549-5469}, mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; CRISPR-Associated Protein 9/metabolism/genetics ; Gene Knockout Techniques/methods ; Drosophila/genetics ; Software ; Drosophila Proteins/genetics/metabolism ; Drosophila melanogaster/genetics ; }, abstract = {A single guide RNA (sgRNA) directs Cas9 nuclease for gene-specific scission of double-stranded DNA. High Cas9 activity is essential for efficient gene editing to generate gene deletions and gene replacements by homologous recombination. However, cleavage efficiency is below 50% for more than half of randomly selected sgRNA sequences in human cell culture screens or model organisms. We used in vitro assays to determine intrinsic molecular parameters for maximal sgRNA activity including correct folding of sgRNAs and Cas9 structural information. From the comparison of over 10 data sets, we find major constraints in sgRNA design originating from defective secondary structure of the sgRNA, sequence context of the seed region, GC context, and detrimental motifs, but we also find considerable variation among different prediction tools when applied to different data sets. To aid selection of efficient sgRNAs, we developed web-based PlatinumCRISPr, an sgRNA design tool to evaluate base-pairing and sequence composition parameters for optimal design of highly efficient sgRNAs for Cas9 genome editing. We applied this tool to select sgRNAs to efficiently generate gene deletions in Drosophila Ythdc1 and Ythdf, that bind to N [6] methylated adenosines (m[6]A) in mRNA. However, we discovered that generating small deletions with sgRNAs and Cas9 leads to ectopic reinsertion of the deleted DNA fragment elsewhere in the genome. These insertions can be removed by standard genetic recombination and chromosome exchange. These new insights into sgRNA design and the mechanisms of CRISPR-Cas9 genome editing advance the efficient use of this technique for safer applications in humans.}, }
@article {pmid39626678, year = {2024}, author = {Aviram, N and Shilton, AK and Lyn, NG and Reis, BS and Brivanlou, A and Marraffini, LA}, title = {Cas10 relieves host growth arrest to facilitate spacer retention during type III-A CRISPR-Cas immunity.}, journal = {Cell host &amp; microbe}, volume = {32}, number = {12}, pages = {2050-2062.e6}, pmid = {39626678}, issn = {1934-6069}, support = {K99 GM148720/GM/NIGMS NIH HHS/United States ; R01 GM149834/GM/NIGMS NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; *Virus Replication ; Bacterial Proteins/metabolism/genetics ; CRISPR-Associated Proteins/metabolism/genetics ; DNA, Viral/genetics ; Staphylococcus aureus/genetics/immunology ; }, abstract = {Cells from all kingdoms of life can enter growth arrest in unfavorable environmental conditions. Key to this process are mechanisms enabling recovery from this state. Staphylococcal type III-A CRISPR-Cas loci encode the Cas10 complex that uses a guide RNA to locate complementary viral transcripts and start an immune response. When the target sequence is expressed late in the viral lytic cycle, defense requires the activity of Csm6, a non-specific RNase that inhibits the growth of the infected cell. How Csm6 protects from infection and whether growth can be restored is not known. Here, we show that growth arrest provides immunity at the population level, preventing viral replication and allowing uninfected cells to propagate. In addition, the ssDNase activity of Cas10 is required for the regrowth of a subset of the arrested cells and the recovery of the infected host, presumably ending the immune response through degradation of the viral DNA.}, }
@article {pmid39626673, year = {2024}, author = {Awasthi, S and Dobrolecki, LE and Sallas, C and Zhang, X and Li, Y and Khazaei, S and Ghosh, S and Jeter, CR and Liu, J and Mills, GB and Westin, SN and Lewis, MT and Peng, W and Sood, AK and Yap, TA and Yi, SS and McGrail, DJ and Sahni, N}, title = {UBA1 inhibition sensitizes cancer cells to PARP inhibitors.}, journal = {Cell reports. Medicine}, volume = {5}, number = {12}, pages = {101834}, doi = {10.1016/j.xcrm.2024.101834}, pmid = {39626673}, issn = {2666-3791}, mesh = {Humans ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; *Ubiquitin-Activating Enzymes/antagonists &amp; inhibitors/metabolism/genetics ; Animals ; Cell Line, Tumor ; Female ; Mice ; Xenograft Model Antitumor Assays ; Ovarian Neoplasms/drug therapy/pathology/genetics ; DNA Damage/drug effects ; CRISPR-Cas Systems/genetics ; Poly(ADP-ribose) Polymerases/metabolism/genetics ; }, abstract = {Therapeutic strategies targeting the DNA damage response, such as poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi), have revolutionized cancer treatment in tumors deficient in homologous recombination (HR). However, overcoming innate and acquired resistance to PARPi remains a significant challenge. Here, we employ a genome-wide CRISPR knockout screen and discover that the depletion of ubiquitin-activating enzyme E1 (UBA1) enhances sensitivity to PARPi in HR-proficient ovarian cancer cells. We show that silencing or pharmacological inhibition of UBA1 sensitizes multiple cell lines and organoid models to PARPi. Mechanistic studies uncover that UBA1 inhibition not only impedes HR repair to sensitize cells to PARP inhibition but also increases PARylation, which may subsequently be targeted by PARP inhibition. In vivo experiments using patient-derived xenografts demonstrate that combining PARP and UBA1 inhibition provided significant survival benefit compared to individual therapies with no detectable signs of toxicity, establishing this combination approach as a promising strategy to extend PARPi benefit.}, }
@article {pmid39625871, year = {2025}, author = {Nguyen, LT and Moutesidi, P and Ziegler, J and Glasneck, A and Khosravi, S and Abel, S and Hensel, G and Krupinska, K and Humbeck, K}, title = {WHIRLY1 regulates aliphatic glucosinolate biosynthesis in early seedling development of Arabidopsis.}, journal = {The Plant journal : for cell and molecular biology}, volume = {121}, number = {1}, pages = {e17181}, pmid = {39625871}, issn = {1365-313X}, support = {400681449/GRK2498//Deutsche Forschungsgemeinschaft/ ; }, mesh = {*Arabidopsis/genetics/growth &amp; development/metabolism ; *Seedlings/genetics/growth &amp; development/metabolism ; *Glucosinolates/metabolism/biosynthesis ; *Arabidopsis Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; Transcription Factors/genetics/metabolism ; Cotyledon/genetics/growth &amp; development/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; }, abstract = {WHIRLY1 belongs to a family of plant-specific transcription factors capable of binding DNA or RNA in all three plant cell compartments that contain genetic materials. In Arabidopsis thaliana, WHIRLY1 has been studied at the later stages of plant development, including flowering and leaf senescence, as well as in biotic and abiotic stress responses. In this study, WHIRLY1 knockout mutants of A. thaliana were prepared by CRISPR/Cas9-mediated genome editing to investigate the role of WHIRLY1 during early seedling development. The loss-of-function of WHIRLY1 in 5-day-old seedlings did not cause differences in the phenotype and the photosynthetic performance of the emerging cotyledons compared with the wild type. Nevertheless, comparative RNA sequencing analysis revealed that the knockout of WHIRLY1 affected the expression of a small but specific set of genes during this critical phase of development. About 110 genes were found to be significantly deregulated in the knockout mutant, wherein several genes involved in the early steps of aliphatic glucosinolate (GSL) biosynthesis were suppressed compared with wild-type plants. The downregulation of these genes in WHIRLY1 knockout lines led to decreased GSL contents in seedlings and in seeds. Since GSL catabolism mediated by myrosinases was not altered during seed-to-seedling transition, the results suggest that AtWHIRLY1 plays a major role in modulation of aliphatic GSL biosynthesis during early seedling development. In addition, phylogenetic analysis revealed a coincidence between the evolution of methionine-derived aliphatic GSLs and the addition of a new WHIRLY in core families of the plant order Brassicales.}, }
@article {pmid39624314, year = {2024}, author = {Panahi, B and Rostampour, M and Ghaffari, MR and Nami, Y}, title = {Genome mining approach reveals the CRISPR-Cas systems features and characteristics in Lactobacillus delbrueckii strains.}, journal = {Heliyon}, volume = {10}, number = {22}, pages = {e39920}, pmid = {39624314}, issn = {2405-8440}, abstract = {This study employed a comprehensive genome-mining approach to characterize CRISPR-Cas systems in Lactobacillus delbrueckii strains. The analysis involved retrieving 105 genome sequences to explore the variety, occurrence, and evolution of CRISPR-Cas systems within the species. Homology analysis of spacer sequences in detected CRISPR arrays was conducted to assess the variety of target phages and plasmids. The evolutionary trajectories of spaceromes in each subtype of CRISPR arrays were determined by analyzing acquisition and deletion events under the selection pressure of foreign plasmids and phages. Among the analyzed strains, 53 contained only one CRISPR-Cas locus, 11 had two loci, and 21 featured three loci with complete CRISPR-Cas systems. Subtype designation of the results of current study revealed that 56 % of these systems belong to subtypes I-E/I-C, 23 % to subtypes III-A/III-D, and 17 % to subtype II-A. Notably, certain plasmids were found to be specifically targeted by distinct CRISPR array systems. The comparison of spacer sequences with phage genomes indicated that strains containing only CRISPR-Cas type-I systems targeted a broader range of phages. In conclusion, this study highlights the diversity of the three identified CRISPR-Cas types in L. delbrueckii strains and emphasizes their significant role in defense against phage invasion.}, }
@article {pmid39623998, year = {2024}, author = {Wu, Y and Zhang, X and Li, J and Xie, J and Wang, L and Sun, H}, title = {[Progress of researches on toxoplasmosis vaccines based on the CRISPR/Cas9 technology].}, journal = {Zhongguo xue xi chong bing fang zhi za zhi = Chinese journal of schistosomiasis control}, volume = {36}, number = {5}, pages = {542-547}, doi = {10.16250/j.32.1374.2024026}, pmid = {39623998}, issn = {1005-6661}, support = {ZR2022MH271//Natural Science Foundation of Shandong Province/ ; 202301011242//Shandong Provincial Medical and Health Science and Technology Project/ ; 202101050153//Shandong Provincial Science and Technology Development Project for Medical Sciences and Health/ ; }, mesh = {*CRISPR-Cas Systems ; Humans ; Animals ; *Toxoplasma/genetics/immunology ; *Toxoplasmosis/prevention &amp; control/immunology/parasitology ; Protozoan Vaccines/immunology ; Gene Editing/methods ; }, abstract = {Toxoplasma gondii is an obligatory intracellular parasite which infects a variety of warm-blooded animals and causes toxoplasmosis. Toxoplasmosis seriously endangers human health and animal husbandry production. As one of the effective gene editing tools, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been widely used for knockout of genes in T. gondii. This review summarizes the applications of the CRISPR/Cas9 technology in vaccines against single- and double-gene deletion strains of T. gondii, so as to provide insights into development of toxoplasmosis vaccines.}, }
@article {pmid39622959, year = {2024}, author = {Fang, B and Wang, C and Yuan, Y and Liu, X and Shi, L and Li, L and Wang, Y and Dai, Y and Yang, H}, title = {Generation and characterization of genetically modified pigs with GGTA1/β4GalNT2/CMAH knockout and human CD55/CD47 expression for xenotransfusion studies.}, journal = {Scientific reports}, volume = {14}, number = {1}, pages = {29870}, pmid = {39622959}, issn = {2045-2322}, support = {81970164//National Natural Science Foundation of China/ ; }, mesh = {Animals ; Humans ; *Animals, Genetically Modified ; *Galactosyltransferases/genetics/metabolism ; Swine ; *CD55 Antigens/genetics/metabolism ; *CD47 Antigen/genetics/metabolism ; *Gene Knockout Techniques/methods ; *N-Acetylgalactosaminyltransferases/genetics/metabolism ; *Mixed Function Oxygenases/genetics/metabolism ; *Transplantation, Heterologous ; CRISPR-Cas Systems ; Erythrocytes/metabolism ; Macaca fascicularis/genetics ; Immunoglobulin G ; }, abstract = {Pig red blood cells (pRBCs) represent a promising alternative to address the shortage in transfusion medicine. Nonetheless, a major obstacle to their clinical implementation is immunological rejection. In this study, we generated transgenic pigs expressing human CD47 (hCD47) and CD55 (hCD55) in α1,3-galactosyltransferase KO/β-1,4-N-acetyl-galactosaminyl transferase 2 KO/cytidine monophosphate-N-acetylneuraminic acid hydroxylase KO (TKO) pigs using CRISPR/Cas9 technology. Compared to wild-type pRBCs, TKO/hCD47/hCD55 pRBCs exhibit significantly reduced human IgG/IgM antibody binding. Moreover, when transfused into Cynomolgus monkeys, TKO/hCD47/hCD55 pRBCs remained detectable for 2 h post-transfusion, whereas wild-type pRBCs were eliminated within 20 min. This study demonstrates the potential of multi-gene edited pigs to provide immunologically compatible pRBCs.}, }
@article {pmid39622625, year = {2024}, author = {Mallozzi, A and Fusco, V and Ragazzini, F and di Bernardo, D}, title = {A Biomolecular Circuit for Automatic Gene Regulation in Mammalian Cells with CRISPR Technology.}, journal = {ACS synthetic biology}, volume = {13}, number = {12}, pages = {3917-3925}, pmid = {39622625}, issn = {2161-5063}, mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Gene Expression Regulation ; HEK293 Cells ; Transcription Factors/genetics/metabolism ; Plasmids/genetics/metabolism ; Gene Regulatory Networks ; Animals ; }, abstract = {We introduce a biomolecular circuit for precise control of gene expression in mammalian cells. The circuit leverages the stochiometric interaction between the artificial transcription factor VPR-dCas9 and the anti-CRISPR protein AcrIIA4, enhanced with synthetic coiled-coil domains to boost their interaction, to maintain the expression of a reporter protein constant across diverse experimental conditions, including fluctuations in protein degradation rates and plasmid concentrations, by automatically adjusting its mRNA level. This capability, known as robust perfect adaptation (RPA), is crucial for the stable functioning of biological systems and has wide-ranging implications for biotechnological applications. This system belongs to a class of biomolecular circuits named antithetic integral controllers, and it can be easily adapted to regulate any endogenous transcription factor thanks to the versatility of the CRISPR-Cas system. Finally, we show that RPA also holds in cells genomically integrated with the circuit, thus paving the way for practical applications in biotechnology that require stable cell lines.}, }
@article {pmid39621555, year = {2024}, author = {Emaldi, M and Rey-Iborra, E and Marín, &#xc1; and Mosteiro, L and Lecumberri, D and Øyjord, T and Roncier, N and Mælandsmo, GM and Angulo, JC and Errarte, P and Larrinaga, G and Pulido, R and López, JI and Nunes-Xavier, CE}, title = {Impact of B7-H3 expression on metastasis, immune exhaustion and JAK/STAT and PI3K/AKT pathways in clear cell renal cell carcinoma.}, journal = {Oncoimmunology}, volume = {13}, number = {1}, pages = {2419686}, pmid = {39621555}, issn = {2162-402X}, mesh = {*Carcinoma, Renal Cell/pathology/immunology/genetics/metabolism/drug therapy ; Humans ; *B7 Antigens/metabolism/genetics ; *Kidney Neoplasms/pathology/immunology/metabolism/genetics/drug therapy ; *Proto-Oncogene Proteins c-akt/metabolism ; *Phosphatidylinositol 3-Kinases/metabolism ; *Signal Transduction/drug effects ; Janus Kinases/metabolism ; Cell Line, Tumor ; STAT Transcription Factors/metabolism ; Male ; Female ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Middle Aged ; Neoplasm Metastasis ; Aged ; Immune System Exhaustion ; }, abstract = {Immune checkpoint inhibitors in combination with tyrosine kinase inhibitors (TKIs) are improving the response rates of advanced renal cancer patients. However, many treated patients do not respond, making novel immune checkpoint-based immunotherapies potentially clinically beneficial only for specific groups of patients. We detected high expression of the immune checkpoint protein B7-H3 in clear cell renal cell carcinomas (ccRCCs) and evaluated B7-H3 immunohistochemistry staining in tissue microarray samples from two distinct renal cancer cohorts. B7-H3 was highly expressed in approximately 50% of primary tumors and in 30% of metastatic lesions. B7-H3 expression in primary tumors correlated with tumor necrosis, sarcomatoid transformation, disease-free survival, and synchronous metastasis, while B7-H3 expression in metastasis correlated with metastases to the lymph nodes. Gene expression analysis revealed the association of B7-H3 expression with gene expression scores involved in T cell exhaustion and myeloid immune evasion, as well as with PI3K/AKT and JAK/STAT pathways. Furthermore, knocking down B7-H3 expression in renal cancer cells by siRNA and CRISPR/Cas resulted in lower 2D and 3D cell proliferation and viability as well as increased sensitivity to TKI axitinib. Together, our findings suggest a pro-oncogenic and immune evasive role for B7-H3 in ccRCC and highlight B7-H3 as an actionable novel immune checkpoint protein in combination with TKI in advanced renal cancer.}, }
@article {pmid39621264, year = {2025}, author = {Gowripalan, A and Smith, SA and Tscharke, DC}, title = {Cas9-Mediated Poxvirus Recombinant Recovery (CASPRR) for Fast Recovery of Recombinant Vaccinia Viruses.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2860}, number = {}, pages = {115-130}, pmid = {39621264}, issn = {1940-6029}, mesh = {*Vaccinia virus/genetics ; Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Recombination, Genetic ; Poxviridae/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; Cell Line ; Transfection/methods ; }, abstract = {Generation of recombinant vaccinia viruses opens many avenues for poxvirus research; however current methods for virus production can be laborious. Traditional methods rely on recombination strategies that produce engineered viruses at a low frequency, which then need to be identified and isolated from a large background of parent virus. For this reason, marker and reporter genes are often included, but in many cases these require removal in subsequent steps and the entire process is relatively inefficient. Cas9-mediated selection is a technique that repurposes Cas9/guide RNA complexes to amplify chosen subsets of vaccinia viruses. We refer to this approach as Cas9-mediated poxvirus recombinant recovery (CASPRR). Transient introduction of appropriately guided Cas9 allows for recovery of marker-free recombinant viruses in just 5 days, and requires no additional virus modification. Following three rounds of purification, pure virus stocks are obtained. A newer method, stable expression of Cas9 and guide RNAs in a permissive cell line, allows for additional process streamlining, removing cell type-specific concerns related to transient transfection of Cas9. Within this chapter, the protocol for CASPRR is described in both a transient and stable expression model. These methods can be utilized to accelerate recovery of recombinant vaccinia viruses and be applied to generation of vaccinia libraries or novel therapeutic agents.}, }
@article {pmid39621263, year = {2025}, author = {Lin, YJ and Evans, DH and Noyce, RS}, title = {Rapid Generation of Recombinant Poxviruses Using CRISPR/Cas9 Gene Editing.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2860}, number = {}, pages = {97-114}, pmid = {39621263}, issn = {1940-6029}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Vaccinia virus/genetics ; *Poxviridae/genetics ; Humans ; Genome, Viral ; DNA, Viral/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; Recombination, Genetic ; }, abstract = {The low-frequency natural recombination that is detected in poxvirus-infected cells has long been used to genetically modify poxviruses. Such recombinant poxviruses have found many applications as vaccines for preventing infectious diseases and as experimental cancer therapeutics. Unfortunately, these methods are time consuming, can leave behind "scars" or selectable markers, and many months of work may be required to generate plaque-purified recombinants bearing multiple virus gene substitutions, deletions, and/or inserted transgenes. Over the last decade, several reports have described how CRISPR/Cas9 technologies can be used to better facilitate genetic manipulation of vaccinia virus (VACV). These protocols use Cas9/gRNA complexes to introduce double-stranded breaks into specific sites in virus genomic DNA either in vivo or in vitro. Recombination-repair reactions are then employed to repair the breaks using transfected DNAs encoding the required homologies and desired mutation(s). Here we describe a method where we combine CRISPR/Cas9 genome editing in vitro, followed by Leporipoxvirus-catalyzed repair and reactivation of the cut VACV DNA using repair fragments provided in trans. This method optimizes several steps in the preparation of the CRISPR/Cas9-cut VACV DNA and can be used to introduce mutations at multiple sites without requiring selectable markers. It also provides some guidance regarding how the position of the CRISPR/Cas9-cuts can affect co-conversion of flanking markers embedded in the repair fragment. The method allows researchers to quickly generate recombinant VACV bearing multiple genetic alterations and using only a single round of reactivation and plating.}, }
@article {pmid39620587, year = {2024}, author = {Cui, Z and Yuan, P and Zhao, Z and Zhao, F and Lu, L}, title = {Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {213}, pages = {}, doi = {10.3791/66966}, pmid = {39620587}, issn = {1940-087X}, mesh = {Animals ; Mice ; *Th17 Cells/immunology/cytology ; *Cell Differentiation/genetics ; *CRISPR-Cas Systems/genetics ; *Retroviridae/genetics ; Gene Targeting/methods ; Flow Cytometry/methods ; }, abstract = {T helper cells that produce IL-17A, known as Th17 cells, play a critical role in immune defense and are implicated in autoimmune disorders. CD4 T cells can be stimulated with antigens and well-defined cytokine cocktails in vitro to mimic Th17 cell differentiation in vivo. Research has been conducted extensively on the Th17 differentiation regulation mechanisms using the in vitro Th17 polarization assay. Conventional Th17 polarization methods typically involve obtaining naïve CD4 T cells from genetically modified mice to study the effects of specific genes on Th17 differentiation and function. These methods can be time-consuming and costly and may be influenced by cell-extrinsic factors from the knockout animals. Thus, a protocol using retroviral transduction of guide RNA to introduce gene knockout in CRISPR/Cas9 knockin primary mouse T cells serves as a very useful alternative approach. This paper presents a protocol to differentiate naïve primary T cells into Th17 cells following retroviral-mediated gene targeting, as well as the subsequent flow cytometry analysis methods for assaying infection and differentiation efficiency.}, }
@article {pmid39620398, year = {2024}, author = {Zhao, Z and Wang, R and Yang, X and Jia, J and Zhang, Q and Ye, S and Man, S and Ma, L}, title = {Machine Learning-Assisted, Dual-Channel CRISPR/Cas12a Biosensor-In-Microdroplet for Amplification-Free Nucleic Acid Detection for Food Authenticity Testing.}, journal = {ACS nano}, volume = {18}, number = {49}, pages = {33505-33519}, doi = {10.1021/acsnano.4c10823}, pmid = {39620398}, issn = {1936-086X}, mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Machine Learning ; Food Analysis/methods ; Animals ; Meat/analysis ; Nucleic Acids/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {The development of novel detection technology for meat species authenticity is imperative. Here, we developed a machine learning-supported, dual-channel biosensor-in-microdroplet platform for meat species authenticity detection named CC-drop (CRISPR/Cas12a digital single-molecule microdroplet biosensor). This strategy allowed us to quickly identify and analyze animal-derived components in foods. This biosensor was enabled by CRISPR/Cas12a-based fluorescence lighting-up detection, and the nucleic acid signals can be recognized by a Cas12a-crRNA binary complex to trigger the trans-cleavage of any by-stander reporter single-stranded (ss) DNA, in which nucleic acid signals can be converted and amplified to fluorescent readouts. The ultralocalized microdroplet reactor was constructed by reducing the reaction volume from up to picoliter to accommodate the aforementioned reaction to further enhance the sensitivity to even render an amplification-free nucleic acid detection. Moreover, we established a smartphone App coupled with a random forest machine learning model based on parameters such as area, fluorescent intensity, and counting number to ensure the accuracy of image recording and processing. The sample-to-result time was within 80 min. Importantly, the proposed biosensor was able to accurately detect the ND1 (pork-specific) and IL-2 (duck-specific) genes in deep processed meat-derived foods that usually had truncated DNA, and the results were more robust and practical than conventional real-time polymerase chain reaction after a side-by-side comparison. All in all, the proposed biosensor can be expected to be used for rapid food authenticity and other nucleic acid detections in the future.}, }
@article {pmid39619730, year = {2024}, author = {Ahmed, MM and Kayode, HH and Okesanya, OJ and Ukoaka, BM and Eshun, G and Mourid, MR and Adigun, OA and Ogaya, JB and Mohamed, ZO and Lucero-Prisno, DE}, title = {CRISPR-Cas Systems in the Fight Against Antimicrobial Resistance: Current Status, Potentials, and Future Directions.}, journal = {Infection and drug resistance}, volume = {17}, number = {}, pages = {5229-5245}, pmid = {39619730}, issn = {1178-6973}, abstract = {BACKGROUND: Antimicrobial resistance (AMR) is a critical global health concern that threatens the efficacy of existing antibiotics and poses significant challenges to public health and the economy worldwide. This review explores the potential of CRISPR-Cas systems as a novel approach to combating AMR and examines current applications, limitations, and prospects.<br><br>METHODS: A comprehensive literature search was conducted across multiple databases, including PubMed, Google Scholar, Scopus, and Web of Science, covering publications published from 2014 to August 2024. This review focuses on CRISPR-Cas technologies and their applications in AMR.<br><br>RESULTS: CRISPR-Cas systems have demonstrated efficacy in combating antimicrobial resistance by targeting and eliminating antibiotic-resistance genes. For example, studies have shown that CRISPR-Cas9 can effectively target and eliminate colistin resistance genes in MCR-1 plasmids, restoring susceptibility to carbapenems in bacteria such as E. coli and Klebsiella pneumoniae. Further molecular findings highlight the impact of CRISPR-Cas systems on various bacterial species, such as Enterococcus faecalis, in which CRISPR systems play a crucial role in preventing the acquisition of resistance genes. The effectiveness of CRISPR-Cas in targeting these genes varies due to differences in CRISPR locus formation among bacterial species. For instance, variations in CRISPR loci influence the targeting of resistance genes in E. faecalis, and CRISPR-Cas9 successfully reduces resistance by targeting genes such as tetM and ermB.<br><br>CONCLUSION: CRISPR-Cas systems are promising for fighting AMR by targeting and eliminating antibiotic-resistant genes, as demonstrated by the effective targeting of colistin resistance genes on MCR-1 plasmids and their similar activities. However, the effectiveness of CRISPR-Cas is affected by variations in the CRISPR loci among bacterial species. Challenges persist, such as optimizing delivery methods and addressing off-target effects to ensure the safety and precision of CRISPR-Cas systems in clinical settings.}, }
@article {pmid39618347, year = {2025}, author = {Yu, C and Liu, Y and Zhang, W and Yao, X}, title = {A three-way junction probe triggered CRISPR/Cas14a1 enhanced EXPonential amplification reaction for sensitive Pseudomonas aeruginosa detection.}, journal = {Analytical methods : advancing methods and applications}, volume = {17}, number = {2}, pages = {284-290}, doi = {10.1039/d4ay01728e}, pmid = {39618347}, issn = {1759-9679}, mesh = {*Pseudomonas aeruginosa/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Humans ; Pseudomonas Infections ; Aptamers, Nucleotide/chemistry ; }, abstract = {Pseudomonas aeruginosa (P. aeruginosa, PA) is a rod-shaped Gram-negative opportunistic bacterium capable of causing nosocomial infections during nursing, such as burn wound infections and meningitis. However, sensitive and rapid PA detection remains a huge challenge. Herein, a new fluorescent biosensor was developed for the sensitive detection of PA using a three-way junction (TWJ) probe for specific identification and CRISPR/Cas14a1 for enhanced exponential amplification. The interaction between PA and its aptamer on a DNA TWJ structure probe triggered the migration of the double-stranded DNA branch, inducing DNA polymerase/endonuclease assisted chain displacement and the generation of single-stranded DNA sequences. The amplification products have the ability to activate CRISPR/Cas14a1, resulting in effective trans-cleavage and the subsequent release of fluorescence from the reporter probes. Under optimized conditions, the proposed biosensor was able to detect a wide range of bacterial concentrations, ranging from 10 to 10[5] cfu mL[-1] within 30 min. The limit of detection, which is the lowest concentration that can be reliably detected, was determined to be 3.4 cfu mL[-1] according to the 3δ rule. The results of the recovery test suggest that the biosensor shows significant potential for clinical applications. The established biosensor utilizing the TWJ probe generated multiple isothermal exponential amplification and the CRISPR/Cas14a1 biosensor is an excellent platform for rapidly detecting pathogenic bacteria in postoperative infection.}, }
@article {pmid39617792, year = {2024}, author = {Hosseinpour, M and Xi, X and Liu, L and Malaver-Ortega, L and Perlaza-Jimenez, L and Joo, JE and York, HM and Beesley, J and Caldon, CE and Dugué, PA and Dowty, JG and Arumugam, S and Southey, MC and Rosenbluh, J}, title = {SAM-DNMT3A, a strategy for induction of genome-wide DNA methylation, identifies DNA methylation as a vulnerability in ER-positive breast cancers.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10449}, pmid = {39617792}, issn = {2041-1723}, support = {2011329//Department of Health | National Health and Medical Research Council (NHMRC)/ ; MCRF20035//Victorian Cancer Agency (VCA)/ ; }, mesh = {*DNA Methylation ; Humans ; *Breast Neoplasms/genetics/metabolism/drug therapy ; *DNA Methyltransferase 3A/metabolism ; Female ; *DNA (Cytosine-5-)-Methyltransferases/metabolism/genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Receptors, Estrogen/metabolism/genetics ; MCF-7 Cells ; Epigenesis, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {DNA methylation is an epigenetic mark that plays a critical role in regulating gene expression. DNA methyltransferase (DNMT) inhibitors, inhibit global DNA methylation and have been a key tool in studies of DNA methylation. A major bottleneck is the lack of tools to induce global DNA methylation. Here, we engineered a CRISPR based approach, that we initially designed, to enable site-specific DNA methylation. Using the synergistic activation mediator (SAM) system, we unexpectedly find that regardless of the targeted sequence any sgRNA induces global genome-wide DNA methylation. We term this method SAM-DNMT3A and show that induction of global DNA methylation is a unique vulnerability in ER-positive breast cancer suggesting a therapeutic approach. Our findings highlight the need of caution when using CRISPR based approaches for inducing DNA methylation and demonstrate a method for global induction of DNA methylation.}, }
@article {pmid39617755, year = {2024}, author = {Wu, Y and Huang, Z and Liu, Y and He, P and Wang, Y and Yan, L and Wang, X and Gao, S and Zhou, X and Yoon, CW and Sun, K and Situ, Y and Ho, P and Zeng, Y and Yuan, Z and Zhu, L and Zhou, Q and Zhao, Y and Liu, T and Kwong, GA and Chien, S and Liu, L and Wang, Y}, title = {Ultrasound Control of Genomic Regulatory Toolboxes for Cancer Immunotherapy.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10444}, pmid = {39617755}, issn = {2041-1723}, support = {EB029122//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; GM140929//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; HL121365//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; HD107206//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; CA262815//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; EB032822//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; K01EB035649//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; }, mesh = {Humans ; *Neoplasms/therapy/genetics/immunology ; Animals ; *CRISPR-Cas Systems ; *Receptors, Chimeric Antigen/metabolism/genetics ; Mice ; Cell Line, Tumor ; Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Immunotherapy, Adoptive/methods ; Dependovirus/genetics ; Immunotherapy/methods ; Genomics/methods ; Ultrasonic Waves ; }, abstract = {There remains a critical need for the precise control of CRISPR (clustered regularly interspaced short palindromic repeats)-based technologies. Here, we engineer a set of inducible CRISPR-based tools controllable by focused ultrasound (FUS), which can penetrate deep and induce localized hyperthermia for transgene activation. We demonstrate the capabilities of FUS-inducible CRISPR, CRISPR activation (CRISPRa), and CRISPR epigenetic editor (CRISPRee) in modulating the genome and epigenome. We show that FUS-CRISPR-mediated telomere disruption primes solid tumours for chimeric antigen receptor (CAR)-T cell therapy. We further deliver FUS-CRISPR in vivo using adeno-associated viruses (AAVs), followed by FUS-induced telomere disruption and the expression of a clinically validated antigen in a subpopulation of tumour cells, functioning as "training centers" to activate synthetic Notch (synNotch) CAR-T cells to produce CARs against a universal tumour antigen to exterminate neighboring tumour cells. The FUS-CRISPR(a/ee) toolbox hence allows the noninvasive and spatiotemporal control of genomic/epigenomic reprogramming for cancer treatment.}, }
@article {pmid39616960, year = {2024}, author = {Xing, W and Zhang, J and Liu, T and Wang, Y and Qian, J and Wang, B and Zhang, Y and Zhang, Q}, title = {An innovative CRISPR/Cas9 mouse model of human isolated microtia indicates the potential contribution of CNVs near HMX1 gene.}, journal = {International journal of pediatric otorhinolaryngology}, volume = {187}, number = {}, pages = {112141}, doi = {10.1016/j.ijporl.2024.112141}, pmid = {39616960}, issn = {1872-8464}, mesh = {Animals ; *Congenital Microtia/genetics ; Humans ; Mice ; *Disease Models, Animal ; *DNA Copy Number Variations ; *CRISPR-Cas Systems ; Female ; Male ; *Pedigree ; *Homeodomain Proteins/genetics ; Mutation ; Gene Editing/methods ; }, abstract = {BACKGROUND: Microtia is a prevalent congenital malformation, the precise etiology and pathogenesis of which remain elusive. Mutations in the non-coding region of the HMX1 gene have been implicated in isolated cases of microtia, emerging as a significant focus of contemporary research. Several pathogenic copy number variations (CNVs) proximal to the HMX1 gene have been documented in wild animal populations, whereas only a single large segmental duplication in this region has been identified in humans. However, the absence of a gene-edited animal model has impeded the investigation of the unclear gene function associated with HMX1 mutations in human isolated microtia. In this study, we sought to precisely identify the pathogenic mutation by analyzing three pedigrees alongside population controls. Subsequently, our objective was to develop a CRISPR/Cas9 gene-edited mouse model to elucidate the functional implications of the identified mutation.<br><br>METHODS: Genomic DNA was collected from 32 affected individuals across three pedigrees, as well as from 2000 control subjects. Comprehensive genomic analyses, including genome-wide linkage analysis, targeted capture, second-generation sequencing, and copy number analysis, were conducted to identify potential mutations associated with congenital auricle malformation. CRISPR/Cas9 gene-edited murine models were generated in response to the identified mutation. The auricular phenotypes of these gene-edited mice were systematically monitored. Small-animal Micro-CT scanning was employed to identify potential craniofacial or skeletal abnormalities. Furthermore, the expression of the HMX1 gene in the PA2 region of mouse embryos was quantified using RT-qPCR.<br><br>RESULTS: A co-segregated 600 base pair duplication located on chromosome 4 (chr4:8701900-8702500, hg19) was identified in affected individuals across three pedigrees, but was absent in healthy controls. Two types of CRISPR/Cas9 gene-edited mice were subsequently generated. The knock-in (KI) mouse model was engineered by inserting one copy of the duplicated sequence directly adjacent to the mutated site, whereas the knockout (KO) mouse model was created by excising the mutation sequence. The phenotypes of different group of CRISPR/Cas9 gene-edited mice demonstrated distinct auricular deformities. Furthermore, an increase in the copy number of the mutated sequence was associated with elevated expression levels of HMX1 in the gene-edited mouse model.<br><br>CONCLUSIONS: In this study, we further narrowed down and identified a 600 base pair copy number variation (CNV) located at chr4:8701900-8702500 (hg19), which is implicated in human bilateral, isolated microtia. Utilizing CRISPR/Cas9 technology, we developed novel mouse models harboring the identified mutation. These models serve as a robust platform for the comprehensive investigation of the underlying mechanisms of the disease.}, }
@article {pmid39616957, year = {2025}, author = {Fu, Z and Zhao, L and Guo, Y and Yang, J}, title = {Gene therapy for hereditary hearing loss.}, journal = {Hearing research}, volume = {455}, number = {}, pages = {109151}, doi = {10.1016/j.heares.2024.109151}, pmid = {39616957}, issn = {1878-5891}, mesh = {Animals ; Humans ; CRISPR-Cas Systems/genetics ; Dependovirus/genetics ; *Disease Models, Animal ; *Gene Editing/methods ; Gene Transfer Techniques ; *Genetic Therapy/methods ; *Genetic Vectors ; Hearing/genetics ; *Hearing Loss/genetics/therapy/physiopathology ; Phenotype ; Treatment Outcome ; }, abstract = {Gene therapy is a technique by which exogenous genetic material is introduced into target cells to treat or prevent diseases caused by genetic mutations. Hearing loss is the most common sensory disorder. Genetic factors contribute to approximately 50 % of all cases of profound hearing loss, and more than 150 independent genes have been reported as associated with hearing loss. Recent advances in CRISPR/Cas based gene-editing tools have facilitated the development of gene therapies for hereditary hearing loss (HHL). Viral delivery vectors, and especially adeno-associated virus (AAV) vectors, have been demonstrated as safe and efficient carriers for the delivery of transgenes into inner ear cells in animal models. More importantly, AAV-mediated gene therapy can restore hearing in some children with hereditary deafness. However, there are many different types of HHL that need to be identified and evaluated to determine appropriate gene therapy options. In the present review, we summarize recent animal model-based advances in gene therapy for HHL, as well as gene therapy strategies, gene-editing tools, delivery vectors, and administration routes. We also discuss the strengths and limitations of different gene therapy methods and describe future challenges for the eventual clinical application of gene therapy for HHL.}, }
@article {pmid39616917, year = {2024}, author = {Nassereddine, ZN and Opara, SD and Coutinho, OA and Qyteti, F and Book, R and Heinicke, MP and Napieralski, J and Tiquia-Arashiro, SM}, title = {Critical perspectives on advancing antibiotic resistant gene (ARG) detection technologies in aquatic ecosystems.}, journal = {The Science of the total environment}, volume = {957}, number = {}, pages = {177775}, doi = {10.1016/j.scitotenv.2024.177775}, pmid = {39616917}, issn = {1879-1026}, mesh = {Anti-Bacterial Agents/analysis ; Drug Resistance, Bacterial/genetics ; *Drug Resistance, Microbial/genetics ; *Ecosystem ; *Environmental Monitoring/methods ; Metagenomics/methods ; *Water Microbiology ; }, abstract = {The spread of antibiotic resistance genes (ARGs) in aquatic ecosystems poses a serious risk to environmental and public health, making advanced detection and monitoring methods essential. This review provides a fresh perspective and a critical evaluation of recent advances in detecting and monitoring ARGs in aquatic environments. It highlights the latest innovations in molecular, bioinformatic, and environmental techniques. While traditional methods like culture-based assays and polymerase chain reaction (PCR) remain important, they are increasingly being supplemented by high-throughput sequencing technologies applied to metagenomics. These technologies offer comprehensive insights into the diversity and distribution of ARGs in aquatic environments. The integration of bioinformatic tools and databases has improved the accuracy and efficiency of ARG detection, enabling the analysis of complex datasets and tracking the evolution of ARGs in aquatic settings. Additionally, new environmental monitoring methods, including novel biosensors, geographic information systems (GIS) applications, and remote sensing technologies, have emerged as powerful tools for real-time ARG surveillance in water systems. This review critically examines the challenges of standardizing these methodologies and emphasizes the need for interdisciplinary approaches to enhance ARG monitoring across different aquatic ecosystems. By assessing the strengths and limitations of various methods, this review aims to guide future research and the development of more effective strategies for managing antibiotic resistance in aquatic environments.}, }
@article {pmid39616576, year = {2025}, author = {Ng, EFY and Meitinger, F}, title = {Genetic Engineering and Screening Using Base Editing and Inducible Gene Knockout.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2872}, number = {}, pages = {167-187}, pmid = {39616576}, issn = {1940-6029}, mesh = {Humans ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; *CRISPR-Cas Systems ; Genetic Engineering/methods ; Genetic Testing/methods ; }, abstract = {Genetic engineering and screening in human cells are powerful techniques for the precise and comprehensive identification and analysis of gene and protein domain functions. Genome-wide knockout screens have been extensively utilized to discover essential genes, tumor suppressors, and genes that regulate responses to various chemicals, including antimitotic and therapeutic drugs. The advent of base editors, which facilitate the targeted mutation of single amino acids, has advanced the identification of critical and functional domains or motifs. In this context, we outline methods for creating efficient base editor and inducible knockout cell lines for targeted gene manipulation and conducting genetic screens to elucidate the roles of genes and their domains within a specific cell biological context.}, }
@article {pmid39616566, year = {2025}, author = {Hatano, Y and Yonezawa, N and Tokoro, M and Yao, T and Yamagata, K}, title = {Chromosome Counting at Meiosis and Mitosis of Mouse Oocytes and Embryos Using Super-Resolution Live-Cell Imaging and CRISPR/dCas9-Mediated Live-FISH.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2872}, number = {}, pages = {21-35}, pmid = {39616566}, issn = {1940-6029}, mesh = {Animals ; Mice ; *Oocytes/cytology/metabolism ; *Meiosis/genetics ; *In Situ Hybridization, Fluorescence/methods ; *CRISPR-Cas Systems ; *Mitosis/genetics ; Female ; Embryo, Mammalian/cytology ; Microscopy, Confocal/methods ; Chromosome Segregation ; Chromosomes, Mammalian/genetics ; }, abstract = {Live cell imaging techniques are now essential for capturing chromosomal segregation in fertilized eggs. Although better spatiotemporal resolution of fluorescence observations could provide more information, higher phototoxicity may occur. Super-resolution microscopy is generally considered unsuitable for live cell imaging because of the considerable cell damage. Here, we developed a method for counting chromosomes in mouse living oocytes and early embryos using super-resolution microscopy based on disk confocal photon reassignment microscopy (OPRA). In this chapter, we describe the imaging conditions for minimally invasive, high-resolution observation of mouse oocytes and embryos and a method to count chromosomes via CRISPR/dCas-mediated live-cell fluorescence in situ hybridization.}, }
@article {pmid39615910, year = {2025}, author = {Amouzadeh Tabrizi, M and Ali, AA and Singuru, MMR and Mi, L and Bhattacharyya, P and You, M}, title = {A portable and sensitive DNA-based electrochemical sensor for detecting piconewton-scale cellular forces.}, journal = {Analytica chimica acta}, volume = {1333}, number = {}, pages = {343392}, pmid = {39615910}, issn = {1873-4324}, support = {R35 GM133507/GM/NIGMS NIH HHS/United States ; }, mesh = {Humans ; *Electrochemical Techniques/instrumentation/methods ; *DNA/chemistry/analysis ; HeLa Cells ; *Biosensing Techniques/instrumentation ; Smartphone ; Electrodes ; Gold/chemistry ; CRISPR-Cas Systems ; }, abstract = {BACKGROUND: Cell-generated forces are a key player in cell biology, especially during cellular shape formation, migration, cancer development, and immune response. The measurement of forces exerted and experienced by cells is fundamental in understanding these mechanosensitive cellular behaviors. While cell-generated forces can now be detected based on techniques like fluorescence microscopy, atomic force microscopy, optical/magnetic tweezers, however, most of these approaches rely on complicated instruments or materials, as well as skilled operators, which could limit their potential broad applications in regular biological laboratories.<br><br>RESULTS: A new type of smartphone-based electrochemical sensor is developed here for cellular force measurement. In this system, a double-stranded DNA-based force probe, known as tension gauge tether, is attached to the surface of a gold screen-printed electrode, which is then incorporated into a portable smartphone-based electrochemical device. Cellular force-induced DNA detachment on the sensor surface results in multiple redox reporters to reach the surface of the electrode and generate enhanced electrochemical signals. To further improve the sensitivity, a CRISPR-Cas12a system has also been incorporated to cleave the remaining surface-attached anchor DNA strand. Using integrin-mediated tension as an example, piconewton-scale adhesion forces generated by&#xa0;&#x2264;&#xa0;10 HeLa cells could now be reliably detected. Meanwhile, the threshold forces of these electrochemical sensors can also be modularly tuned to detect different levels of cellular forces.<br><br>SIGNIFICANCE: These novel DNA-based highly sensitive, portable, cost-efficient, and easy-to-use electrochemical sensors can be potentially powerful tools for detecting different cell-generated molecular forces. Functioning as complementary tools with traction force microscopy and fluorescent probes, these electrochemical sensors can be straightforwardly applied in regular biological laboratories for understanding the basic mechanical principles of cell signaling and for developing novel strategies and materials in tissue engineering, regenerative medicine, and cell therapy.}, }
@article {pmid39615707, year = {2024}, author = {Kadkhoda, H and Gholizadeh, P and Ghotaslou, R and Nabizadeh, E and Pirzadeh, T and Ahangarzadeh Rezaee, M and Feizi, H and Samadi Kafil, H and Aghazadeh, M}, title = {Role of CRISPR-cas system on virulence traits and carbapenem resistance in clinical Klebsiella pneumoniae isolates.}, journal = {Microbial pathogenesis}, volume = {199}, number = {}, pages = {107151}, doi = {10.1016/j.micpath.2024.107151}, pmid = {39615707}, issn = {1096-1208}, abstract = {BACKGROUND AND OBJECTIVES: The bacterial adaptive immune system known as CRISPR-Cas (clustered regularly interspersed short palindromic repeats-CRISPR-associated protein) is engaged in defense against various mobile genetic elements (MGEs) such as plasmids and bacteriophages. The purpose of this study was to characterize the CRISPR-Cas systems in carbapenem-resistant Klebsiella pneumoniae isolates and assess any possible correlation between these systems with antibiotic susceptibility, biofilm formation, and bacterial virulence.<br><br>MATERIALS AND METHODS: A total of 156 CRKP isolates were collected from different specimens of the inpatients. Biofilm formation and antibiotic susceptibility testing were evaluated using standard methods. Furthermore, the CRISPR-Cas system subtype genes, 11 carbapenemase genes, and 17 virulence genes were identified using separate standard PCR reactions. The diversity of the isolates was determined by random amplified polymorphic DNA (RAPD)-PCR.<br><br>RESULTS: The development of biofilms and antibiotic susceptibility of several CRKP isolates were significantly correlated with the absence or presence of the CRISPR-Cas system. PCR analysis of carbapenemase genes revealed that the frequency of the blaNDM-1 gene was significantly higher in the isolates with the subtype I-E CRISPR-Cas system. Moreover, the isolates with the subtype I-E CRISPR-Cas system exhibited a propensity to possess more virulence genes such as allS, k2A, wcaG, aerobactin, rmpA, iroN, magA, rmpA2, kfu, iutA, iucB, ybtS, repA, and terW.<br><br>CONCLUSION: CRISPR-Cas systems could affect the antibiotic susceptibility, capacity for biofilm formation, and virulence of Klebsiella pneumoniae. Our findings showed that the isolates containing the CRISPR-Cas system were moderate or strong biofilm producers and had a higher frequency of virulence genes.}, }
@article {pmid39615461, year = {2024}, author = {Li, CY and Chen, LW and Tsai, MC and Chou, YY and Lin, PX and Chang, YM and Hwu, WL and Chien, YH and Lin, JL and Chen, HA and Lee, NC and Su, PH and Hsieh, TC and Klinkhammer, H and Wang, YC and Huang, YT and Krawitz, PM and Lin, SH and Huang, LLH and Chiang, PM and Shih, MH and Chen, PC}, title = {Homozygous variant in translocase of outer mitochondrial membrane 7 leads to metabolic reprogramming and microcephalic osteodysplastic dwarfism with moyamoya disease.}, journal = {EBioMedicine}, volume = {110}, number = {}, pages = {105476}, pmid = {39615461}, issn = {2352-3964}, mesh = {Humans ; Animals ; *Zebrafish ; *Moyamoya Disease/genetics/metabolism/pathology ; *Microcephaly/genetics/metabolism ; Male ; *Homozygote ; Female ; Mitochondrial Precursor Protein Import Complex Proteins ; Proteomics/methods ; Mitochondria/metabolism/genetics ; Induced Pluripotent Stem Cells/metabolism ; Endothelial Cells/metabolism ; Dwarfism/genetics/metabolism/pathology ; Child ; Pedigree ; CRISPR-Cas Systems ; Child, Preschool ; Phenotype ; Disease Models, Animal ; Metabolic Reprogramming ; }, abstract = {BACKGROUND: Impaired mitochondrial protein import machinery leads to phenotypically heterogeneous diseases. Here, we report a recurrent homozygous missense variant in the gene that encodes the translocase of outer mitochondrial membrane 7 (TOMM7) in nine patients with microcephaly, short stature, facial dysmorphia, atrophic macular scarring, and moyamoya disease from seven unrelated families.<br><br>METHODS: To prove the causality of the TOMM7 variant, mitochondrial morphology, proteomics, and respiration were investigated in CRISPR/Cas9-edited iPSCs-derived endothelial cells. Cerebrovascular defects and mitochondrial respiration were also examined in CRISPR/Cas9-edited zebrafish.<br><br>FINDINGS: iPSC-derived endothelial cells with homozygous TOMM7 p.P29L showed increased TOM7 stability, enlarged mitochondria, increased senescence, and defective tube formation. In addition, proteomic analysis revealed a reduced abundance of mitochondrial proteins involved in ATP synthesis or coordinating TCA cycle and gluconeogenesis. Moreover, mitochondrial respiration was slightly decreased while ATP production from glycolysis was significantly increased. Furthermore, deletion of tomm7 in zebrafish caused craniofacial and cerebrovascular defects that recapitulated human phenotypes. Notably, homozygous iPSCs differentially expressed genes involved in glycolysis and response to hypoxia. Finally, the metabolic imbalance was evidenced by decreased oxygen consumption, increased level of hexokinase 2, and enhanced glycolysis in endothelial cells derived from the patient's iPSCs.<br><br>INTERPRETATION: These results revealed the essential role of TOMM7 in balancing cellular sources of energy production at both proteomic and transcriptomic levels and provided the molecular mechanisms through which TOMM7 p.P29L variant leads to an autosomal recessive microcephalic osteodysplastic dwarfism with moyamoya disease.<br><br>FUNDING: This work is supported by National Science and Technology Council grants and National Cheng Kung University Hospital.}, }
@article {pmid39614269, year = {2024}, author = {Wang, Y and Yu, F and Zhang, K and Shi, K and Chen, Y and Li, J and Li, X and Zhang, L}, title = {End-point RPA-CRISPR/Cas12a-based detection of Enterocytozoon bieneusi nucleic acid: rapid, sensitive and specific.}, journal = {BMC veterinary research}, volume = {20}, number = {1}, pages = {540}, pmid = {39614269}, issn = {1746-6148}, support = {22A310019//Key Scientific Research Foundation of the Higher Education Institutions of Henan Province/ ; 2023YFD1801200//National Key Research and Development Program of China/ ; 231111111500//Key Research and Development Special Project of Henan Province of China/ ; }, mesh = {*Enterocytozoon/genetics/isolation &amp; purification ; *Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Microsporidiosis/veterinary/diagnosis/microbiology ; Animals ; DNA, Fungal/genetics ; Limit of Detection ; Feces/microbiology ; Dogs ; }, abstract = {Enterocytozoon bieneusi is a common species of microsporidia that infects humans and animals. Current methods for detecting E. bieneusi infections have trade-offs in sensitivity, specificity, simplicity, cost and speed and are thus unacceptable for clinical application. We tested the effectiveness of a previously reported CRISPR/Cas12a-based method (ReCTC) when used for the nucleic acid detection of E. bieneusi. The limit of detection (LOD) and the specificity of the expanded ReCTC were evaluated using prepared target DNA, and the accuracy of the ReCTC-based detection of E. bieneusi in clinical samples was validated. The ReCTC method was successfully used for the nucleic acid detection of E. bieneusi. The sensitivity test indicated an LOD of 3.7 copies/µl for the ReCTC-based fluorescence and lateral flow strip methods. In specificity test involving other common enteric pathogens, a fluorescent signal and/or test line appeared only when the sample was positive for E. bieneusi. These results demonstrated that the ReCTC method can successfully detect E. bieneusi in clinical samples. The ReCTC method was successfully used to detect E. bieneusi nucleic acid with high sensitivity and specificity. It had excellent performance in clinical DNA samples and was superior to nested polymerase chain reaction. Furthermore, the ReCTC method demonstrated its capability for use in on-site detection.}, }
@article {pmid39614172, year = {2024}, author = {Yang, W and Zhu, JK and Jin, W}, title = {A catalog of gene editing sites and genetic variations in editing sites in model organisms.}, journal = {BMC genomics}, volume = {25}, number = {1}, pages = {1153}, pmid = {39614172}, issn = {1471-2164}, support = {2021YFA1300404//National Key R&amp;D Program of China/ ; 2021YFA0909300//National Key R&amp;D Program of China/ ; 32188102//National Natural Science Foundation of China/ ; 32170646//National Natural Science Foundation of China/ ; }, mesh = {*Gene Editing ; Animals ; Humans ; *CRISPR-Cas Systems ; *Genetic Variation ; Mice ; Receptors, CCR5/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; Oryza/genetics ; Polymorphism, Single Nucleotide ; Arabidopsis/genetics ; }, abstract = {BACKGROUND: CRISPR-Cas systems require a protospacer adjacent motif (PAM), which plays an essential role in self/non-self discrimination in their natural context, to cleave DNA for genome editing. Unfortunately, common genetic variation is distributed throughout genomes, which can block recognition of target sites by Cas proteins. However, little information is available about the distribution of editing sites in model organisms and how often common variation overlaps with those PAM sites.<br><br>RESULTS: Herein, we characterized six representative Cas proteins (Cas9, Cas12a, Cas12b, Cas12i, Cas12j and Cas12l) genomic editing sites in ten model organisms (yeast, flatworms, flies, zebrafish, mice, humans, rice, maize, Arabidopsis and tomato). We demonstrated that there were more than 34 editing sites per kilobase on average in these genomes. In each genome, 91.69-99.83% and 95.4-99.73% of genes had at least one unique editing site in exon and promoter, respectively. Depending on publicly available genomic diversity data, we identified the variations (SNPs and InDels) in editing sites in humans and rice, indicating the risk in the application of CRISPR/Cas technology. Finally, using CCR5 and BCL11A as examples, we revealed variation site was a factor that must be considered when designing sgRNA.<br><br>CONCLUSIONS: Our findings not only revealed the distribution characteristics of editing sites of six representative Cas proteins in ten model organism genomes but also shed light on the adverse effect of variation sites on target site recognition. Our current work will serve as a reminder of the risks of CRISPR application.}, }
@article {pmid39614043, year = {2025}, author = {Lei, T and Zhang, G}, title = {Generation of Cdc20 RNAi-Sensitive Cell Lines to Study Mitotic Exit.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2874}, number = {}, pages = {9-20}, pmid = {39614043}, issn = {1940-6029}, mesh = {*Cdc20 Proteins/genetics/metabolism ; *Mitosis/genetics ; *RNA Interference ; *CRISPR-Cas Systems ; Humans ; Cell Line ; Kinetochores/metabolism ; M Phase Cell Cycle Checkpoints/genetics ; }, abstract = {Accurate mitotic progression ensures the fidelity of genome passage. Cdc20 is a key mitotic regulator. It promotes mitotic exit by activating the anaphase-promoting complex or cyclosome (APC/C) and monitors kinetochore-microtubule attachment through activating the spindle assembly checkpoint (SAC). Precise characterization of Cdc20 requires efficient depletion of endogenous Cdc20, which is extremely difficult to achieve by RNA interference (RNAi). This chapter describes the methodology to generate Cdc20 RNAi-sensitive cell lines with the help of CRISPR/Cas9 technology. These cell lines are highly sensitive to Cdc20 RNAi and provide a very useful tool for Cdc20 functionality investigation without the interference of endogenous Cdc20 protein. Similar strategy could be applied to other genes.}, }
@article {pmid39612312, year = {2024}, author = {Liu, Y and Liu, Q and Yi, C and Liu, C and Shi, Q and Wang, M and Han, F}, title = {Past innovations and future possibilities in plant chromosome engineering.}, journal = {Plant biotechnology journal}, volume = {}, number = {}, pages = {}, doi = {10.1111/pbi.14530}, pmid = {39612312}, issn = {1467-7652}, support = {31991212//National Natural Science Foundation of China/ ; 2023ZD04025//Biological Breeding-National Science and Technology Major Project/ ; }, abstract = {Plant chromosome engineering has emerged as a pivotal tool in modern plant breeding, facilitating the transfer of desirable traits through the incorporation of alien chromosome fragments into plants. Here, we provide a comprehensive overview of the past achievements, current methodologies and future prospects of plant chromosome engineering. We begin by examining the successful integration of specific examples such as the incorporation of rye chromosome segments (e.g. the 1BL/1RS translocation), Dasypyrum villosum segments (e.g. the 6VS segment for powdery mildew resistance), Thinopyrum intermedium segments (e.g. rust resistance genes) and Thinopyrum elongatum segments (e.g. Fusarium head blight resistance genes). In addition to trait transfer, advancements in plant centromere engineering have opened new possibilities for chromosomal manipulation. This includes the development of plant minichromosomes via centromere-mediated techniques, the generation of haploids through CENH3 gene editing, and the induction of aneuploidy using KaryoCreate. The advent of CRISPR/Cas technology has further revolutionized chromosome engineering, enabling large-scale chromosomal rearrangements, such as inversions and translocations, as well as enabling targeted insertion of large DNA fragments and increasing genetic recombination frequency. These advancements have significantly expanded the toolkit for genetic improvement in plants, opening new horizons for the future of plant breeding.}, }
@article {pmid39611587, year = {2024}, author = {Sayid, R and van den Hurk, AWM and Rothschild-Rodriguez, D and Herrema, H and de Jonge, PA and Nobrega, FL}, title = {Characteristics of phage-plasmids and their impact on microbial communities.}, journal = {Essays in biochemistry}, volume = {68}, number = {5}, pages = {583-592}, pmid = {39611587}, issn = {1744-1358}, support = {AUF 5188//Amsterdam University Fund/ ; PhD fellowship//Amsterdam Cardiovascular Sciences, Amsterdam University Medical Centers (ACS)/ ; //Bowel Research UK (BRUK)/ ; Aspasia premium (015.017.050)//Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO)/ ; BB/T008768/1//Biotechnology and Biological Sciences Research Council (BBSRC)/ ; Black Futures Research Studentship//University of Southampton (University of Southampton UK)/ ; }, mesh = {*Bacteriophages/physiology ; *Plasmids/genetics ; *Bacteria/virology/genetics ; *Microbiota ; CRISPR-Cas Systems ; }, abstract = {Bacteria host various foreign genetic elements, most notably plasmids and bacteriophages (or phages). Historically, these two classes were seen as separate, but recent research has shown considerable interplay between them. Phage-plasmids (P-Ps) exhibit characteristics of both phages and plasmids, allowing them to exist extrachromosomally within bacterial hosts as plasmids, but also to infect and lyse bacteria as phages. This dual functionality enables P-Ps to utilize the modes of transmission of both phage and plasmids, facilitating the rapid dissemination of genetic material, including antibiotic resistance and virulence genes, throughout bacterial populations. Additionally, P-Ps have been found to encode toxin-antitoxin and CRISPR-Cas adaptive immune systems, which enhance bacterial survival under stress and provide immunity against other foreign genetic elements. Despite a growing body of literature on P-Ps, large gaps remain in our understanding of their ecological roles and environmental prevalence. This review aims to synthesise existing knowledge and identify research gaps on the impacts of P-Ps on microbial communities.}, }
@article {pmid39610939, year = {2024}, author = {Chu, LL and Sohng, JK and Bae, H and Dhakal, D}, title = {Editorial: Recent advances in application of synthetic biology for production of bioactive compounds, volume II.}, journal = {Frontiers in bioengineering and biotechnology}, volume = {12}, number = {}, pages = {1517610}, doi = {10.3389/fbioe.2024.1517610}, pmid = {39610939}, issn = {2296-4185}, }
@article {pmid39609592, year = {2024}, author = {Geuverink, WP and Houtman, D and Retel Helmrich, IRA and Kist, JD and Henneman, L and Cornel, MC and Riedijk, SR and , }, title = {A decade of public engagement regarding human germline gene editing: a systematic scoping review.}, journal = {European journal of human genetics : EJHG}, volume = {}, number = {}, pages = {}, pmid = {39609592}, issn = {1476-5438}, abstract = {Following the discovery of the CRISPR-Cas technology in 2012, there has been a growing global call for public engagement regarding the potential use of human germline gene editing (HGGE). In this systematic scoping review, we aim to evaluate public engagement studies considering the following questions based on three points of attention: 1) Inclusion of underrepresented groups: who have been engaged? 2) Gathering values: what output has been reported? 3) Reaching societal impact: what objectives of public engagement have been reported? A systematic literature search from 2012 to 2023 identified 3464 articles reporting on public engagement studies regarding HGGE retrieved from 12 databases. After screening, 52 full-text articles were assessed for eligibility, resulting in 36 articles that cover 31 public engagement studies. We conclude that co-created efforts are needed to engage underrepresented groups as well as to yield values rather than acceptance levels, and to concretise how engagement might result in societal impact.}, }
@article {pmid39609418, year = {2024}, author = {Miskalis, A and Shirguppe, S and Winter, J and Elias, G and Swami, D and Nambiar, A and Stilger, M and Woods, WS and Gosstola, N and Gapinske, M and Zeballos, A and Moore, H and Maslov, S and Gaj, T and Perez-Pinera, P}, title = {SPLICER: a highly efficient base editing toolbox that enables in vivo therapeutic exon skipping.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10354}, pmid = {39609418}, issn = {2041-1723}, support = {R01 GM131272/GM/NIGMS NIH HHS/United States ; MDA602798//Muscular Dystrophy Association (Muscular Dystrophy Association Inc.)/ ; UL1 TR001422/TR/NCATS NIH HHS/United States ; 17SDG33650087//American Heart Association (American Heart Association, Inc.)/ ; 1U01NS122102//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 1R01NS123556//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; 1R01GM127497//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01 GM141296/GM/NIGMS NIH HHS/United States ; 1R01GM141296//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01 GM127497/GM/NIGMS NIH HHS/United States ; U01 NS122102/NS/NINDS NIH HHS/United States ; R01 NS123556/NS/NINDS NIH HHS/United States ; T32 EB019944/EB/NIBIB NIH HHS/United States ; 1R01GM131272//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; }, mesh = {*Exons/genetics ; Animals ; *Gene Editing/methods ; Humans ; Mice ; *Alzheimer Disease/genetics/therapy/metabolism ; Disease Models, Animal ; HEK293 Cells ; CRISPR-Cas Systems ; Mice, Transgenic ; RNA Splice Sites/genetics ; Amyloid beta-Peptides/metabolism/genetics ; RNA Splicing ; }, abstract = {Exon skipping technologies enable exclusion of targeted exons from mature mRNA transcripts, which have broad applications in medicine and biotechnology. Existing techniques including antisense oligonucleotides, targetable nucleases, and base editors, while effective for specific applications, remain hindered by transient effects, genotoxicity, and inconsistent exon skipping. To overcome these limitations, here we develop SPLICER, a toolbox of next-generation base editors containing near-PAMless Cas9 nickase variants fused to adenosine or cytosine deaminases for the simultaneous editing of splice acceptor (SA) and splice donor (SD) sequences. Synchronized SA and SD editing improves exon skipping, reduces aberrant splicing, and enables skipping of exons refractory to single splice site editing. To demonstrate the therapeutic potential of SPLICER, we target APP exon 17, which encodes amino acids that are cleaved to form Aβ plaques in Alzheimer's disease. SPLICER reduces the formation of Aβ42 peptides in vitro and enables efficient exon skipping in a mouse model of Alzheimer's disease. Overall, SPLICER is a widely applicable and efficient exon skipping toolbox.}, }
@article {pmid39609269, year = {2024}, author = {Li, C and Xiao, Y and Zhou, J and Liu, S and Zhang, L and Song, X and Guo, X and Song, Q and Zhao, J and Deng, N}, title = {Knockout of onecut2 inhibits proliferation and promotes apoptosis of tumor cells through SKP2-mediated p53 acetylation in hepatocellular carcinoma.}, journal = {Cellular and molecular life sciences : CMLS}, volume = {81}, number = {1}, pages = {469}, pmid = {39609269}, issn = {1420-9071}, support = {81972705//National Natural Science Foundation of China/ ; }, mesh = {*S-Phase Kinase-Associated Proteins/metabolism/genetics ; *Carcinoma, Hepatocellular/pathology/metabolism/genetics ; Humans ; *Liver Neoplasms/pathology/metabolism/genetics ; *Apoptosis/genetics ; *Cell Proliferation/genetics ; Acetylation ; Animals ; *Tumor Suppressor Protein p53/metabolism/genetics ; Mice ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Mice, Nude ; Gene Knockout Techniques ; CRISPR-Cas Systems/genetics ; }, abstract = {Onecut2 (OC2) plays a vital regulatory role in tumor growth, metastasis and angiogenesis. In this study, we report the regulatory role and specific molecular mechanism of OC2 in the apoptosis of hepatocellular carcinoma (HCC) cells. We found that OC2 knockout via the CRISPR/CAS9 system not only significantly inhibited the proliferation and angiogenesis of HCC cells but also significantly promoted apoptosis. The apoptosis rate of the OC2 knockout HCC cell line reached 30.514%. In a mouse model, the proliferation inhibition rate of tumor cells reached 98.8%. To explore the mechanism of apoptosis, ChIP-Seq and dual-luciferase reporter assays were carried out. The results showed that OC2 could directly bind to the promotor of SKP2 and regulate its expression. Moreover, downregulating the expression of OC2 and SKP2 could release p300, promote the acetylation of p53, increase the expression of p21 and p27, and promote the apoptosis of HCC cells. Moreover, the overexpression of OC2 or SKP2 in the knockout HCC cell line clearly inhibited the acetylation level of p53 and reduced cell apoptosis. This study revealed that OC2 could regulate the apoptosis of HCC cells through the SKP2/p53/p21 axis, which may provide some therapeutic targets for HCC in the clinic.}, }
@article {pmid39608317, year = {2025}, author = {Nguyen, HV and Hwang, S and Lee, SW and Jin, E and Lee, MH}, title = {Detection of HPV 16 and 18 L1 genes by a nucleic acid amplification-free electrochemical biosensor powered by CRISPR/Cas9.}, journal = {Bioelectrochemistry (Amsterdam, Netherlands)}, volume = {162}, number = {}, pages = {108861}, doi = {10.1016/j.bioelechem.2024.108861}, pmid = {39608317}, issn = {1878-562X}, mesh = {*Biosensing Techniques/methods ; *Human papillomavirus 16/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *Human papillomavirus 18/genetics ; *Electrochemical Techniques/methods ; Humans ; *Oncogene Proteins, Viral/genetics ; Capsid Proteins/genetics ; Limit of Detection ; Papillomavirus Infections/diagnosis/virology ; Nucleic Acid Amplification Techniques/methods ; Female ; }, abstract = {Cervical cancer, closely linked to Human Papillomavirus (HPV) infection, remains a significant health threat for women worldwide. Conventional HPV detection methods, such as reverse transcription polymerase chain reaction (RT-PCR), rely on nucleic acid amplification (NAA), which can be costly and time-consuming. This study introduces an NAA-free electrochemical Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based biosensor designed to detect HPV 16 and HPV 18 L1 genes simultaneously. The system utilizes a Cas9-single guided RNA complex to initiate a selective cleavage reaction, releasing Methylene blue or Ferrocene-labeled fragments correlate to L1 gene concentrations. These fragments then interact with modified gold electrodes immobilized with a complementary probe, allowing precise electrochemical signal measurement during hybridization. The biosensor offers a wide detection range from 1 fM to 10 nM, with detection limits as low as 0.4 fM for HPV 16 L1 and 0.51 fM for HPV 18 L1, providing a sensitive and efficient solution for L1 gene detection. Additionally, its specificity and sensitivity closely match RT-PCR results in clinical testing, highlighting its potential for molecular diagnostics and point-of-care applications.}, }
@article {pmid39607706, year = {2024}, author = {Deng, C and Xin, R and Li, X and Zhang, J and Fan, L and Qiu, Y and Zhao, L}, title = {Optogenetic control of Corynebacterium glutamicum gene expression.}, journal = {Nucleic acids research}, volume = {52}, number = {22}, pages = {14260-14276}, pmid = {39607706}, issn = {1362-4962}, support = {32301214//National Natural Science Foundation of China/ ; //Shanghai Municipal Education Commission/ ; }, mesh = {*Corynebacterium glutamicum/genetics/metabolism ; *Gene Expression Regulation, Bacterial ; *Optogenetics/methods ; Light ; CRISPR-Cas Systems ; Transcription Factors/metabolism/genetics ; Bioreactors ; Bacterial Proteins/metabolism/genetics ; Oligosaccharides/metabolism/biosynthesis ; Metabolic Engineering/methods ; }, abstract = {Corynebacterium glutamicum is a key industrial workhorse for producing amino acids and high-value chemicals. Balancing metabolic flow between cell growth and product synthesis is crucial for enhancing production efficiency. Developing dynamic, broadly applicable, and minimally toxic gene regulation tools for C. glutamicum remains challenging, as optogenetic tools ideal for dynamic regulatory strategies have not yet been developed. This study introduces an advanced light-controlled gene expression system using light-controlled RNA-binding proteins (RBP), a first for Corynebacterium glutamicum. We established a gene expression regulation system, 'LightOnC.glu', utilizing the light-controlled RBP to construct light-controlled transcription factors in C. glutamicum. Simultaneously, we developed a high-performance light-controlled gene interference system using CRISPR/Cpf1 tools. The metabolic flow in the synthesis network was designed to enable the production of chitin oligosaccharides (CHOSs) and chondroitin sulphate oligosaccharides A (CSA) for the first time in C. glutamicum. Additionally, a light-controlled bioreactor was constructed, achieving a CHOSs production concentration of 6.2 g/L, the highest titer recorded for CHOSs biosynthesis to date. Herein, we have established a programmable light-responsive genetic circuit in C. glutamicum, advancing the theory of dynamic regulation based on light signaling. This breakthrough has potential applications in optimizing metabolic modules in other chassis cells and synthesizing other compounds.}, }
@article {pmid39607705, year = {2024}, author = {Kadam, A and Shilo, S and Naor, H and Wainstein, A and Brilon, Y and Feldman, T and Minden, M and Kaushansky, N and Chapal-Ilani, N and Shlush, L}, title = {Utilizing insights of DNA repair machinery to discover MMEJ deletions and novel mechanisms.}, journal = {Nucleic acids research}, volume = {52}, number = {22}, pages = {e106}, pmid = {39607705}, issn = {1362-4962}, support = {isf-nsfc 2427/18//Rising Tide Foundation/ ; //Steven B. Rubenstein research fund/ ; //Applebaum Foundation/ ; //Ernest and Bonnie Beutler Research Program/ ; //Sagol Institute for Longevity Research/ ; //Barry and Eleanore Reznik Family Cancer Research fund/ ; 1123/21//ISF-IPMP-Israel Precision Medicine Program/ ; }, mesh = {Humans ; *Sequence Deletion ; *DNA End-Joining Repair ; Algorithms ; DNA Repair/genetics ; Exome/genetics ; CRISPR-Cas Systems ; Leukemia, Myeloid, Acute/genetics ; Female ; G-Quadruplexes ; Breast Neoplasms/genetics ; }, abstract = {We developed Del-read, an algorithm targeting medium-sized deletions (6-100 bp) in short-reads, which are challenging for current variant callers relying on alignment. Our focus was on Micro-Homolog mediated End Joining deletions (MMEJ-dels), prevalent in myeloid malignancies. MMEJ-dels follow a distinct pattern, occurring between two homologies, allowing us to generate a comprehensive list of MMEJ-dels in the exome. Using Del-read, we identified numerous novel germline and somatic MMEJ-dels in BEAT-AML and TCGA-breast datasets. Validation in 672 healthy individuals confirmed their presence. These novel MMEJ-dels were linked to genomic features associated with replication stress, like G-quadruplexes and minisatellite. Additionally, we observed a new category of MMEJ-dels with an imperfect-match at the flanking sequences of the homologies, suggesting a mechanism involving mispairing in homology alignment. We demonstrated robustness of the repair system despite CRISPR/Cas9-induced mismatches in the homologies. Further analysis of the canonical ASXL1 deletion revealed a diverse array of these imperfect-matches. This suggests a potentially more flexible and error-prone MMEJ repair system than previously understood. Our findings highlight Del-read's potential in uncovering previously undetected deletions and deepen our understanding of repair mechanisms.}, }
@article {pmid39607301, year = {2024}, author = {Du, B and Chen, M and Chang, L and Zhang, X and Zhang, X and Wang, X and Gong, P and Zhang, N and Zhang, X and Li, X and Li, J}, title = {Immunization with the NcMYR1 gene knockout strain effectively protected C57BL/6 mice and their pups against the Neospora caninum challenge.}, journal = {Virulence}, volume = {15}, number = {1}, pages = {2427844}, pmid = {39607301}, issn = {2150-5608}, mesh = {Animals ; *Neospora/genetics/immunology/pathogenicity ; *Coccidiosis/prevention &amp; control/immunology/parasitology/veterinary ; *Mice, Inbred C57BL ; Mice ; *Protozoan Proteins/genetics/immunology ; *Protozoan Vaccines/immunology/genetics ; *Vaccines, Attenuated/immunology/genetics/administration &amp; dosage ; *Gene Knockout Techniques ; Female ; Immunization ; Virulence ; CRISPR-Cas Systems ; }, abstract = {Neospora caninum is an important protozoan parasite that causes abortion in cattle and nervous system dysfunction in dogs. No effective drugs and vaccines for neosporosis are available. Further elucidation of proteins related to N. caninum virulence will provide potential candidates for vaccine development against neosporosis. In the present study, N. caninum c-Myc regulatory protein (NcMYR1) gene knockout strains (ΔNcMYR1-1, ΔNcMYR1-2, and ΔNcMYR1-3) were generated using the CRISPR-Cas9 gene editing system to investigate phenotype changes and the potential of the ΔNcMYR1-1 strain as an attenuated vaccine, and this is the first time of using the N. caninum CRISPR-Cas9 gene knockout strain as an attenuated vaccine. NcMYR1 was determined to be a cytoplasmic protein in N. caninum tachyzoites. The deficiency of NcMYR1 decreased the plaque area and the rate of invasion, replication, and egression of the parasites. ΔNcMYR1-1 strain-infected C57BL/6 mice had 100% survival rate, reduced parasite burden, and alleviated pathological changes in tissues compared with those in Nc-1 strain-infected mice. Immunization with ΔNcMYR1-1 tachyzoites increased the productions of cytokines in mice, with a survival rate reaching 80%, and the parasite burdens in the liver and spleen were greatly reduced when challenged with the Nc-1 strain with a lethal dose after 40 days of ΔNcMYR1-1 tachyzoite immunization. ΔNcMYR1 immunization could decrease the abortion rate of female mice from 71.4% to 12.5% and increase the survival rate of pups from 12.5% to 83.3% against the N. caninum challenge. Above all, NcMYR1 is a virulence factor and the ΔNcMYR1-1 strain could be used as a candidate vaccine against N. caninum infection and vertical transmission.}, }
@article {pmid39606944, year = {2024}, author = {Gautier, LJ}, title = {CRISPR in 3D: Innovations in Disease Modelling and Personalized Medicine.}, journal = {BioTechniques}, volume = {}, number = {}, pages = {1-6}, doi = {10.1080/07366205.2024.2418748}, pmid = {39606944}, issn = {1940-9818}, abstract = {The CRISPR-Cas system of genetic engineering has had a significant impact on science and society since its advent in 2013. CRISPR integration with 3D culture systems such as organ-on-a-chips, as well as fast emerging commercial technologies, has encouraged translation of more complex pathophysiological modelling and personalized medicine.[Formula: see text].}, }
@article {pmid39606390, year = {2024}, author = {Orosco, C and Rananaware, SR and Huang, B and Hanna, MP and Ahmadimashhadi, MR and Lewis, JG and Baugh, MP and Bodin, AP and Flannery, SJ and Lange, IH and Fang, ZR and Karalkar, VN and Meister, KS and Jain, PK}, title = {DNA-guided CRISPR/Cas12 for RNA targeting.}, journal = {medRxiv : the preprint server for health sciences}, volume = {}, number = {}, pages = {}, pmid = {39606390}, support = {R21 AI156321/AI/NIAID NIH HHS/United States ; R21 AI168795/AI/NIAID NIH HHS/United States ; R35 GM147788/GM/NIGMS NIH HHS/United States ; R61 AI181016/AI/NIAID NIH HHS/United States ; }, abstract = {CRISPR-Cas nucleases are transforming genome editing, RNA editing, and diagnostics but have been limited to RNA-guided systems. We present ΨDNA, a DNA-based guide for Cas12 enzymes, engineered for specific and efficient RNA targeting. ΨDNA mimics a crRNA but with a reverse orientation, enabling stable Cas12-RNA assembly and activating trans-cleavage without RNA components. ΨDNAs are effective in sensing short and long RNAs and demonstrated 100% accuracy for detecting HCV RNA in clinical samples. We discovered that ΨDNAs can guide certain Cas12 enzymes for RNA targeting in cells, enhancing mRNA degradation via ribosome stalling and enabling multiplex knockdown of multiple RNA transcripts. This study establishes ΨDNA as a robust alternative to RNA guides, augmenting the potential of CRISPR-Cas12 for diagnostic applications and targeted RNA modulation in cellular environments.}, }
@article {pmid39605323, year = {2024}, author = {Alves, CRR and Das, S and Krishnan, V and Ha, LL and Fox, LR and Stutzman, HE and Shamber, CE and Kalailingam, P and McCarthy, S and Lino Cardenas, CL and Fong, CE and Imai, T and Mitra, S and Yun, S and Wood, RK and Benning, FMC and Lawton, J and Kim, N and Silverstein, RA and da Silva, JF and de la Cruz, D and Richa, R and Malhotra, R and Chung, DY and Chao, LH and Tsai, SQ and Maguire, CA and Lindsay, ME and Kleinstiver, BP and Musolino, PL}, title = {In vivo Treatment of a Severe Vascular Disease via a Bespoke CRISPR-Cas9 Base Editor.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, pmid = {39605323}, issn = {2692-8205}, support = {R35 GM142553/GM/NIGMS NIH HHS/United States ; R01 DC017117/DC/NIDCD NIH HHS/United States ; R01 HL162928/HL/NHLBI NIH HHS/United States ; K08 NS112601/NS/NINDS NIH HHS/United States ; P01 HL142494/HL/NHLBI NIH HHS/United States ; DP2 CA281401/CA/NCI NIH HHS/United States ; R01 NS125353/NS/NINDS NIH HHS/United States ; K01 NS134784/NS/NINDS NIH HHS/United States ; U01 AI176470/AI/NIAID NIH HHS/United States ; U01 AI176471/AI/NIAID NIH HHS/United States ; }, abstract = {Genetic vascular disorders are prevalent diseases that have diverse etiologies and few treatment options. Pathogenic missense mutations in the alpha actin isotype 2 gene (ACTA2) primarily affect smooth muscle cell (SMC) function and cause multisystemic smooth muscle dysfunction syndrome (MSMDS), a genetic vasculopathy that is associated with stroke, aortic dissection, and death in childhood. Here, we explored genome editing to correct the most common MSMDS-causative mutation ACTA2 R179H. In a first-in-kind approach, we performed mutation-specific protein engineering to develop a bespoke CRISPR-Cas9 enzyme with enhanced on-target activity against the R179H sequence. To directly correct the R179H mutation, we screened dozens of configurations of base editors (comprised of Cas9 enzymes, deaminases, and gRNAs) to develop a highly precise corrective A-to-G edit with minimal deleterious bystander editing that is otherwise prevalent when using wild-type SpCas9 base editors. We then created a murine model of MSMDS that exhibits phenotypes consistent with human patients, including vasculopathy and premature death, to explore the in vivo therapeutic potential of this base editing strategy. Delivery of the customized base editor via an engineered SMC-tropic adeno-associated virus (AAV-PR) vector substantially prolonged survival and rescued systemic phenotypes across the lifespan of MSMDS mice, including in the vasculature, aorta, and brain. Together, our optimization of a customized base editor highlights how bespoke CRISPR-Cas enzymes can enhance on-target correction while minimizing bystander edits, culminating in a precise editing approach that may enable a long-lasting treatment for patients with MSMDS.}, }
@article {pmid39605316, year = {2024}, author = {Weber, R and Vasella, F and Klimko, A and Silginer, M and Lamfers, M and Neidert, MC and Regli, L and Schwank, G and Weller, M}, title = {Targeting the IDH1 [R132H] mutation in gliomas by CRISPR/Cas precision base editing.}, journal = {Neuro-oncology advances}, volume = {6}, number = {1}, pages = {vdae182}, pmid = {39605316}, issn = {2632-2498}, abstract = {BACKGROUND: Gliomas, the most frequent malignant primary brain tumors, lack curative treatments. Understanding glioma-specific molecular alterations is crucial to develop novel therapies. Among them, the biological consequences of the isocitrate dehydrogenase 1 gene mutation (IDH1 [R132H]) remain inconclusive despite its early occurrence and widespread expression.<br><br>METHODS: We thus employed CRISPR/Cas adenine base editors, which allow precise base pair alterations with minimal undesirable effects, to correct the IDH1 [R132H] mutation.<br><br>RESULTS: Successful correction of the IDH1 [R132H] mutation in primary patient-derived cell models led to reduced IDH1 [R132H] protein levels and decreased production of 2-hydroxyglutarate, but increased proliferation. A dual adeno-associated virus split intein system was used to successfully deliver the base editor in vitro and in vivo.<br><br>CONCLUSIONS: Taken together, our study provides a strategy for a precise genetic intervention to target the IDH1 [R132H] mutation, enabling the development of accurate models to study its impact on glioma biology and serving as a framework for an in vivo gene therapy.}, }
@article {pmid39604746, year = {2024}, author = {Saha, UB and Dixit, KK and Jadhav, SV and Pathak, KN and Gupta, NS and Saroj, SD}, title = {Genomic Insights of Multidrug-Resistant Enterococcus faecalis and Acinetobacter baumannii Isolated from a Sepsis Patient with Pauci-Immune Crescentic Glomerulonephritis, India.}, journal = {Current microbiology}, volume = {82}, number = {1}, pages = {16}, pmid = {39604746}, issn = {1432-0991}, support = {SR/PURSE/2023/181//Department of Science and Technology, Ministry of Science and Technology, India/ ; }, mesh = {Humans ; Acinetobacter baumannii/genetics/drug effects/isolation &amp; purification ; *Acinetobacter Infections/microbiology ; *Anti-Bacterial Agents/pharmacology ; Bacteremia/microbiology ; Coinfection/microbiology ; *Drug Resistance, Multiple, Bacterial/genetics ; Enterococcus faecalis/genetics/drug effects/isolation &amp; purification ; *Genome, Bacterial ; Genomics ; *Glomerulonephritis/microbiology/genetics ; *Gram-Positive Bacterial Infections/microbiology ; India ; Microbial Sensitivity Tests ; *Sepsis/microbiology ; Whole Genome Sequencing ; }, abstract = {Acinetobacter baumannii and Enterococcus faecalis are opportunistic bacteria frequently associated with hospital-acquired infections. A. baumannii nosocomial infections in intensive care units are a worldwide problem, with high mortality rates. It may also develop rapidly multidrug resistance (MDR), extensive drug resistance (XDR), and even pan-drug resistance (PDR). Colistin resistance which is an example of pan-drug resistance, is highly alarming as it's used as a last-line antibiotic. Microbes capable of crossing epithelial barriers such as E. faecalis have developed novel strategies to counter antimicrobial agents and cause bacteremia in immunocompromised patients. However, the coinfection of these bacteria in the same patient is unusual. Here, we report a genomic investigation of the extensively drug-resistant E. faecalis and A. baumannii isolated from the blood sample of a patient diagnosed with pauci-immune crescentic glomerulonephritis (PICGN). Identification of cultures isolated from blood sample was carried out using whole-genome sequencing and resistome profiles were mapped. Whole genome sequencing revealed that E. faecalis SVJ-EF01 had a genome size of 2,935,226 bp and GC content of 37.4%, whereas A. baumannii SVJ-AC01 had a genome size of 3,730,857 bp and GC content of 39%. Draft genomes were functionally annotated demonstrating that the organism harbors multiple virulence factors and antimicrobial-resistant mechanisms including MDR efflux pumps. A. baumannii genome possessed a CRISPR-Cas system which might contribute to antimicrobial resistance. This highlights the significance of polymicrobial nature in ESKAPE pathogenesis research. This genomic investigation helps to gain insights into the virulence, resistance profile, and functional potential of these pathogens.}, }
@article {pmid39604418, year = {2024}, author = {Qiao, L and Niu, L and Wang, M and Wang, Z and Kong, D and Yu, G and Ye, H}, title = {A sensitive red/far-red photoswitch for controllable gene therapy in mouse models of metabolic diseases.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10310}, pmid = {39604418}, issn = {2041-1723}, support = {no.32430064//National Natural Science Foundation of China (National Science Foundation of China)/ ; no.32261160373//National Natural Science Foundation of China (National Science Foundation of China)/ ; }, mesh = {Animals ; *Genetic Therapy/methods ; *Optogenetics/methods ; Mice ; *Dependovirus/genetics ; Humans ; *Disease Models, Animal ; Metabolic Diseases/therapy/genetics ; Light ; Insulin/metabolism ; Cytokines/metabolism ; Obesity/therapy/genetics/metabolism ; Diabetes Mellitus, Type 1/therapy/genetics/metabolism ; CRISPR-Cas Systems ; HEK293 Cells ; Male ; Mice, Inbred C57BL ; Blood Glucose/metabolism ; Gene Expression Regulation ; Tumor Necrosis Factor Ligand Superfamily Member 14 ; }, abstract = {Red light optogenetic systems are in high demand for the precise control of gene expression for gene- and cell-based therapies. Here, we report a red/far-red light-inducible photoswitch (REDLIP) system based on the chimeric photosensory protein FnBphP (Fn-REDLIP) or PnBphP (Pn-REDLIP) and their interaction partner LDB3, which enables efficient dynamic regulation of gene expression with a timescale of seconds without exogenous administration of a chromophore in mammals. We use the REDLIP system to establish the REDLIP-mediated CRISPR-dCas9 (REDLIPcas) system, enabling optogenetic activation of endogenous target genes in mammalian cells and mice. The REDLIP system is small enough to support packaging into adeno-associated viruses (AAVs), facilitating its therapeutic application. Demonstrating its capacity to treat metabolic diseases, we show that an AAV-delivered Fn-REDLIP system achieved optogenetic control of insulin expression to effectively lower blood glucose levels in type 1 diabetes model mice and control an anti-obesity therapeutic protein (thymic stromal lymphopoietin, TSLP) to reduce body weight in obesity model mice. REDLIP is a compact and sensitive optogenetic tool for reversible and non-invasive control that can facilitate basic biological and biomedical research.}, }
@article {pmid39604381, year = {2024}, author = {Goldberg, GW and Kogenaru, M and Keegan, S and Haase, MAB and Kagermazova, L and Arias, MA and Onyebeke, K and Adams, S and Beyer, DK and Fenyö, D and Noyes, MB and Boeke, JD}, title = {Engineered transcription-associated Cas9 targeting in eukaryotic cells.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10287}, pmid = {39604381}, issn = {2041-1723}, support = {F32GM137482//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01 GM133936/GM/NIGMS NIH HHS/United States ; RM1HG009491//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; RM1 HG009491/HG/NHGRI NIH HHS/United States ; R01 GM118851/GM/NIGMS NIH HHS/United States ; F32 GM137482/GM/NIGMS NIH HHS/United States ; K99 GM147604/GM/NIGMS NIH HHS/United States ; R01GM118851//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; K99GM147604//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; }, mesh = {Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics ; *Transcription, Genetic ; *Saccharomyces cerevisiae/genetics/metabolism ; Gene Editing/methods ; DNA/metabolism/genetics ; Eukaryotic Cells/metabolism ; HEK293 Cells ; Genetic Engineering/methods ; }, abstract = {DNA targeting Class 2 CRISPR-Cas effector nucleases, including the well-studied Cas9 proteins, evolved protospacer-adjacent motif (PAM) and guide RNA interactions that sequentially license their binding and cleavage activities at protospacer target sites. Both interactions are nucleic acid sequence specific but function constitutively; thus, they provide intrinsic spatial control over DNA targeting activities but naturally lack temporal control. Here we show that engineered Cas9 fusion proteins which bind to nascent RNAs near a protospacer can facilitate spatiotemporal coupling between transcription and DNA targeting at that protospacer: Transcription-associated Cas9 Targeting (TraCT). Engineered TraCT is enabled in eukaryotic yeast or human cells when suboptimal PAM interactions limit basal activity and when one or more nascent RNA substrates are still tethered to the actively transcribed target DNA in cis. Using yeast, we further show that this phenomenon can be applied for selective editing at one of two identical targets in distinct gene loci, or, in diploid allelic loci that are differentially transcribed. Our work demonstrates that temporal control over Cas9's targeting activity at specific DNA sites may be engineered without modifying Cas9's core domains and guide RNA components or their expression levels. More broadly, it establishes co-transcriptional RNA binding as a cis-acting mechanism that can conditionally stimulate CRISPR-Cas DNA targeting in eukaryotic cells.}, }
@article {pmid39604306, year = {2024}, author = {Kim, Y and Lee, E and Kang, BC}, title = {Etiolation promotes protoplast transfection and genome editing efficiency.}, journal = {Physiologia plantarum}, volume = {176}, number = {6}, pages = {e14637}, doi = {10.1111/ppl.14637}, pmid = {39604306}, issn = {1399-3054}, support = {RS-2022-NR072144//National Research Foundation of Korea (NRF)/ ; }, mesh = {*Protoplasts/metabolism ; *Gene Editing/methods ; *Lactuca/genetics/growth &amp; development ; *Transfection/methods ; Brassica/genetics/growth &amp; development ; Genome, Plant/genetics ; CRISPR-Cas Systems ; Green Fluorescent Proteins/metabolism/genetics ; }, abstract = {In plants, DNA-free genome editing using preassembled clustered regularly interspaced short palindromic repeats (CRISPR)-ribonucleoprotein (RNP) has the advantage of avoiding transgene integration and limiting off-target effects. The efficiency of this gene editing strategy can vary, so optimization of protoplast transfection conditions is necessary to achieve maximum yield. In this study, we examined the effects of etiolation, or increased exposure to darkness during cultivation, on the transfection efficiency of protoplasts from lettuce and Chinese cabbage. Seedlings were grown under three different conditions: non-etiolated, etiolated, and de-etiolated. First, we tested PEG-mediated transfection after etiolation using a plasmid DNA for green fluorescent protein (GFP)-expression. Etiolated protoplasts had the highest percentage of GFP-expressing cells, with a 3.1-fold and 4.8-fold improvement in lettuce and Chinese cabbage, respectively, compared with non-etiolated protoplasts. We also assessed gene editing of endogenous genes after etiolation using CRISPR-RNP. Using targeted deep sequencing, we observed the highest editing efficiency in etiolated protoplasts from both plant species, for the LsPDS and LsFT genes in lettuce, this led to an 8.7-fold and 4.4-fold improvement compared with non-etiolated protoplasts, respectively. These results suggest that etiolation during seedling growth can improve transfection efficiency and DNA-free gene editing in protoplasts.}, }
@article {pmid39603754, year = {2024}, author = {Maladan, Y and Retnaningrum, E and Daryono, BS and Sarassari, R and Sari, RF and Balqis, SA and Wahid, GA and Safari, D}, title = {A New Serotyping Method of Streptococcus pneumoniae Based on CRISPR/Cas9-Targeted Sequencing.}, journal = {The Journal of molecular diagnostics : JMD}, volume = {26}, number = {12}, pages = {1045-1054}, doi = {10.1016/j.jmoldx.2024.08.009}, pmid = {39603754}, issn = {1943-7811}, mesh = {*Streptococcus pneumoniae/genetics/classification ; *CRISPR-Cas Systems/genetics ; *Serotyping/methods ; Humans ; High-Throughput Nucleotide Sequencing/methods ; Sequence Analysis, DNA/methods ; Pneumococcal Infections/microbiology/genetics ; Genome, Bacterial ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) application for targeted sequencing has made a breakthrough in the genomic research era. High diversity in the capsular polysaccharide (cps) locus of Streptococcus pneumoniae has hampered identification of the serotype. This study developed a new serotyping method for S. pneumoniae using CRISPR/Cas9-targeted sequencing with the Oxford Nanopore Technologies platform. A probe was designed at the position of the cps locus using an excision approach on two sides flanking genes between the dexB and aliA genes with approximately 20 kb. A native barcoding method was used for multiplexing. The probe will attach to a specific side followed by attachment of CRISPR/Cas9 to cut the recognition area. The study used de novo assembly to reconstruct sequence reads, which were analyzed using PneumoCRISPR, a new serotyping pipeline for Oxford Nanopore Technologies sequencing data output. Four CRISPR/Cas9 probes have been designed and recognize the cps locus of S. pneumoniae. Serotyping results align precisely with serotyping data from whole-genome sequencing. This serotyping method also allows researchers to use multiple samples in a single run. The new serotyping method based on CRISPR/Cas9-targeted sequencing holds immense promise for serotype identification of S. pneumoniae.}, }
@article {pmid39603242, year = {2024}, author = {Pahl, V and Lubrano, P and Troßmann, F and Petras, D and Link, H}, title = {Intact protein barcoding enables one-shot identification of CRISPRi strains and their metabolic state.}, journal = {Cell reports methods}, volume = {4}, number = {12}, pages = {100908}, pmid = {39603242}, issn = {2667-2375}, mesh = {*Mass Spectrometry/methods ; Ubiquitin/metabolism/genetics ; CRISPR-Cas Systems/genetics ; Escherichia coli/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Metabolome ; }, abstract = {Detecting strain-specific barcodes with mass spectrometry can facilitate the screening of genetically engineered bacterial libraries. Here, we introduce intact protein barcoding, a method to measure protein-based library barcodes and metabolites using flow injection mass spectrometry (FI-MS). Protein barcodes are based on ubiquitin with N-terminal tags of six amino acids. We demonstrate that FI-MS detects intact ubiquitin proteins and identifies the mass of N-terminal barcodes. In the same analysis, we measured relative concentrations of primary metabolites. We constructed six ubiquitin-barcoded CRISPR interference (CRISPRi) strains targeting metabolic enzymes and analyzed their metabolic profiles and ubiquitin barcodes. FI-MS detected barcodes and distinct metabolome changes in CRISPRi-targeted pathways. We demonstrate the scalability of intact protein barcoding by measuring 132 ubiquitin barcodes in microtiter plates. These results show that intact protein barcoding enables fast and simultaneous detection of library barcodes and intracellular metabolites, opening up new possibilities for mass spectrometry-based barcoding.}, }
@article {pmid39603211, year = {2025}, author = {Yan, J and Yin, B and Zhang, Q and Li, C and Chen, J and Huang, Y and Hao, J and Yi, C and Zhang, Y and Wong, SHD and Yang, M}, title = {A CRISPR-Cas12a-mediated dual-mode luminescence and colorimetric nucleic acid biosensing platform based on upconversion nanozyme.}, journal = {Biosensors &amp; bioelectronics}, volume = {270}, number = {}, pages = {116963}, doi = {10.1016/j.bios.2024.116963}, pmid = {39603211}, issn = {1873-4235}, mesh = {*Biosensing Techniques/methods ; *Colorimetry/methods ; *CRISPR-Cas Systems ; *Graphite/chemistry ; DNA, Single-Stranded/chemistry ; Limit of Detection ; Silicon Dioxide/chemistry ; Cerium/chemistry ; CRISPR-Associated Proteins/chemistry ; Luminescent Measurements/methods ; Benzidines/chemistry ; Bacterial Proteins/chemistry/genetics ; Luminescence ; Endodeoxyribonucleases ; }, abstract = {In this study, a CRISPR-Cas12a-mediated dual-mode upconversion luminescence/colorimetric nucleic acid biosensing platform is developed based on UCNP@SiO2/CeO2 (UNSC) nanozyme. Here, UNSC is conjugated with single-stranded DNA (ssDNA) probes used as both peroxidase-like nanozyme and upconversion luminescence donors. When no target nucleic acid is present, ssDNA-conjugated UNSC attaches on magnetic graphene oxide (MGO) via pi-pi stacking force, resulting in upconversion luminescence quenching (OFF) and no color change after magnetic removal of nanozymes attached on the MGO. In the presence of target nucleic acid, Cas12a is specifically activated by targeted nucleic acid and indiscriminately cleaves the ssDNA probes on UNSCs. UNSCs then detach from the MGO surface due to the weakening of binding force, leading to upconversion luminescence recovery (ON) and colorimetric change due to the existence of free nanozyme in the 3,3',5,5'-tetramethyl-benzidine assay. As a proof-of-concept, this biosensing platform shows a limit of detection of around 320 fM in the upconversion luminescence mode and ∼28.4 pM in the colorimetric mode for nucleic acid detection, respectively. This UNSC nanozyme-based CRISPR-Cas12a dual-mode biosensing system also demonstrates high selectivity, good repeatability, and facile operation, which allows easy adaption to other nucleic acid-based detection only by redesigning the sequence of CRISPR RNA.}, }
@article {pmid39603092, year = {2024}, author = {Zhang, S and Li, J and Pan, W and Ren, Q and Zhou, Y and Chen, M and Qu, J and Li, S}, title = {Establishment of a CPAMD8-GFP reporter human embryonic stem cell line, IBBDe001-B, using CRISPR/Cas9 editing.}, journal = {Stem cell research}, volume = {81}, number = {}, pages = {103615}, doi = {10.1016/j.scr.2024.103615}, pmid = {39603092}, 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 ; Genes, Reporter ; }, abstract = {The human CPAMD8 gene encodes proteins in the A2M/C3 (alpha-2-macroglobulin/complement 3) family, predominantly expressed in the distal tips of the retinal neuroepithelium that form the iris and ciliary body. Mutations in CPAMD8 have been linked to anterior segment dysplasia and congenital glaucoma. Using CRISPR/Cas9 editing, we inserted a 3*EAAAK-EGFP fluorescent tag into the CPAMD8 gene, enabling real-time observation of its expression and providing insights into its biological functions. The resulting gene-edited cell line retained normal stem cell morphology and karyotype, expressed essential pluripotency markers, and exhibited differentiation potential.}, }
@article {pmid39602282, year = {2025}, author = {Lim, RM and Lu, A and Chuang, BM and Anaraki, C and Chu, B and Halbrook, CJ and Edinger, AL}, title = {CARMIL1-AA selectively inhibits macropinocytosis while sparing autophagy.}, journal = {Molecular biology of the cell}, volume = {36}, number = {1}, pages = {ar4}, doi = {10.1091/mbc.E24-09-0434}, pmid = {39602282}, issn = {1939-4586}, mesh = {*Pinocytosis/physiology ; Humans ; *Autophagy/physiology ; Cell Line, Tumor ; Sodium-Hydrogen Exchangers/metabolism/genetics ; Amiloride/pharmacology/analogs &amp; derivatives ; Cell Proliferation ; CRISPR-Cas Systems ; }, abstract = {Macropinocytosis is reported to fuel tumor growth and drug resistance by allowing cancer cells to scavenge extracellular macromolecules. However, accurately defining the role of macropinocytosis in cancer depends on our ability to selectively block this process. 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) is widely used to inhibit macropinocytosis but affects multiple Na[+]/H[+] exchangers (NHE) that regulate cytoplasmic and organellar pH. Consistent with this, we report that EIPA slows proliferation to a greater extent than can be accounted for by macropinocytosis inhibition and triggers conjugation of ATG8 to single membranes (CASM). Knocking down only NHE1 would not avoid macropinocytosis-independent effects on pH. Moreover, contrary to published reports, NHE1 loss did not block macropinocytosis in multiple cell lines. Knocking down CARMIL1 with CRISPR-Cas9 editing limited macropinocytosis, but only by 50%. In contrast, expressing the CARMIL1-AA mutant inhibits macropinocytosis induced by a wide range of macropinocytic stimuli to a similar extent as EIPA. CARMIL1-AA expression did not inhibit proliferation, highlighting the shortcomings of EIPA as a macropinocytosis inhibitor. Importantly, autophagy, another actin dependent, nutrient-producing process, was not affected by CARMIL1-AA expression. In sum, constitutive or inducible CARMIL1-AA expression reduced macropinocytosis without affecting proliferation, RAC activation, or autophagy, other processes that drive tumor initiation and progression.}, }
@article {pmid39601843, year = {2024}, author = {Cao, L and Chen, W and Kang, W and Lei, C and Nie, Z}, title = {Engineering stimuli-responsive CRISPR-Cas systems for versatile biosensing.}, journal = {Analytical and bioanalytical chemistry}, volume = {}, number = {}, pages = {}, pmid = {39601843}, issn = {1618-2650}, support = {22034002//National Natural Science Foundation of China/ ; 92253304//National Natural Science Foundation of China/ ; 2020YFA0907500//National Key Research and Development Program of China/ ; 2021YFA0910100//National Key Research and Development Program of China/ ; }, abstract = {The precise target recognition and nuclease-mediated effective signal amplification capacities of CRISPR-Cas systems have attracted considerable research interest within the biosensing field. Guided by insights into their structural and biochemical mechanisms, researchers have endeavored to engineer the key biocomponents of CRISPR-Cas systems with stimulus-responsive functionalities. By the incorporation of protein/nucleic acid engineering techniques, a variety of conditional CRISPR-Cas systems whose activities depend on the presence of target triggers have been established for the efficient detection of diverse types of non-nucleic acid analytes. In this review, we summarized recent research progress in engineering Cas proteins, guide RNA, and substrate nucleic acids to possess target analyte-responsive abilities for diverse biosensing applications. Furthermore, we also discussed the challenges and future possibilities of the stimulus-responsive CRISPR-Cas systems in versatile biosensing.}, }
@article {pmid39601618, year = {2024}, author = {Liu, X and Peng, H and Gong, L and Zhang, H and Zhao, C and Lai, W and An, G and Zhao, X}, title = {Reliable and precise lipoprotein detection based on a self-priming hairpin-triggered Cas12a/crRNA based signaling strategy.}, journal = {The Analyst}, volume = {150}, number = {1}, pages = {46-54}, doi = {10.1039/d4an01167h}, pmid = {39601618}, issn = {1364-5528}, mesh = {Humans ; *Limit of Detection ; *Lipoproteins, LDL/blood/analysis ; Lipoproteins, HDL/blood/analysis ; Antibodies, Immobilized/immunology ; CRISPR-Cas Systems ; CRISPR-Associated Proteins/metabolism ; Lipoproteins/chemistry/blood ; }, abstract = {Cardiovascular disease, intimately linked to dyslipidemia, is one of the leading global causes of mortality. Dyslipidaemia often presents as an elevated concentration of low-density lipoprotein (LDL) and a decreased concentration of high-density lipoprotein (HDL). Therefore, accurately measuring the levels of LDL and HDL particles is crucial for assessing the risk of developing cardiovascular diseases. However, conventional approaches can commonly quantify HDL/LDL particles by detecting cholesterol or protein molecules within them, which may fail to reflect the number of intact particles. In addition, these approaches are sometimes tedious and time-consuming, highlighting the need for a novel method for precise and effective identification of intact HDL and LDL particles. We have devised a technique that allows accurately and sensitively determining the levels of intact HDL and LDL in a sample without the need for isolation. This method relies on antibody-based immobilization and a self-priming hairpin-triggered Cas12a/crRNA signaling strategy. Based on the elegant design, this technique can be employed to directly and precisely measure the concentration of "actual" HDL and LDL particles, rather than the cholesterol content inside HDL and LDL. The approach has detection limits of 12.3 mg dL[-1] and 5.4 mg dL[-1] for HDL and LDL, respectively, and is also suitable for analyzing lipoproteins in clinical samples. Hence, this platform exhibits immense potential for clinical applications and health management.}, }
@article {pmid39601564, year = {2025}, author = {Zhang, X and Hao, S and Feng, Z and Ning, K and Aksu Kuz, C and McFarlin, S and Richart, D and Cheng, F and Zhang-Chen, A and McFarlane, R and Yan, Z and Qiu, J}, title = {Identification of SLC35A1 as an essential host factor for the transduction of multi-serotype recombinant adeno-associated virus (AAV) vectors.}, journal = {mBio}, volume = {16}, number = {1}, pages = {e0326824}, pmid = {39601564}, issn = {2150-7511}, support = {AI166293//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R21 AI166293/AI/NIAID NIH HHS/United States ; R01 HL174593/HL/NHLBI NIH HHS/United States ; P30 GM103326/GM/NIGMS NIH HHS/United States ; S10 OD032207/OD/NIH HHS/United States ; S10 OD023625/OD/NIH HHS/United States ; YAN23G0//Cystic Fibrosis Foundation (CFF)/ ; HL174593//HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; AI150877//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; P30 CA168524/CA/NCI NIH HHS/United States ; R21 AI180416/AI/NIAID NIH HHS/United States ; AI180416//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; AI156448//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01 AI150877/AI/NIAID NIH HHS/United States ; R21 AI156448/AI/NIAID NIH HHS/United States ; }, mesh = {*Dependovirus/genetics ; Humans ; *Nucleotide Transport Proteins/genetics/metabolism ; *Genetic Vectors/genetics/metabolism ; *Transduction, Genetic ; HEK293 Cells ; CRISPR-Cas Systems ; Golgi Apparatus/metabolism ; Serogroup ; Gene Knockout Techniques ; }, abstract = {We conducted a genome-wide CRISPR/Cas9 screen in suspension 293 F cells transduced with rAAV5. The highly selected genes revealed after two rounds of screening included the previously reported KIAA0319L, TM9SF2, and RNF121, along with a cluster of genes involved in glycan biogenesis, Golgi apparatus localization, and endoplasmic reticulum penetration. In this report, we focused on solute carrier family 35 member A1 (SLC35A1), a Golgi apparatus-localized cytidine 5'-monophosphate-sialic acid (CMP-SIA) transporter. We confirmed that SLC35A1 knockout (KO) significantly decreased rAAV5 transduction to a level lower than that observed in KIAA0319L or TM9SF2 KO cells. Although SLC35A1 KO drastically reduced the expression of α2,6-linked SIA on the cell surface, the expression of α2,3-linked SIA, as well as the cell binding and internalization of rAAV5, was only moderately affected. Moreover, SLC35A1 KO significantly diminished the transduction of AAV multi-serotypes, including rAAV2 and rAAV3, which do not utilize SIAs for primary attachment. Notably, the SLC35A1 KO markedly increased transduction of rAAV9 and rAAV11, which primarily attach to cells via binding to galactose. Further analyses revealed that SLC35A1 KO significantly decreased vector nuclear import. More importantly, although the C-terminal cytoplasmic tail deletion (∆C Tail) mutant of SLC35A1 did not drastically decrease SIA expression, it significantly decreased rAAV transduction, as well as vector nuclear import, suggesting that the C-tail is critical in these processes. Furthermore, the T128A mutant significantly decreased SIA expression but still supported rAAV transduction and nuclear import. These findings highlight the involvement of the CMP-SIA transporter in the intracellular trafficking of rAAV vectors post-internalization.IMPORTANCErAAV is an essential tool for gene delivery in the treatment of genetic disorders; however, the mechanisms of rAAV transduction remain partially understood. GPR108 is vital for the transduction of most rAAV vectors, but not for rAAV5. We aimed to identify host factors that impact AAV5 transduction akin to GPR108. Using a genome-wide CRISPR/Cas9 screen in 293 F cells, we identified SLC35A1, a Golgi apparatus-localized CMP-sialic acid transporter that transports CMP-sialic acid from the cytoplasm into the Golgi apparatus for sialylation, is essential to rAAV transduction. Further studies across various AAV serotypes showed SLC35A1 significantly affects vector nuclear import post-internalization. These results underscore the crucial role of SLC35A1 in intracellular trafficking beyond the initial cell attachment of rAAV.}, }
@article {pmid39601181, year = {2024}, author = {Bamundo, M and Palumbo, S and D'Auria, L and Missero, C and Di Girolamo, D}, title = {CRISPR/Cas9 Ribonucleoprotein Nucleofection for Genome Editing in Primary Human Keratinocytes: Knockouts, Deletions, and Homology-Directed Repair Mutagenesis.}, journal = {Current protocols}, volume = {4}, number = {11}, pages = {e70056}, pmid = {39601181}, issn = {2691-1299}, mesh = {Humans ; *Keratinocytes/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; Ribonucleoproteins/genetics/metabolism ; Transfection/methods ; Recombinational DNA Repair ; Cells, Cultured ; Mutagenesis ; }, abstract = {Keratinocytes are the most abundant cell type in the human epidermis, the outermost layer of the skin. For years, primary human keratinocytes (HKs) have been used as a crucial tool for studying the pathogenesis of a wide range of skin-related diseases. To mimic the physiological and pathological behavior of human skin, organotypic 3D skin models can be generated by in vitro differentiation of HKs. However, manipulation of HKs is notoriously difficult. Liposome-mediated gene delivery often results in low transfection rates, and conventional electroporation results in high mortality, is difficult to optimize, and requires high cell numbers without necessarily achieving maximum efficiency. Additionally, HKs have a short lifespan in vitro, with a limited number of cell divisions before senescence, even when cultured on a feeder layer. Therefore, the possibility to use an efficient CRISPR/Cas9 system in HKs is not without challenge in terms of transfection technology and clonal selection. In this article, we provide detailed protocols to perform efficient gene knock-out (KO) or genomic deletion in a small number of HKs without clonal selection of edited cells. By nucleofecting ribonucleoprotein complexes, we efficiently generate KO cells as well as deletion of specific genomic regions. Moreover, we describe an optimized protocol for generating site-specific mutations in immortalized keratinocytes (N/TERT2G) by exploiting the homology-directed repair system combined with rapid single-clone screening. These methods can also be applied to other immortalized cells and tumoral cells of epithelial origin. Together, these protocols provide a comprehensive and powerful tool that can be used to better understand the molecular mechanisms underlying different skin diseases. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Knock-out generation by indel mutation in primary human keratinocytes using nucleofection of ribonucleoprotein (RNP) complex Basic Protocol 2: Deletion of specific genomic region using RNPs via nucleofection Basic Protocol 3: Use of homology-directed repair system to introduce site-specific mutations.}, }
@article {pmid39599550, year = {2024}, author = {Mikhaylova, Y and Tyumentseva, M and Karbyshev, K and Tyumentsev, A and Slavokhotova, A and Smirnova, S and Akinin, A and Shelenkov, A and Akimkin, V}, title = {Interrelation Between Pathoadaptability Factors and Crispr-Element Patterns in the Genomes of Escherichia coli Isolates Collected from Healthy Puerperant Women in Ural Region, Russia.}, journal = {Pathogens (Basel, Switzerland)}, volume = {13}, number = {11}, pages = {}, pmid = {39599550}, issn = {2076-0817}, support = {075-15-2019-1666//Ministry of Science and Higher Education of Russian Federation/ ; }, abstract = {Escherichia coli is a commensal and opportunistic bacterium widely distributed around the world in different niches including intestinal of humans and animals, and its extraordinary genome plasticity led to the emergence of pathogenic strains causing a wide range of diseases. E. coli is one of the monitored species in maternity hospitals, being the main etiological agent of urogenital infections, endometriosis, puerperal sepsis, and neonatal diseases. This study presents a comprehensive analysis of E. coli isolates obtained from the maternal birth canal of healthy puerperant women 3-4 days after labor. According to whole genome sequencing data, 31 sequence types and six phylogenetic groups characterized the collection containing 53 isolates. The majority of the isolates belonged to the B2 phylogroup. The data also includes phenotypic and genotypic antibiotic resistance profiles, virulence factors, and plasmid replicons. Phenotypic and genotypic antibiotic resistance testing did not demonstrate extensive drug resistance traits except for two multidrug-resistant E. coli isolates. The pathogenic factors revealed in silico were assessed with respect to CRISPR-element patterns. Multiparametric and correlation analyses were conducted to study the interrelation of different pathoadaptability factors, including antimicrobial resistance and virulence genomic determinants carried by the isolates under investigation. The data presented will serve as a valuable addition to further scientific investigations in the field of bacterial pathoadaptability, especially in studying the role of CRISPR/Cas systems in the E. coli genome plasticity and evolution.}, }
@article {pmid39597141, year = {2024}, author = {Xie, S and Yue, Y and Yang, F}, title = {Recent Advances in CRISPR/Cas System-Based Biosensors for the Detection of Foodborne Pathogenic Microorganisms.}, journal = {Micromachines}, volume = {15}, number = {11}, pages = {}, pmid = {39597141}, issn = {2072-666X}, support = {32102633//National Natural Science Foundation of China/ ; }, abstract = {Foodborne pathogens pose significant risks to food safety. Conventional biochemical detection techniques are facing a series of challenges. In recent years, with the gradual development of CRISPR (clustered regularly interspaced short palindromic repeats) technology, CRISPR/Cas system-based biosensors, a newly emerging technology, have received much attention from researchers because of their supreme flexibility, sensitivity, and specificity. While numerous CRISPR-based biosensors have a broad application in the field of environmental monitoring, food safety, and point-of-care diagnosis, they remain in high demand to summarize recent advances in CRISPR/Cas system-based biosensors for foodborne pathogen detection. In this paper, we briefly classify and discuss the working principles of CRISPR/Cas systems with trans-cleavage activity in applications for the detection of foodborne pathogenic microorganisms. We highlight the current status, the unique feature of each CRISPR system and CRISPR-based biosensing platforms, and the integration of CRISPR-Cas with other techniques, concluding with a discussion of the advantages, disadvantages, and future directions.}, }
@article {pmid39596692, year = {2024}, author = {Chen, J and Miao, Z and Kong, D and Zhang, A and Wang, F and Liu, G and Yu, X and Luo, L and Liu, Y}, title = {Application of CRISPR/Cas9 Technology in Rice Germplasm Innovation and Genetic Improvement.}, journal = {Genes}, volume = {15}, number = {11}, pages = {}, pmid = {39596692}, issn = {2073-4425}, support = {23N11900100//Shanghai Municipal Commission of Science and technology/ ; INV-033236-3//The Bill and Melinda Gates Foundation/ ; }, mesh = {*Oryza/genetics/growth &amp; development ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Plant Breeding/methods ; Plants, Genetically Modified/genetics ; Crops, Agricultural/genetics ; Genome, Plant ; }, abstract = {Improving the efficiency of germplasm innovation has always been the aim of rice breeders. Traditional hybrid breeding methods for variety selection rarely meet the practical needs of rice production. The emergence of genome-editing technologies, such as CRISPR/Cas9, provides a new approach to the genetic improvement of crops such as rice. The number of published scientific papers related to "gene editing" and "CRISPR/Cas9" retrievable on websites both from China and other countries exhibited an increasing trend, year by year, from 2014 to 2023. Research related to gene editing in rice accounts for 33.4% and 12.3% of all the literature on gene editing published in China and other countries, respectively, much higher than that on maize and wheat. This article reviews recent research on CRISPR/Cas9 gene-editing technology in rice, especially germplasm innovation and genetic improvement of commercially promoted varieties with improved traits such as disease, insect, and herbicide resistance, salt tolerance, quality, nutrition, and safety. The aim is to provide a reference for the precise and efficient development of new rice cultivars that meet market demand.}, }
@article {pmid39596534, year = {2024}, author = {Kim, JY and Lee, YJ and Lee, HJ and Go, JY and Lee, HM and Park, JS and Cho, YG and Jung, YJ and Kang, KK}, title = {Knockout of OsGAPDHC7 Gene Encoding Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenase Affects Energy Metabolism in Rice Seeds.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596534}, issn = {1422-0067}, mesh = {*Oryza/genetics/metabolism/growth &amp; development/enzymology ; *Glyceraldehyde-3-Phosphate Dehydrogenases/genetics/metabolism ; *Energy Metabolism/genetics ; *Gene Expression Regulation, Plant ; *Seeds/metabolism/genetics ; Plant Proteins/genetics/metabolism ; Gene Knockout Techniques ; Cytosol/metabolism ; CRISPR-Cas Systems ; Starch/metabolism ; Carbohydrate Metabolism/genetics ; }, abstract = {Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a major glycolytic enzyme that plays an important role in several cellular processes, including plant hormone signaling, plant development, and transcriptional regulation. In this study, we divided it into four groups through structural analysis of eight GAPDH genes identified in the rice genome. Among them, the expression level of five genes of cytosolic GAPDH was shown to be different for each organ. The mutation induction of the GAPDHC7 gene by the CRISPR/Cas9 system revealed that the 7 bp and 2 bp deletion, early end codon, was used in protein production. In addition, the selected mutants showed lower plant heights compared to the wild-type plants. To investigate the effect on carbohydrate metabolism, the expression of the genes of starch-branched enzyme I (SbeI), sucrose synthase (SS), and 3-phosphoglycer phosphokinase (PGK) increased the expression of the SBeI gene threefold in the knockout lines compared to the wild-type (WT) plant, while the expression of the SS and PGK genes decreased significantly. And the starch and soluble sugar content of the knockout lines increased by more than 60% compared to the WT plant. Also, the free amino acid content was significantly increased in the Gln and Asn contents of the knockout lines compared to the WT plants, while the contents of Gly and Ser were decreased. Our results suggest that OsGAPDHC7 has a great influence on energy metabolism, such as pre-harvested sprouting and amino acid content.}, }
@article {pmid39596521, year = {2024}, author = {Sheveleva, O and Protasova, E and Grigor'eva, E and Butorina, N and Kuziaeva, V and Antonov, D and Melnikova, V and Medvedev, S and Lyadova, I}, title = {The Generation of Genetically Engineered Human Induced Pluripotent Stem Cells Overexpressing IFN-β for Future Experimental and Clinically Oriented Studies.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596521}, issn = {1422-0067}, support = {&#x2116;075-15-2021-1075//Ministry of Science and Higher Education of the Russian Federation/ ; }, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Interferon-beta/metabolism/genetics ; *Cell Differentiation/genetics ; *CRISPR-Cas Systems ; Genetic Engineering/methods ; Embryoid Bodies/metabolism/cytology ; Cell Line ; Gene Editing/methods ; }, abstract = {Induced pluripotent stem cells (iPSCs) can be generated from various adult cells, genetically modified and differentiated into diverse cell populations. Type I interferons (IFN-Is) have multiple immunotherapeutic applications; however, their systemic administration can lead to severe adverse outcomes. One way of overcoming the limitation is to introduce cells able to enter the site of pathology and to produce IFN-Is locally. As a first step towards the generation of such cells, here, we aimed to generate human iPSCs overexpressing interferon-beta (IFNB, IFNB-iPSCs). IFNB-iPSCs were obtained by CRISPR/Cas9 editing of the previously generated iPSC line K7-4Lf. IFNB-iPSCs overexpressed IFNB RNA and produced a functionally active IFN-β. The cells displayed typical iPSC morphology and expressed pluripotency markers. Following spontaneous differentiation, IFNB-iPSCs formed embryoid bodies and upregulated endoderm, mesoderm, and some ectoderm markers. However, an upregulation of key neuroectoderm markers, PAX6 and LHX2, was compromised. A negative effect of IFN-β on iPSC neuroectoderm differentiation was confirmed in parental iPSCs differentiated in the presence of a recombinant IFN-β. The study describes new IFN-β-producing iPSC lines suitable for the generation of various types of IFN-β-producing cells for future experimental and clinical applications, and it unravels an inhibitory effect of IFN-β on stem cell neuroectoderm differentiation.}, }
@article {pmid39596457, year = {2024}, author = {Sakovina, L and Vokhtantsev, I and Akhmetova, E and Vorobyeva, M and Vorobjev, P and Zharkov, DO and Novopashina, D}, title = {Photocleavable Guide crRNAs for a Light-Controllable CRISPR/Cas9 System.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596457}, issn = {1422-0067}, support = {22-14-00294//Russian Science Foundation/ ; 121031300042-1//Russian state-funded project/ ; }, mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; CRISPR-Associated Protein 9/metabolism/genetics ; Humans ; Ultraviolet Rays ; Photolysis ; Light ; }, abstract = {The design of controllable and precise RNA-targeted CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) systems is an important problem of modern molecular biology and genetic technology. Herein, we have designed a series of photocleavable guide CRISPR RNAs (crRNA) and their 2'-modified (2'-fluoro and locked nucleic acid) analogs containing one or two 1-(2-nitrophenyl)-1,2-ethanediol photolabile linkers (PL). We have demonstrated that these crRNAs can be destroyed by relatively mild UVA irradiation with the rate constants 0.24-0.77 min[-1] and that the photocleavage markedly slows down the action of Cas9 nuclease in the model in vitro system. Two PLs provide more rapid crRNA destruction than a single linker. PLs in the crRNA structure improve the specificity of DNA cleavage by Cas9 nuclease for the fully complementary target. The application of photocleavable crRNA in CRISPR/Cas9 genome editing permits the system to be switched off in a spatiotemporally controlled manner, thus alleviating its off-target effects.}, }
@article {pmid39596448, year = {2024}, author = {Chang, H and Sha, H and Gao, S and Liu, Q and Liu, Y and Ma, C and Shi, B and Nie, S}, title = {A Novel Gene, OsRLCK191, Involved in Culm Strength Improving Lodging Resistance in Rice.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596448}, issn = {1422-0067}, support = {LH2021C092, CZKYF2023-1-A005, CXSTOP2021005, HTRC202209//Heilongjiang Provincial Natural Science Foundation of China, Fundamental Research Funds for the Research Institutes of Heilongjiang Province, the Opening Foundation of the Key Laboratory of Germplasm Enhancement, Physiology and Ecology of Food Crops in Co/ ; }, mesh = {*Oryza/genetics/growth &amp; development ; *Plant Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; Cell Wall/metabolism/genetics ; CRISPR-Cas Systems ; Genes, Plant ; Mutation ; }, abstract = {Lodging is one of the major problems in rice production. However, few genes that can explain the culm strength within the temperate japonica subspecies have been identified. In this study, we identified OsRLCK191, which encodes receptor-like cytoplasmic kinase and plays critical roles in culm strength. OsRLCK191 mutants were produced by the CRISPR-Cas9 DNA-editing system. Compared with wild types (WTs), the bending moment of the whole plant (WP), the bending moment at breaking (BM), and the section modulus (SM) were decreased in rlck191 significantly. Although there is no significant decrease in the culm length of rlck191 compared with the WT; in the mutant, except the length of the fourth internode being significantly increased, the lengths of other internodes are significantly shortened. In addition, the yield traits of panicle length, thousand-seed weight, and seed setting rate decreased significantly in rlck191. Moreover, RNA-seq experiments were performed at an early stage of rice panicle differentiation in shoot apex. The differentially expressed genes (DEGs) are mainly involved in cell wall biogenesis, cell wall polysaccharide metabolic processes, cellar component biogenesis, and DNA-binding transcription factors. Transcriptome analysis of the cell wall biological process pathways showed that major genes that participated in the cytokinin oxidase/dehydrogenase family, cellulose synthase catalytic subunit genes, and ethylene response factor family transcription factor were related to culm strength. Our research provides an important theoretical basis for analyzing the lodging resistance mechanism and lodging resistance breeding of temperate japonica.}, }
@article {pmid39596447, year = {2024}, author = {Meng, L and Zhang, J and Clarke, N}, title = {A Critical Review of Recent Advances in Maize Stress Molecular Biology.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596447}, issn = {1422-0067}, support = {C200937//Natural Science Foundation of Heilongjiang Province/ ; }, mesh = {*Zea mays/genetics/metabolism ; *Stress, Physiological/genetics ; *Gene Expression Regulation, Plant ; Genome-Wide Association Study ; Quantitative Trait Loci ; Droughts ; }, abstract = {With the intensification of global climate change and environmental stress, research on abiotic and biotic stress resistance in maize is particularly important. High temperatures and drought, low temperatures, heavy metals, salinization, and diseases are widespread stress factors that can reduce maize yields and are a focus of maize-breeding research. Molecular biology provides new opportunities for the study of maize and other plants. This article reviews the physiological and biochemical responses of maize to high temperatures and drought, low temperatures, heavy metals, salinization, and diseases, as well as the molecular mechanisms associated with them. Special attention is given to key transcription factors in signal transduction pathways and their roles in regulating maize stress adaptability. In addition, the application of transcriptomics, genome-wide association studies (GWAS), and QTL technology provides new strategies for the identification of molecular markers and genes for maize-stress-resistance traits. Crop genetic improvements through gene editing technologies such as the CRISPR/Cas system provide a new avenue for the development of new stress-resistant varieties. These studies not only help to understand the molecular basis of maize stress responses but also provide important scientific evidence for improving crop tolerance through molecular biological methods.}, }
@article {pmid39596426, year = {2024}, author = {Niu, D and Zhao, Q and Xu, L and Lin, K}, title = {Physiological and Molecular Mechanisms of Lepidopteran Insects: Genomic Insights and Applications of Genome Editing for Future Research.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596426}, issn = {1422-0067}, support = {2022YFD1400600//Research on Mechanisms of Grassland Moth Catastrophes and Sustainable Control Technologies/ ; }, mesh = {Animals ; *Gene Editing/methods ; *Lepidoptera/genetics ; *Genomics/methods ; *Insecticide Resistance/genetics ; CRISPR-Cas Systems ; Genome, Insect ; Insecticides/pharmacology ; }, abstract = {Lepidopteran insects are a major threat to global agriculture, causing significant crop losses and economic damage. Traditional pest control methods are becoming less effective due to the rapid evolution of insecticide resistance. This study explores the current status and genomic characteristics of 1315 Lepidopteran records, alongside an overview of relevant research, utilizing advanced functional genomics techniques, including RNA-seq and CRISPR/Cas9 gene-editing technologies to uncover the molecular mechanisms underlying insecticide resistance. Our genomic analysis revealed significant variability in genome size, assembly quality, and chromosome number, which may influence species' biology and resistance mechanisms. We identified key resistance-associated genes and pathways, including detoxification and metabolic pathways, which help these insects evade chemical control. By employing CRISPR/Cas9 gene-editing techniques, we directly manipulated resistance-associated genes to confirm their roles in resistance, demonstrating their potential for targeted interventions in pest management. These findings emphasize the value of integrating genomic data into the development of effective and sustainable pest control strategies, reducing reliance on chemical insecticides and promoting environmentally friendly integrated pest management (IPM) approaches. Our study highlights the critical role of functional genomics in IPM and its potential to provide long-term solutions to the growing challenge of Lepidopteran resistance.}, }
@article {pmid39596412, year = {2024}, author = {Sosnovtseva, AO and Demidova, NA and Klimova, RR and Kovalev, MA and Kushch, AA and Starodubova, ES and Latanova, AA and Karpov, DS}, title = {Control of HSV-1 Infection: Directions for the Development of CRISPR/Cas-Based Therapeutics and Diagnostics.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596412}, issn = {1422-0067}, support = {22-14-00377//Russian Science Foundation/ ; 075-15-2019-1660//Ministry of Science and Higher Education of the Russian Federation/ ; }, mesh = {*CRISPR-Cas Systems ; Humans ; *Herpesvirus 1, Human/genetics ; *Herpes Simplex/diagnosis/therapy/virology ; Animals ; Antiviral Agents/therapeutic use/pharmacology ; Gene Editing/methods ; Drug Resistance, Viral/genetics ; Genetic Therapy/methods ; }, abstract = {It is estimated that nearly all individuals have been infected with herpesviruses, with herpes simplex virus type 1 (HSV-1) representing the most prevalent virus. In most cases, HSV-1 causes non-life-threatening skin damage in adults. However, in patients with compromised immune systems, it can cause serious diseases, including death. The situation is further complicated by the emergence of strains that are resistant to both traditional and novel antiviral drugs. It is, therefore, imperative that new methods of combating HSV-1 and other herpesviruses be developed without delay. CRISPR/Cas systems may prove an effective means of controlling herpesvirus infections. This review presents the current understanding of the underlying molecular mechanisms of HSV-1 infection and discusses four potential applications of CRISPR/Cas systems in the fight against HSV-1 infections. These include the search for viral and cellular genes that may serve as effective targets, the optimization of anti-HSV-1 activity of CRISPR/Cas systems in vivo, the development of CRISPR/Cas-based HSV-1 diagnostics, and the validation of HSV-1 drug resistance mutations.}, }
@article {pmid39596282, year = {2024}, author = {Khmeleva, SA and Kurbatov, LK and Ptitsyn, KG and Timoshenko, OS and Morozova, DD and Suprun, EV and Radko, SP and Lisitsa, AV}, title = {Detection of Potato Pathogen Clavibacter sepedonicus by CRISPR/Cas13a Analysis of NASBA Amplicons.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596282}, issn = {1422-0067}, support = {075-15-2021-1345, unique identifier RF-193021X0012//Ministry of Science and Higher Education of the Russian Federation/ ; }, mesh = {*Solanum tuberosum/microbiology ; *CRISPR-Cas Systems ; *Clavibacter/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Plant Diseases/microbiology ; RNA, Ribosomal, 16S/genetics ; Plant Tubers/microbiology ; Molecular Diagnostic Techniques ; }, abstract = {The ring rot of potato caused by the bacterial pathogen Clavibacter sepedonicus is a quarantine disease posing a threat to the potato industry worldwide. The sensitive and selective detection of C. sepedonicus is of a high importance for its effective control. Here, the detection system is reported to determine viable bacteria of C. sepedonicus in potato tubers, based on the coupling of CRISPR/Cas13a nuclease with NASBA (Nucleic Acid Sequence Based Amplification)-the method of isothermal amplification of RNA. Detection can be conducted using both instrumental and non-instrumental (visual inspection of test tubes under blue light) modes. When NASBA and Cas13a analyses were carried out in separate test tubes, the limit of detection (LOD) for the system was 1000 copies of purified target 16S rRNA per NASBA reaction or about 24 colony-forming units (CFUs) of C. sepedonicus per 1 g of tuber tissue. The testing can also be conducted in the "one-pot" format (a single test tube), though with lower sensitivity: LOD was 10,000 copies of target RNA or about 100 CFU per 1 g of tuber tissue for both instrumental and visual detection modes. The overall time of NASBA/Cas13a analysis did not exceed 2 h. The developed NASBA/Cas13a detection system has the potential to be employed as a routine test of C. sepedonicus, especially for on-site testing.}, }
@article {pmid39596246, year = {2024}, author = {Shmelev, ME and Pilnik, AA and Shved, NA and Penkova, AO and Gulaia, VS and Kumeiko, VV}, title = {IDH1 R132H and TP53 R248Q Mutations Modulate Glioma Cell Migration and Adhesion on Different ECM Components.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596246}, issn = {1422-0067}, support = {20-15-00378&#x43f;//Russian Science Foundation/ ; }, mesh = {Humans ; *Isocitrate Dehydrogenase/genetics ; *Cell Movement/genetics ; *Tumor Suppressor Protein p53/genetics/metabolism ; *Extracellular Matrix/metabolism/genetics ; Cell Line, Tumor ; *Cell Adhesion/genetics ; *Glioma/genetics/pathology/metabolism ; Mutation ; Brain Neoplasms/genetics/pathology/metabolism ; }, abstract = {Mutations in IDH1 and TP53 have a significant impact on glioma prognosis and progression; however, their roles in tumor cell invasion in terms of interactions with particular components of the extracellular matrix (ECM) are still unclear. Using gene editing protocol based on CRISPR-Cas 9 with cytidine deaminase, we introduced point mutations into U87MG glioblastoma cells to establish modified cell lines with heterozygous IDH1 R132H, homozygous TP53 R248Q and heterozygous IDH1 R132H, homozygous TP53 R248Q genotypes. A comparative study of cell migration on major ECM components was carried out by high-content microscopy. IDH1 R132H mutation introduced to U87MG glioblastoma cells was shown to decrease the migration speed on Matrigel and collagen IV substrates compared to the wild-type. This data were supported by cell adhesion quantification via the lateral shift assay performed by atomic force microscopy (AFM). TP53 R248Q mutation increased cell adhesion to various substrates and significantly promoted cell migration on hyaluronic acid and chondroitin sulfate but did not change the migration rates on laminin and collagens IV and I. A double-mutant genotype produced by consequently introducing IDH1 R132H and TP53 R248Q to parental glioblastoma cells was characterized by the highest migration among all the cell lines, with particularly faster motility on chondroitin sulfate. These findings underscore the complex interactions between glioma cells, with the most important driver mutations and specific ECM components regulating cancer cell migration, offering valuable insights for potential therapeutic targets in glioma treatment.}, }
@article {pmid39596176, year = {2024}, author = {Wang, Z and Wang, C and Zhai, Y and Bai, Y and Wang, H and Rong, X}, title = {Loss of Brcc3 in Zebrafish Embryos Increases Their Susceptibility to DNA Damage Stress.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596176}, issn = {1422-0067}, support = {2022YFC2804400//the National Key R &amp; D Program of China/ ; }, mesh = {Animals ; *Zebrafish/genetics/embryology ; *Zebrafish Proteins/genetics/metabolism ; *DNA Damage ; Tumor Suppressor Protein p53/genetics/metabolism ; Ultraviolet Rays/adverse effects ; Apoptosis/genetics ; CRISPR-Cas Systems ; Embryo, Nonmammalian/metabolism ; DNA Breaks, Double-Stranded ; DNA Repair ; Etoposide/pharmacology ; }, abstract = {DNA double-strand breaks (DSBs) represent one of the most severe forms of genetic damage in organisms, yet vertebrate models capable of monitoring DSBs in real-time remain scarce. BRCA1/BRCA2-containing complex subunit 3 (BRCC3), also known as BRCC36, functions within various multiprotein complexes to mediate diverse biological processes. However, the physiological role of BRCC3 in vertebrates, as well as the underlying mechanisms that govern its activity, are not well understood. To explore these questions, we generated brcc3-knockout zebrafish using CRISPR/Cas9 gene-editing technology. While brcc3 mutant zebrafish appear phenotypically normal and remain fertile, they exhibit significantly increased rates of mortality and deformity following exposure to DNA damage. Furthermore, embryos lacking Brcc3 display heightened p53 signaling, elevated γ-H2AX levels, and increased apoptosis in response to DNA-damaging agents such as ultraviolet (UV) light and Etoposide (ETO). Notably, genetic inactivation of p53 or pharmacological inhibition of Ataxia-telangiectasia mutated (ATM) activity rescues the hypersensitivity to UV and ETO observed in Brcc3-deficient embryos. These findings suggest that Brcc3 plays a critical role in DNA damage response (DDR), promoting cell survival during embryogenesis. Additionally, brcc3-null mutant zebrafish offer a promising vertebrate model for real-time monitoring of DSBs.}, }
@article {pmid39596160, year = {2024}, author = {de Jonge, AV and Csikós, T and Eken, M and Bulthuis, EP and Poddighe, PJ and Roemer, MGM and Chamuleau, MED and Mutis, T}, title = {Delineating MYC-Mediated Escape Mechanisms from Conventional and T Cell-Redirecting Therapeutic Antibodies.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596160}, issn = {1422-0067}, mesh = {Humans ; *T-Lymphocytes/immunology/metabolism ; Cell Line, Tumor ; Proto-Oncogene Proteins c-myc/genetics/metabolism/immunology ; Killer Cells, Natural/immunology/metabolism ; Antibodies, Bispecific/pharmacology ; Apoptosis ; Rituximab/pharmacology/therapeutic use ; Multiple Myeloma/therapy/immunology/genetics/pathology ; CRISPR-Cas Systems ; Tumor Escape/drug effects ; Antigens, CD20/immunology/metabolism/genetics ; Antibodies, Monoclonal/pharmacology/therapeutic use ; }, abstract = {In B-cell malignancies, the overexpression of MYC is associated with poor prognosis, but its mechanism underlying resistance to immunochemotherapy remains less clear. In further investigations of this issue, we show here that the pharmacological inhibition of MYC in various lymphoma and multiple myeloma cell lines, as well as patient-derived primary tumor cells, enhances their susceptibility to NK cell-mediated cytotoxicity induced by conventional antibodies targeting CD20 (rituximab) and CD38 (daratumumab), as well as T cell-mediated cytotoxicity induced by the CD19-targeting bispecific T-cell engager blinatumomab. This was associated with upregulation of the target antigen only for rituximab, suggesting additional escape mechanisms. To investigate these mechanisms, we targeted the MYC gene in OCI-LY18 cells using CRISPR-Cas9 gene-editing technology. CRISPR-Cas9-mediated MYC targeting not only upregulated CD20 but also triggered broader apoptotic pathways, upregulating pro-apoptotic PUMA and downregulating anti-apoptotic proteins BCL-2, XIAP, survivin and MCL-1, thereby rendering tumor cells more prone to apoptosis, a key tumor-lysis mechanism employed by T-cells and NK-cells. Moreover, MYC downregulation boosted T-cell activation and cytokine release in response to blinatumomab, revealing a MYC-mediated T-cell suppression mechanism. In conclusion, MYC overexpressing tumor cells mitigated the efficacy of therapeutic antibodies through several non-overlapping mechanisms. Given the challenges associated with direct MYC inhibition due to toxicity, successful modulation of MYC-mediated immune evasion mechanisms may improve the outcome of immunotherapeutic approaches in B-cell malignancies.}, }
@article {pmid39596103, year = {2024}, author = {Begovic, M and Schneider, L and Zhou, X and Hamdani, N and Akin, I and El-Battrawy, I}, title = {The Role of Human-Induced Pluripotent Stem Cells in Studying Cardiac Channelopathies.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39596103}, issn = {1422-0067}, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Channelopathies/genetics/pathology ; *Myocytes, Cardiac/metabolism/pathology ; Brugada Syndrome/genetics/pathology/metabolism ; Gene Editing ; Animals ; Tachycardia, Ventricular/genetics/pathology ; CRISPR-Cas Systems ; }, abstract = {Cardiac channelopathies are inherited diseases that increase the risk of sudden cardiac death. While different genes have been associated with inherited channelopathies, there are still subtypes, e.g., catecholaminergic polymorphic ventricular tachycardia and Brugada syndrome, where the genetic cause remains unknown. Various models, including animal models, heterologous expression systems, and the human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSCs-CMs) model, have been used to study the pathophysiological mechanisms of channelopathies. Recently, researchers have focused on using hiPSCs-CMs to understand the genotype-phenotype correlation and screen drugs. By combining innovative techniques such as Clustered Regularly Interspaced Short Palindromic Repeats/Clustered Regularly Interspaced Short Palindromic Repeats associated protein 9 (CRISPR/Cas9)-mediated genome editing, and three-dimensional (3D) engineered heart tissues, we can gain new insights into the pathophysiological mechanisms of channelopathies. This approach holds promise for improving personalized drug treatment. This review highlights the role of hiPSCs-CMs in understanding the pathomechanism of Brugada syndrome and catecholaminergic polymorphic ventricular tachycardia and how these models can be utilized for drug screening.}, }
@article {pmid39595965, year = {2024}, author = {Fernández, JP and Petersen, B and Hassel, P and Lucas Hahn, A and Kielau, P and Geibel, J and Kues, WA}, title = {Comparison Between Electroporation at Different Voltage Levels and Microinjection to Generate Porcine Embryos with Multiple Xenoantigen Knock-Outs.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39595965}, issn = {1422-0067}, support = {TRR 127//Deutsche Forschungsgemeinschaft/ ; }, mesh = {Animals ; *Electroporation/methods ; *Microinjections/methods ; Swine ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; Animals, Genetically Modified ; Antigens, Heterophile/genetics/immunology ; Galactosyltransferases/genetics ; Embryo, Mammalian/metabolism ; Mosaicism ; Blastocyst/metabolism ; Zygote/metabolism ; Mixed Function Oxygenases ; }, abstract = {In the context of xenotransplantation, the production of genetically modified pigs is essential. For several years, knock-out pigs were generated through somatic cell nuclear transfer employing donor cells with the desired genetic modifications, which resulted in a lengthy and cumbersome procedure. The CRISPR/Cas9 system enables direct targeting of specific genes in zygotes directly through microinjection or electroporation. However, these techniques require improvement to minimize mosaicism and low mutation rates without compromising embryo survival. This study aimed to determine the gene editing potential of these two techniques to deliver multiplexed ribonucleotide proteins (RNPs) to generate triple-knock-out porcine embryos with a multi-transgenic background. We designed RNP complexes targeting the major porcine xenoantigens GGTA1, CMAH, and B4GALNT2. We then compared the development of mosaicism and gene editing efficiencies between electroporation and microinjection. Our results indicated a significant effect of voltage increase on molecule intake in electroporated embryos, without it notably affecting the blastocyst formation rate. Our gene editing analysis revealed differences among delivery approaches and gene loci. Notably, employing electroporation at 35 V yielded the highest frequency of biallelic disruptions. However, mosaicism was the predominant genetic variant in all RNP delivery methods, underscoring the need for further research to optimize multiplex genome editing in porcine zygotes.}, }
@article {pmid39595944, year = {2024}, author = {Li, P and Shang, H and Xu, X and Gong, J and Wu, JL and Zhang, X}, title = {A Novel Single Base Mutation in OsSPL42 Leads to the Formation of Leaf Lesions in Rice.}, journal = {International journal of molecular sciences}, volume = {25}, number = {22}, pages = {}, pmid = {39595944}, issn = {1422-0067}, support = {LZ24C130004//the Zhejiang Provincial Natural Science Foundation of China/ ; LQ24C130008//the Zhejiang Provincial Natural Science Foundation of China/ ; 2022YFF1003301//The National Key Research and Development Program of China/ ; }, mesh = {*Oryza/genetics/metabolism ; *Plant Leaves/genetics/metabolism ; *Plant Diseases/genetics/microbiology ; *Plant Proteins/genetics/metabolism ; *Disease Resistance/genetics ; *Plants, Genetically Modified ; Mutation ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; Cloning, Molecular ; Xanthomonas/pathogenicity ; }, abstract = {Rice spotted-leaf mutants serve as valuable resources for studying plant programmed cell death (PCD) and disease resistance mechanisms, making them crucial for research on disease resistance in rice. Map-based cloning was used to identify and clone the spotted-leaf gene OsSPL42. Then, functional complementation and CRISPR/Cas9 techniques were also employed to further validate the function of this gene. By applying leaf clippings for bacterial blight (BB) inoculation, the BB resistance of different rice lines was assessed. The results in this study were as follows: The OsSPL42 behaved as a recessive nuclear gene and was narrowed down to a 111 kb region on chromosome 8. All T0 transgenic rice plants in the complementation experiments exhibited a wild-type phenotype, without any lesion spots on the rice leaves. This suggests that the LOC_Os08g06100 encoding O-methyltransferase is the candidate gene for the mutant spl42. The OsSpl42 is widely expressed and the OsSPL42-GFP protein is mainly localized in the cytoplasm. OsSPL42 overexpression lines are more susceptible to BBs, which indicates that OsSPL42 may act as a negative regulator of rice resistance to BB. In summary, we speculate that OsSPL42 plays an important role in the regulation of pathogen response, providing new insights into plant defense mechanisms.}, }
@article {pmid39595588, year = {2024}, author = {Benati, D and Cattin, E and Corradi, F and Ferrari, T and Pedrazzoli, E and Patrizi, C and Marchionni, M and Bertorelli, R and De Sanctis, V and Merlini, L and Ferlini, A and Sabatelli, P and Gualandi, F and Recchia, A}, title = {Restored Collagen VI Microfilaments Network in the Extracellular Matrix of CRISPR-Edited Ullrich Congenital Muscular Dystrophy Fibroblasts.}, journal = {Biomolecules}, volume = {14}, number = {11}, pages = {}, pmid = {39595588}, issn = {2218-273X}, support = {Million Dollar Bike Ride Award 2018//Cure CMD/ ; }, mesh = {*Collagen Type VI/genetics/metabolism ; Humans ; *Fibroblasts/metabolism ; *Gene Editing ; *Extracellular Matrix/metabolism ; *Muscular Dystrophies/genetics/metabolism/therapy/pathology ; *CRISPR-Cas Systems/genetics ; Sclerosis/genetics ; Mutation ; }, abstract = {Collagen VI is an essential component of the extracellular matrix (ECM) composed by α1, α2 and α3 chains and encoded by COL6A1, COL6A2 and COL6A3 genes. Dominant negative pathogenic variants in COL6A genes result in defects in collagen VI protein and are implicated in the pathogenesis of muscular diseases, including Ullrich congenital muscular dystrophy (UCMD). Here, we designed a CRISPR genome editing strategy to tackle a dominant heterozygous deletion c.824_838del in exon 9 of the COL6A1 gene, causing a lack of secreted collagen VI in a patient's dermal fibroblasts. The evaluation of efficiency and specificity of gene editing in treating patient's fibroblasts revealed the 32% efficiency of editing the mutated allele but negligible editing of the wild-type allele. CRISPR-treated UCMD skin fibroblasts rescued the secretion of collagen VI in the ECM, which restored the ultrastructure of the collagen VI microfibril network. By using normal melanocytes as surrogates of muscle cells, we found that collagen VI secreted by the corrected patient's skin fibroblasts recovered the anchorage to the cell surface, pointing to a functional improvement of the protein properties. These results support the application of the CRISPR editing approach to knock out COL6A1 mutated alleles and rescue the UCMD phenotype in patient-derived fibroblasts.}, }
@article {pmid39595585, year = {2024}, author = {Fu, B and Ma, H and Huo, X and Zhu, Y and Liu, D}, title = {CRISPR Technology Acts as a Dual-Purpose Tool in Pig Breeding: Enhancing Both Agricultural Productivity and Biomedical Applications.}, journal = {Biomolecules}, volume = {14}, number = {11}, pages = {}, pmid = {39595585}, issn = {2218-273X}, support = {CZKYF2024-1-B006//Heilongjiang Provincial Research Institutes Research Business Fund Project/ ; U20A2052//National Natural Science Foundation of China/ ; }, mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Swine/genetics ; *Gene Editing/methods ; *Agriculture/methods ; Breeding/methods ; Animals, Genetically Modified/genetics ; Humans ; }, abstract = {Pigs have long been integral to human society for their roles in agriculture and medicine. Consequently, there is an urgent need for genetic improvement of pigs to meet human dual needs for medicine and food. In agriculture, gene editing can improve productivity traits, such as growth rate and disease resistance, which could lower farming costs and benefit consumers through enhanced meat quality. In biomedical research, gene-edited pigs offer invaluable resources as disease models and in xenotransplantation, providing organs compatible with human physiology. Currently, with CRISPR technology, especially the CRISPR/Cas9 system emerging as a transformative force in modern genetics, pigs are not only sources of sustenance but also cornerstones of biomedical innovation. This review aims to summarize the applications of CRISPR/Cas9 technology in developing pigs that serve dual roles in agriculture and biomedical applications. Compared to ZFNs and TALENs, the CRISPR/Cas9 system offers several advantages, including higher efficiency, greater specificity, ease of design and implementation, and the capability to target multiple genes simultaneously, significantly streamlining the process of genetic modifications in complex genomes. Therefore, CRISPR technology supports the enhancement of traits beneficial for agricultural productivity and facilitates applications in medicine. Furthermore, we must acknowledge the inherent deficiencies and technical challenges of the CRISPR/Cas9 technology while also anticipating emerging technologies poised to surpass CRISPR/Cas9 as the next milestones in gene editing. We hypothesize that with the continuous advancements in gene editing technologies and successful integration of traits beneficial to both agricultural productivity and medical applications, the goal of developing dual-purpose pigs for both agricultural and medical use can ultimately be achieved.}, }
@article {pmid39593080, year = {2024}, author = {Lam, S and Thomas, JC and Jackson, SP}, title = {Genome-aware annotation of CRISPR guides validates targets in variant cell lines and enhances discovery in screens.}, journal = {Genome medicine}, volume = {16}, number = {1}, pages = {139}, pmid = {39593080}, issn = {1756-994X}, mesh = {Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Molecular Sequence Annotation ; Genome, Human ; Cell Line ; Algorithms ; Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; Exome ; }, abstract = {BACKGROUND: CRISPR-Cas9 technology has revolutionised genetic screens and can inform on gene essentiality and chemo-genetic interactions. It is easily deployed and widely supported with many pooled CRISPR libraries available commercially. However, discrepancies between the reference genomes used in the design of those CRISPR libraries and the cell line under investigation can lead to loss of signal or introduction of bias. The problem is particularly acute when dealing with variant cell lines such as cancer cell lines.<br><br>RESULTS: Here, we present an algorithm, EXOme-guided Re-annotation of nuCleotIde SEquences (Exorcise), which uses sequence search to detect and correct mis-annotations in CRISPR libraries. Exorcise verifies the presence of CRISPR targets in the target genome and applies corrections to CRISPR libraries using existing exome annotations. We applied Exorcise to re-annotate guides in pooled CRISPR libraries available on Addgene and found that libraries designed on a more permissive reference sequence had more mis-annotations. In simulated CRISPR screens, we modelled common mis-annotations and found that they adversely affect discovery of hits in the intermediate range. We then confirmed this by applying Exorcise on datasets from Dependency Map (DepMap) and the DNA Damage Response CRISPR Screen Viewer (DDRcs), where we found improved discovery power upon Exorcise while retaining the strongest hits.<br><br>CONCLUSIONS: Pooled CRISPR libraries map guide sequences to genes and these mappings might not be ready to use due to permissive library design or investigating a variant cell line. By re-annotating CRISPR guides, Exorcise focuses CRISPR experiments towards the genome of the cell line under investigation. Exorcise can be applied at the library design stage or the analysis stage and allows post hoc re-analysis of completed screens. It is available under a Creative Commons Zero v1.0 Universal licence at https://github.com/SimonLammmm/exorcise .}, }
@article {pmid39592946, year = {2024}, author = {Bovet, L and Battey, J and Lu, J and Sierro, N and Dewey, RE and Goepfert, S}, title = {Nitrate assimilation pathway is impacted in young tobacco plants overexpressing a constitutively active nitrate reductase or displaying a defective CLCNt2.}, journal = {BMC plant biology}, volume = {24}, number = {1}, pages = {1132}, pmid = {39592946}, issn = {1471-2229}, mesh = {*Nitrates/metabolism ; *Nitrate Reductase/metabolism/genetics ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; Chloride Channels/metabolism/genetics ; Plants, Genetically Modified/genetics ; Plant Leaves/genetics/metabolism ; Tobacco Products ; }, abstract = {BACKGROUND: We have previously shown that the expression of a constitutively active nitrate reductase variant and the suppression of CLCNt2 gene function (belonging to the chloride channel (CLC) gene family) in field-grown tobacco reduces tobacco-specific nitrosamines (TSNA) accumulation in cured leaves and cigarette smoke. In both cases, TSNA reductions resulted from a strong diminution of free nitrate in the leaf, as nitrate is a precursor of the TSNA-producing nitrosating agents formed during tobacco curing and smoking. These nitrosating agents modify tobacco alkaloids to produce TSNAs, the most problematic of which are NNN (N-nitrosonornicotine) and NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone). The expression of a deregulated nitrate reductase enzyme (DNR) that is no longer responsive to light regulation is believed to diminish free nitrate pools by immediately channeling incoming nitrate into the nitrate assimilation pathway. The reduction in nitrate observed when the two tobacco gene copies encoding the vacuolar nitrate transporter CLCNt2 were down-regulated by RNAi-mediated suppression or knocked out using the CRISPR-Cas technology was mechanistically distinct; likely attributable to the inability of the tobacco cell to efficiently sequester nitrate into the vacuole where this metabolite is protected from further assimilation. In this study, we used transcriptomic and metabolomic analyses to compare the nitrate assimilation response in tobacco plants either expressing DNR or lacking CLCNt2 function.<br><br>RESULTS: When grown in a controlled environment, both DNR and CLCNt2-KO (CLCKO) plants exhibited (1) reduced nitrate content in the leaf; (2) increased N-assimilation into the amino acids Gln and Asn; and (3) a similar pattern of differential regulation of several genes controlling stress responses, including water stress, and cell wall metabolism in comparison to wild-type plants. Differences in gene regulation were also observed between DNR and CLCKO plants, including genes encoding nitrite reductase and asparagine synthetase.<br><br>CONCLUSIONS: Our data suggest that even though both DNR and CLCKO plants display common characteristics with respect to nitrate assimilation, cellular responses, water stress, and cell wall remodeling, notable differences in gene regulatory patterns between the two low nitrate plants are also observed. These findings open new avenues in using plants fixing more nitrogen into amino acids for plant improvement or nutrition perspectives.}, }
@article {pmid39592573, year = {2024}, author = {Pallaseni, A and Peets, EM and Girling, G and Crepaldi, L and Kuzmin, I and Moor, M and Muñoz-Subirana, N and Schimmel, J and Serçin, &#xd6; and Mardin, BR and Tijsterman, M and Peterson, H and Kosicki, M and Parts, L}, title = {The interplay of DNA repair context with target sequence predictably biases Cas9-generated mutations.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10271}, pmid = {39592573}, issn = {2041-1723}, support = {/WT_/Wellcome Trust/United Kingdom ; R01 HG003988/HG/NHGRI NIH HHS/United States ; 220540/Z/20/A//Wellcome Trust (Wellcome)/ ; }, mesh = {Mice ; Animals ; *DNA Repair/genetics ; *CRISPR-Cas Systems ; *DNA-Activated Protein Kinase/genetics/metabolism ; *Mutation ; DNA Breaks, Double-Stranded ; Mouse Embryonic Stem Cells/metabolism ; CRISPR-Associated Protein 9/metabolism/genetics ; Cell Line ; DNA-Directed DNA Polymerase/metabolism/genetics ; Nuclear Proteins/genetics/metabolism ; Gene Editing/methods ; DNA-Binding Proteins ; }, abstract = {Repair of double-stranded breaks generated by CRISPR/Cas9 is highly dependent on the flanking DNA sequence. To learn about interactions between DNA repair and target sequence, we measure frequencies of over 236,000 distinct Cas9-generated mutational outcomes at over 2800 synthetic target sequences in 18 DNA repair deficient mouse embryonic stem cells lines. We classify the outcomes in an unbiased way, finding a specialised role for Prkdc (DNA-PKcs protein) and Polm in creating 1 bp insertions matching the nucleotide on the protospacer-adjacent motif side of the break, a variable involvement of Nbn and Polq in the creation of different deletion outcomes, and uni-directional deletions dependent on both end-protection and end-resection. Using our dataset, we build predictive models of the mutagenic outcomes of Cas9 scission that outperform the current standards. This work improves our understanding of DNA repair gene function, and provides avenues for more precise modulation of Cas9-generated mutations.}, }
@article {pmid39592077, year = {2024}, author = {Shen, JY and Mao, FH and Wang, Q and Ou, PP and Liu, JK and Zhao, Q and He, QL}, title = {Efficient genome editing using CRISPR/Cas9 technology and its application for identifying Sesquiterpene synthases involved in the biosynthesis of Steperoxides in Steccherinum ochraceum.}, journal = {Fungal genetics and biology : FG &amp; B}, volume = {175}, number = {}, pages = {103944}, doi = {10.1016/j.fgb.2024.103944}, pmid = {39592077}, issn = {1096-0937}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Sesquiterpenes/metabolism ; Promoter Regions, Genetic/genetics ; Alkyl and Aryl Transferases/genetics ; Fungal Proteins/genetics/metabolism ; Agaricales/genetics/enzymology ; }, abstract = {CRISPR technology has been widely used for gene editing in various species，but the genetic manipulation in basidiomycete mushrooms is still notoriously difficult for unknown endogenous promoters and inefficient DNA delivery. Steccherinum ochraceum is a white rot basidiomycete fungus with abundant secondary metabolites and plays an important ecological role worldwide. To facilitate the study of gene function in S. ochraceum, an effective CRISPR/Cas9 system was successfully developed by identifying highly efficient endogenous promoters, and utilizing the Agrobacterium-transformation method. Two efficient endogenous RNA polymerase II promoters (Psogpd and Psotef1) and one efficient RNA polymerase III promoter (Pu6-d) were identified and characterized, with an editing efficiency of 61.5 % at the ura3 locus. Using this optimized system, the sesquiterpene gene A0064, which could produce 10 possible sesquiterpenes in the heterologous expression system of A. oryzae, was knocked out to obtain A0064 knockout strain S. ochraceum (∆A0064). Steperoxide A could not be detected in S. ochraceum (∆A0064), demonstrating that A0064 was the only enzyme responsible for the biosynthesis of β-chamigrene (the sesquiterpene skeleton of steperoxide A) in S. ochraceum. This efficient system will enable precise targeting and multiplex editing of S. ochraceum genes, facilitating functional studies of genes involved in lignin degradation and natural product biosynthesis in S. ochraceum, and providing some valuable guidance for gene editing in tens of thousands of macrofungi.}, }
@article {pmid39592070, year = {2025}, author = {Donaka, R and Zheng, H and Ackert-Bicknell, CL and Karasik, D}, title = {Early life lipid overload in Native American Myopathy is phenocopied by stac3 knockout in zebrafish.}, journal = {Gene}, volume = {936}, number = {}, pages = {149123}, doi = {10.1016/j.gene.2024.149123}, pmid = {39592070}, issn = {1879-0038}, mesh = {Animals ; *Zebrafish/genetics ; *Zebrafish Proteins/genetics/metabolism ; Disease Models, Animal ; Muscle, Skeletal/metabolism/pathology ; Gene Knockout Techniques ; Muscular Diseases/genetics/pathology/metabolism ; Humans ; Calcium/metabolism ; MyoD Protein/genetics/metabolism ; Muscle Development/genetics ; CRISPR-Cas Systems ; Myogenic Regulatory Factor 5/genetics/metabolism ; Actins/metabolism/genetics ; Larva/genetics ; Lipid Metabolism/genetics ; Adaptor Proteins, Signal Transducing ; }, abstract = {Understanding the early stages of human congenital myopathies is critical for proposing strategies for improving musculoskeletal muscle performance, such as restoring the functional integrity of the cytoskeleton. SH3 and cysteine-rich domain 3 (STAC3) are proteins involved in nutrient regulation and are an essential component of the excitation-contraction (EC) coupling machinery for Ca[2+] releasing. A mutation in STAC3 causes debilitating Native American Myopathy (NAM) in humans, while loss of this gene in mice and zebrafish (ZF) results in premature death. Clinically, NAM patients demonstrated increased lipids in skeletal muscle, but it is unclear if neutral lipids are associated with altered muscle function in NAM. Using a CRISPR/Cas9 induced stac3[-/-] knockout (KO) zebrafish model, we determined that loss of stac3 leads to delayed larval hatching which corresponds with muscle weakness and decreased whole-body Ca[2+] level during early skeletal development. Specifically, we observed defects in the cytoskeleton in F-actin and slow muscle fibers at 5 and 7 days post-fertilizations (dpf). Myogenesis regulators such as myoD and myf5, mstnb were significantly altered in stac3[-/-] larvae. These muscle alterations were associated with elevated neutral lipid levels starting at 5 dpf and persisting beyond 7 dpf. Larva lacking stac3 had reduced viability with no larva knockouts surviving past 11 dpf. This data suggests that our stac3[-/-] zebrafish serve as an alternative model to study the diminished muscle function seen in NAM patients. The data gathered from this new model over time supports a mechanistic view of lipotoxicity as a critical part of the pathology of NAM and the associated loss of function in muscle.}, }
@article {pmid39591548, year = {2024}, author = {Wu, R and Cao, Y and Liu, Y and Zhou, Y and Chen, Z and Zhu, JJ}, title = {Alkynyl Ligands Templated Assemblies of Silver Nanoclusters with Exceptional Electrochemiluminescence Activity for Pancreatic Cancer Specific tsRNAs Measurement.}, journal = {Analytical chemistry}, volume = {96}, number = {49}, pages = {19396-19403}, doi = {10.1021/acs.analchem.4c03758}, pmid = {39591548}, issn = {1520-6882}, mesh = {*Silver/chemistry ; *Pancreatic Neoplasms ; Ligands ; *Metal Nanoparticles/chemistry ; Humans ; *Electrochemical Techniques ; *Alkynes/chemistry ; *Luminescent Measurements ; Biosensing Techniques/methods ; RNA, Small Interfering/chemistry ; }, abstract = {Proper manipulation of the ligand complex on the motifs of metal nanoclusters (MNCs) to form an ordered self-assembly is an effective approach to enhance the electrochemiluminescence (ECL) emission of MNCs. We report a facile approach for the preparation of self-assembled AgNCs (AgNCsAssy) induced by alkynyl ligands with enhanced ECL and stability. The formation of these AgNCsAssy was simultaneously driven by the diverse coordination modes of alkynyl ligands with Ag and intercluster interactions, for which it was found that the para-substituted alkynyl ligands exhibited apparently irregular nanoparticles, while the monosubstituted counterparts were present in the form of ribbons. The calculations revealed that the energy gap between the highest occupied molecular orbitals (HOMOs) and the lowest unoccupied molecular orbitals (LUMOs) played a crucial role in their ECL emissions because of the substituent effects, especially, the low-lying LUMO levels could help to enhance the ECL emission. Moreover, mechanistic studies revealed that both the coreactant and alkynyl ligands made significant contributions to the ECL performance. Concurrently, the CRISPR-associated proteins (CRISPR-Cas) 12a system shows great potential in biosensing applications due to the advantages of easy design and precise targeting. As a proof of concept, we integrated the cascade amplification of catalytic hairpin assembly (CHA) circuit and the collateral cleavage activity of CRISPR-Cas12a to construct an ultrasensitive ECL biosensor for pancreatic cancer (PC)-specific tsRNAs, with a detection limit of 3.33 fM. This work is not only instructive for the synthesis of self-assembled MNCs with high ECL activities but also contributes to the understanding of the ECL mechanism of self-assembled MNCs.}, }
@article {pmid39590667, year = {2024}, author = {Leal, K and Rojas, E and Madariaga, D and Contreras, MJ and Nuñez-Montero, K and Barrientos, L and Goméz-Espinoza, O and Iturrieta-González, I}, title = {Unlocking Fungal Potential: The CRISPR-Cas System as a Strategy for Secondary Metabolite Discovery.}, journal = {Journal of fungi (Basel, Switzerland)}, volume = {10}, number = {11}, pages = {}, pmid = {39590667}, issn = {2309-608X}, support = {11230475//FONDECYT Iniciaci&#xf3;n/ ; 3230521//ANID Fondecyt Postdoctorado/ ; }, abstract = {Natural products (NPs) are crucial for the development of novel antibiotics, anticancer agents, and immunosuppressants. To highlight the ability of fungi to produce structurally diverse NPs, this article focuses on the impact of genome mining and CRISPR-Cas9 technology in uncovering and manipulating the biosynthetic gene clusters (BGCs) responsible for NP synthesis. The CRISPR-Cas9 system, originally identified as a bacterial adaptive immune mechanism, has been adapted for precise genome editing in fungi, enabling targeted modifications, such as gene deletions, insertions, and transcription modulation, without altering the genomic sequence. This review elaborates on various CRISPR-Cas9 systems used in fungi, notably the Streptococcus pyogenes type II Cas9 system, and explores advancements in different Cas proteins for fungal genome editing. This review discusses the methodologies employed in CRISPR-Cas9 genome editing of fungi, including guide RNA design, delivery methods, and verification of edited strains. The application of CRISPR-Cas9 has led to enhanced production of secondary metabolites in filamentous fungi, showcasing the potential of this system in biotechnology, medical mycology, and plant pathology. Moreover, this article emphasizes the integration of multi-omics data (genomics, transcriptomics, proteomics, and metabolomics) to validate CRISPR-Cas9 editing effects in fungi. This comprehensive approach aids in understanding molecular changes, identifying off-target effects, and optimizing the editing protocols. Statistical and machine learning techniques are also crucial for analyzing multi-omics data, enabling the development of predictive models and identification of key molecular pathways affected by CRISPR-Cas9 editing. In conclusion, CRISPR-Cas9 technology is a powerful tool for exploring fungal NPs with the potential to accelerate the discovery of novel bioactive compounds. The integration of CRISPR-Cas9 with multi-omics approaches significantly enhances our ability to understand and manipulate fungal genomes for the production of valuable secondary metabolites and for promising new applications in medicine and industry.}, }
@article {pmid39590528, year = {2024}, author = {Kaupbayeva, B and Tsoy, A and Safarova Yantsen, Y and Nurmagambetova, A and Murata, H and Matyjaszewski, K and Askarova, S}, title = {Unlocking Genome Editing: Advances and Obstacles in CRISPR/Cas Delivery Technologies.}, journal = {Journal of functional biomaterials}, volume = {15}, number = {11}, pages = {}, pmid = {39590528}, issn = {2079-4983}, support = {AP14871448//Kazakhstan MSHE/ ; }, abstract = {CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats associated with protein 9) was first identified as a component of the bacterial adaptive immune system and subsequently engineered into a genome-editing tool. The key breakthrough in this field came with the realization that CRISPR/Cas9 could be used in mammalian cells to enable transformative genetic editing. This technology has since become a vital tool for various genetic manipulations, including gene knockouts, knock-in point mutations, and gene regulation at both transcriptional and post-transcriptional levels. CRISPR/Cas9 holds great potential in human medicine, particularly for curing genetic disorders. However, despite significant innovation and advancement in genome editing, the technology still possesses critical limitations, such as off-target effects, immunogenicity issues, ethical considerations, regulatory hurdles, and the need for efficient delivery methods. To overcome these obstacles, efforts have focused on creating more accurate and reliable Cas9 nucleases and exploring innovative delivery methods. Recently, functional biomaterials and synthetic carriers have shown great potential as effective delivery vehicles for CRISPR/Cas9 components. In this review, we attempt to provide a comprehensive survey of the existing CRISPR-Cas9 delivery strategies, including viral delivery, biomaterials-based delivery, synthetic carriers, and physical delivery techniques. We underscore the urgent need for effective delivery systems to fully unlock the power of CRISPR/Cas9 technology and realize a seamless transition from benchtop research to clinical applications.}, }
@article {pmid39590000, year = {2024}, author = {Guermonprez, P and Nioche, P and Renaud, L and Battaglini, N and Sanaur, S and Krejci, E and Piro, B}, title = {CRISPR-Cas Systems Associated with Electrolyte-Gated Graphene-Based Transistors: How They Work and How to Combine Them.}, journal = {Biosensors}, volume = {14}, number = {11}, pages = {}, pmid = {39590000}, issn = {2079-6374}, support = {ANR-21-CE19-0039.//ANR/ ; PhD scholarship//Universit&#xe9; Paris Cit&#xe9;/ ; }, mesh = {*Graphite/chemistry ; *Transistors, Electronic ; *Biosensing Techniques ; *CRISPR-Cas Systems ; Humans ; SARS-CoV-2 ; COVID-19 ; Electrolytes ; RNA, Viral ; }, abstract = {In this review, recent advances in the combination of CRISPR-Cas systems with graphene-based electrolyte-gated transistors are discussed in detail. In the first part, the functioning of CRISPR-Cas systems is briefly explained, as well as the most common ways to convert their molecular activity into measurable signals. Other than optical means, conventional electrochemical transducers are also developed. However, it seems that the incorporation of CRISPR/Cas systems into transistor devices could be extremely powerful, as the former provides molecular amplification, while the latter provides electrical amplification; combined, the two could help to advance in terms of sensitivity and compete with conventional PCR assays. Today, organic transistors suffer from poor stability in biological media, whereas graphene materials perform better by being extremely sensitive to their chemical environment and being stable. The need for fast and inexpensive sensors to detect viral RNA arose on the occasion of the COVID-19 crisis, but many other RNA viruses are of interest, such as dengue, hepatitis C, hepatitis E, West Nile fever, Ebola, and polio, for which detection means are needed.}, }
@article {pmid39589995, year = {2024}, author = {Yang, JW and Kim, H and Hyeon, LS and Yoo, JS and Kang, S}, title = {Development of a Recombinase Polymerase Amplification-Coupled CRISPR/Cas12a Platform for Rapid Detection of Antimicrobial-Resistant Genes in Carbapenem-Resistant Enterobacterales.}, journal = {Biosensors}, volume = {14}, number = {11}, pages = {}, pmid = {39589995}, issn = {2079-6374}, support = {2022-NI-002-00//National Institute of Health/ ; 2024-NI-023-00//National Institute of Health/ ; }, mesh = {*CRISPR-Cas Systems ; *Recombinases ; *Bacterial Proteins/genetics ; beta-Lactamases/genetics ; Humans ; Carbapenem-Resistant Enterobacteriaceae/genetics ; Nucleic Acid Amplification Techniques ; Carbapenems/pharmacology ; Biosensing Techniques ; Enterobacteriaceae/genetics ; }, abstract = {The worldwide spread of carbapenemase-producing Enterobacterales (CPE) represents a significant threat owing to the high mortality and morbidity rates. Traditional diagnostic methods are often too slow and complex for rapid point-of-care testing. Therefore, we developed a recombinase polymerase amplification (RPA)-coupled CRISPR/Cas12a system (RCCS), a rapid, accurate, and simple diagnostic platform for detecting antimicrobial-resistant genes. The RCCS detected carbapenemase genes (blaKPC and blaNDM) within 50 min, including 10 min for DNA extraction and 30-40 min for RCCS reaction (a 20 min RPA reaction with a 10-20-min CRISPR/Cas12a assay). Fluorescence signals obtained from the RCCS platform were visualized using lateral-flow test strips (LFSs) and real-time and endpoint fluorescence. The LFS clearly displayed test lines while detecting carbapenemase genes. Furthermore, the RCCS platform demonstrated high sensitivity by successfully detecting blaKPC and blaNDM at the attomolar and picomolar levels, respectively. The accuracy of the RCCS platform was validated with clinical isolates of Klebsiella pneumoniae and Escherichia coli; a 100% detection accuracy was achieved, which has not been reported when using conventional PCR. Overall, these findings indicate that the RCCS platform is a powerful tool for rapid and reliable detection of carbapenemase-encoding genes, with significant potential for implementation in point-of-care settings and resource-limited environments.}, }
@article {pmid39589913, year = {2024}, author = {Wang, Q and Yang, G and Jia, R and Wang, F and Wang, G and Xu, Z and Li, J and Li, B and Yu, L and Zhang, Y and Alariqi, M and Cao, J and Liang, S and Zhang, X and Nie, X and Jin, S}, title = {Utilizing the mutant library to investigate the functional characterization of GhGLR3.4 regulating jasmonic acid to defense pest infestation.}, journal = {The Plant journal : for cell and molecular biology}, volume = {120}, number = {6}, pages = {2889-2903}, doi = {10.1111/tpj.17152}, pmid = {39589913}, issn = {1365-313X}, support = {32272128//National Natural Science Foundation of China/ ; 32401780//National Natural Science Foundation of China/ ; 027Y2022-021//Science and Technology Project of Hubei Province of China National Tobacco Corporation/ ; 2022YFF1001403//National Key Research and Development Program of China/ ; 32325039//National Natural Science Fund of China for Distinguished Young Scholars/ ; }, mesh = {*Cyclopentanes/metabolism ; *Oxylipins/metabolism ; *Gossypium/genetics/parasitology ; *Plant Proteins/genetics/metabolism ; Animals ; Receptors, Glutamate/genetics/metabolism ; Gene Expression Regulation, Plant ; Signal Transduction ; Plant Growth Regulators/metabolism ; Arabidopsis/genetics/physiology ; Mutation ; CRISPR-Cas Systems ; Plant Diseases/parasitology/genetics/immunology ; }, abstract = {The glutamate receptor (GLR) serves as a ligand-gated ion channel that plays a vital role in plant growth, development, and stress response. Nevertheless, research on GLRs in cotton is still very limited. The present study conducted a comprehensive analysis of GLRs gene family in cotton. In total, 41 members of the GLR family were identified in cotton unveiling distinct subgroups in comparison to Arabidopsis. Among these members, the third subgroup highlights its pivotal role in cotton's defense against insect infestation. Furthermore, the CRISPR/Cas9 system was utilized to create a mutant library of GLR members, which consisted of a total of 135 independent mutant lines, resulting in the production of novel cotton materials with valuable breeding potential for pest control. Further, this study elucidates the influence of GhGLR3.4 on jasmonic acid (JA) pathway signal transduction and demonstrated its participation in the influx of intracellular Ca[2+], which regulates "calcium transients" following stimulation, thereby influencing multiple intracellular reactions. The study also found that GhGLR3.4 influences the synthesis of the JA pathway and actively partakes in long-distance signal transmission among plants, facilitating the transfer of defense signals to neighbor leaves and thereby triggering systemic defense. Consequently, this research advances our knowledge of plants' comprehensive defense mechanism against insect pest infestation.}, }
@article {pmid39589873, year = {2024}, author = {Chen, X and Koo, J and Kumar Arya, S and Palli, SR}, title = {Chronologically inappropriate morphogenesis (Chinmo) is required for maintenance of larval stages of fall armyworm.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {49}, pages = {e2411286121}, pmid = {39589873}, issn = {1091-6490}, support = {2019-67013-29351//U.S. Department of Agriculture (USDA)/ ; 2353057000//U.S. Department of Agriculture (USDA)/ ; }, mesh = {Animals ; *Larva/growth &amp; development/metabolism/genetics ; *Metamorphosis, Biological ; *Gene Expression Regulation, Developmental ; Drosophila Proteins/genetics/metabolism ; Morphogenesis/genetics ; Transcription Factors/genetics/metabolism ; Pupa/growth &amp; development/genetics/metabolism ; CRISPR-Cas Systems ; Drosophila melanogaster/genetics/growth &amp; development ; Signal Transduction ; Kruppel-Like Transcription Factors ; }, abstract = {Broad complex (Br-C) and eip93F (E93) transcription factors promote insect metamorphosis from larva to pupa and from pupa to adult, respectively. Recently, chronologically inappropriate morphogenesis (Chinmo) has been proposed as a larval specifier in Drosophila melanogaster. However, whether Chinmo is required for larval maintenance in lepidopteran insects, the underlying mechanisms involved in maintaining the larval stage, and its interactions with the JH signaling pathway are not well understood. Here, we used a binary transgenic CRISPR/Cas9 system to knockout Chinmo and Kr-h1 (primary response gene in the JH signaling pathway) in the fall armyworm (FAW). Kr-h1 knockout induced premature metamorphosis only after L5 (penultimate), whereas Chinmo and Kr-h1 double knockout induced premature metamorphosis in L3. Sequencing and differential gene expression (DEG) analysis of RNA isolated from mutants and single-cell multiome ATAC analysis of Chinmo, Kr-h1, and Chinmo and Kr-h1 double knockout Sf9 cells revealed that Chinmo participates in chromatin modifications that prevent the promoter accessibility and expression of metamorphosis promoting genes. These results suggest that Chinmo is a larval specifier that plays a major role in preventing metamorphosis in early larval stages by controlling chromatin accessibility near the promoters of genes such as Br-C and E93 required for pupal and adult development.}, }
@article {pmid39588774, year = {2024}, author = {Zhang, J and Guan, X and Moon, J and Zhang, S and Jia, Z and Yang, R and Hou, C and Guo, C and Pei, M and Liu, C}, title = {Interpreting CRISPR-Cas12a enzyme kinetics through free energy change of nucleic acids.}, journal = {Nucleic acids research}, volume = {52}, number = {22}, pages = {14077-14092}, pmid = {39588774}, issn = {1362-4962}, support = {U01 CA269147/CA/NCI NIH HHS/United States ; R33AI154642/NH/NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; Kinetics ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *Thermodynamics ; Endodeoxyribonucleases/metabolism/chemistry/genetics ; DNA/metabolism/chemistry/genetics ; Bacterial Proteins/metabolism/genetics/chemistry ; DNA, Single-Stranded/metabolism/chemistry ; Machine Learning ; }, abstract = {While CRISPR has revolutionized biotechnology, predicting CRISPR-Cas nuclease activity remains a challenge. Herein, through the trans-cleavage feature of CRISPR-Cas12a, we investigate the correlation between CRISPR enzyme kinetics and the free energy change of crRNA and DNA targets from their initial thermodynamic states to a presumed transition state before hybridization. By subjecting computationally designed CRISPR RNAs (crRNAs), we unravel a linear correlation between the trans-cleavage kinetics of Cas12a and the energy barrier for crRNA spacer and single-stranded DNA target unwinding. This correlation shifts to a parabolic relationship with the energy consumption required for double-stranded DNA target separation. We further validate these correlations using ∼100 randomly selected crRNA/DNA pairs from viral genomes. Through machine learning methods, we reveal the synergistic effect of free energy change of crRNA and DNA on categorizing Cas12a activity on a two-dimensional map. Furthermore, by examining other potential factors, we find that the free energy change is the predominant factor governing Cas12a kinetics. This study will not only empower sequence design for numerous applications of CRISPR-Cas12a systems, but can also extend to activity prediction for a variety of enzymatic reactions driven by nucleic acid dynamics.}, }
@article {pmid39588656, year = {2024}, author = {Jiang, Y and Wang, Y and Luo, W and Luan, X and Zhang, Z and Pan, Y and He, B and Gao, Y and Song, Y}, title = {Detecting telomerase activity at the single-cell level using a CRISPR-Cas12a-based chip.}, journal = {Lab on a chip}, volume = {25}, number = {1}, pages = {49-56}, doi = {10.1039/d4lc00619d}, pmid = {39588656}, issn = {1473-0189}, mesh = {*Telomerase/metabolism ; *CRISPR-Cas Systems ; Humans ; *Single-Cell Analysis/instrumentation ; Lab-On-A-Chip Devices ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {The intimate association between telomerase activity and cancer has driven the exploration of diverse methodologies for its precise detection. However, detecting telomerase activity at the single-cell level remains a significant challenge. Herein, we present a MOF-DNA barcode-amplified CRISPR-Cas12a strategy integrated with a single-cell microfluidic chip for ultrasensitive detection of telomerase activity. DNA-functionalized UiO-66 nanoparticles act as signal transducers, effectively converting telomerase activity into DNA activation strands, which subsequently trigger the trans-cleavage activity of CRISPR-Cas12a. This amplification-based assay could be integrated with a microfluidic chip to enable highly sensitive detection of telomerase activity at the single-cell level, offering promising advancements in early cancer diagnosis.}, }
@article {pmid39588089, year = {2024}, author = {Rasmussen, SK and Jain, SM}, title = {Editorial: Mutational breeding: from induced mutations to site-directed mutagenesis.}, journal = {Frontiers in plant science}, volume = {15}, number = {}, pages = {1511363}, pmid = {39588089}, issn = {1664-462X}, }
@article {pmid39587072, year = {2024}, author = {Montepeloso, A and Mattioli, D and Pellin, D and Peviani, M and Genovese, P and Biffi, A}, title = {Haploinsufficiency at the CX3CR1 locus of hematopoietic stem cells favors the appearance of microglia-like cells in the central nervous system of transplant recipients.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10192}, pmid = {39587072}, issn = {2041-1723}, mesh = {*CX3C Chemokine Receptor 1/metabolism/genetics ; Animals ; *Microglia/metabolism ; *Hematopoietic Stem Cells/metabolism/cytology ; *Hematopoietic Stem Cell Transplantation ; Humans ; *Haploinsufficiency ; Mice ; *Central Nervous System/metabolism ; CRISPR-Cas Systems ; Female ; Male ; Genetic Therapy/methods ; Genetic Loci ; }, abstract = {Transplantation of engineered hematopoietic stem/progenitor cells (HSPCs) showed curative potential in patients affected by neurometabolic diseases treated in early stage. Favoring the engraftment and maturation of the engineered HSPCs in the central nervous system (CNS) could allow enhancing further the therapeutic potential of this approach. Here we unveil that HSPCs haplo-insufficient at the Cx3cr1 (Cx3cr1[-/+]) locus are favored in central nervous system (CNS) engraftment and generation of microglia-like progeny cells (MLCs) as compared to wild type (Cx3cr1[+/+]) HSPCs upon transplantation in mice. Based on this evidence, we have developed a CRISPR-based targeted gene addition strategy at the human CX3CR1 locus resulting in an enhanced ability of the edited human HSPCs to generate mature MLCs upon transplantation in immunodeficient mice, and in lineage specific, regulated and robust transgene expression. This approach, which benefits from the modulation of pathways involved in microglia maturation and migration in haplo-insufficient cells, may broaden the application of HSPC gene therapy to a larger spectrum of neurometabolic and neurodegenerative diseases.}, }
@article {pmid39587061, year = {2024}, author = {Qu, J and Wang, Y and Xiong, C and Wang, M and He, X and Jia, W and Li, CY and Zhang, T and Wang, Z and Li, W and Kuang, BY and Shi, P}, title = {In vivo gene editing of T-cells in lymph nodes for enhanced cancer immunotherapy.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10218}, pmid = {39587061}, issn = {2041-1723}, mesh = {Animals ; *Gene Editing/methods ; *Lymph Nodes/immunology ; Mice ; *T-Lymphocytes/immunology ; *CRISPR-Cas Systems/genetics ; *Immunotherapy/methods ; *Programmed Cell Death 1 Receptor/metabolism/genetics/antagonists &amp; inhibitors ; Mice, Inbred C57BL ; Cell Line, Tumor ; Immune Checkpoint Inhibitors/therapeutic use/pharmacology ; Humans ; Female ; Melanoma, Experimental/therapy/immunology/genetics ; Melanoma/therapy/immunology/genetics ; Electroporation/methods ; }, abstract = {Immune checkpoint blockade (ICB) therapy, while promising for cancer treatment, faces challenges like unexpected side effects and limited objective responses. Here, we develop an in vivo gene-editing strategy for improving ICB cancer therapy in a lastingly effective manner. The approach uses a conductive hydrogel-based electroporation system to enable nucleofection of programmed cell death protein 1 (PD1) targeted CRISPR-Cas9 DNAs into T-cells directly within the lymph nodes, and subsequently produces PD1-deficient T-cells to combat tumor growth, metastasis and recurrence in different melanoma models in mice. Following in vivo gene editing, animals show enhanced cellular and humoral immune responses along with multi-fold increases of effector T-cells infiltration to the solid tumors, preventing tumor recurrence and prolonging their survival. These findings provide a proof-of-concept for direct in vivo T-cell engineering via localized gene-editing for enhanced cancer immunotherapy, and also unlock the possibilities of using this method to treat more complex human diseases.}, }
@article {pmid39586398, year = {2025}, author = {Tachibana, Y and Yamamoto, M}, title = {Recent advances in identifying and characterizing secretory proteins of Toxoplasma gondii by CRISPR-based screening.}, journal = {Parasitology international}, volume = {105}, number = {}, pages = {102997}, doi = {10.1016/j.parint.2024.102997}, pmid = {39586398}, issn = {1873-0329}, mesh = {Animals ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; Host-Parasite Interactions ; *Protozoan Proteins/genetics/metabolism ; *Toxoplasma/genetics/metabolism/pathogenicity ; }, abstract = {The apicomplexan parasite, Toxoplasma gondii, develops unique secretory organelles, such as micronemes, rhoptries, and dense granules, which do not exist in other well-studied eukaryotic organisms. These secretory organelles are key features of apicomplexan parasites and discharge various proteins that are essential for invasion, replication, egress, host-parasite interactions, and virulence. Many studies have therefore focused on identifying and characterizing the proteins secreted by T. gondii that play essential roles in pathology and that can be targeted for therapeutics and vaccine development. The recent development of functional genetic screens based on CRISPR/Cas9 technology has revolutionized this field and has enabled the identification of genes that contribute to parasite fitness in vitro and in vivo. Consequently, characterization of genes identified by unbiased CRISPR screens has revealed novel aspects of apicomplexan biology. In this review, we describe the development of CRIPSR-based screening technology for T. gondii, and recent advances in our understanding of secretory proteins identified and characterized by CRISPR-based screening.}, }
@article {pmid39586143, year = {2025}, author = {Jiang, H and Zhu, X and Jiao, J and Yan, C and Liu, K and Chen, W and Qin, P}, title = {CRISPR/dCas9-based hotspot self-assembling SERS biosensor integrated with a smartphone for simultaneous, ultrasensitive and robust detection of multiple pathogens.}, journal = {Biosensors &amp; bioelectronics}, volume = {270}, number = {}, pages = {116974}, doi = {10.1016/j.bios.2024.116974}, pmid = {39586143}, issn = {1873-4235}, mesh = {*Biosensing Techniques/instrumentation ; *Spectrum Analysis, Raman ; *Smartphone ; *Gold/chemistry ; *Pseudomonas aeruginosa/isolation &amp; purification/genetics ; *Metal Nanoparticles/chemistry ; *Staphylococcus aureus/isolation &amp; purification/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Escherichia coli O157/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/instrumentation ; DNA, Bacterial/genetics/analysis ; Limit of Detection ; Molecular Diagnostic Techniques ; }, abstract = {Accurate detection of multiple pathogens in the early stages of infection is critical for guiding treatment and saving patients' lives, but current methods are still challenged by low sensitivity, poor robustness and long turnaround times. Here, we report a CRISPR/dCas9-based hotspot self-assembling surface-enhanced Raman scattering (SERS) biosensor (called dCasSERS) and its integration with a smartphone to address these challenges. In this design, bacterial DNA was pre-amplified by loop-mediated isothermal amplification (LAMP), and the repeat sequences of the amplicons were recognized by CRISPR/dCas9, providing abundant sites for the assembly of gold nanoparticles (AuNPs) and forming numerous hotspots for SERS analysis. Using AuNPs labeled with different Raman molecules as reporters, the presence of Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa) and Escherichia coli O157:H7 (E. coli O157:H7) could be dexterously converted into distinguishable SERS signals. CRISPR/dCas9-based amplicon-specific recognition and SERS hotspot self-assembly improved the specificity and sensitivity, enabling the biosensor to simultaneously detect three target pathogens down to 1 CFU/mL without any cross-reactivity. By introducing a rapid extraction procedure and a smartphone-integrated handheld Raman spectrometer, rapid on-site analysis of multiple pathogens could be achieved in less than 50 min. The accuracy and robustness of the biosensor were demonstrated by 500 real urine specimens. This study displays a new paradigm for CRISPR/Cas-based self-assembly of SERS hotspots and provides insight into the future development of pathogen screening tools.}, }
@article {pmid39585990, year = {2024}, author = {Li, L and Scott, WS and Khristich, AN and Armenia, JF and Mirkin, SM}, title = {Recurrent DNA nicks drive massive expansions of (GAA)n repeats.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {49}, pages = {e2413298121}, pmid = {39585990}, issn = {1091-6490}, support = {R35 GM130322/GM/NIGMS NIH HHS/United States ; R35GM130322//HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 2153071//NSF | BIO | Division of Molecular and Cellular Biosciences (MCB)/ ; }, mesh = {Humans ; *Trinucleotide Repeat Expansion/genetics ; Friedreich Ataxia/genetics ; CRISPR-Cas Systems ; DNA/genetics ; DNA Breaks, Single-Stranded ; }, abstract = {Over 50 hereditary degenerative disorders are caused by expansions of short tandem DNA repeats (STRs). (GAA)n repeat expansions are responsible for Friedreich's ataxia as well as late-onset cerebellar ataxias (LOCAs). Thus, the mechanisms of (GAA)n repeat expansions attract broad scientific attention. To investigate the role of DNA nicks in this process, we utilized a CRISPR-Cas9 nickase system to introduce targeted nicks adjacent to the (GAA)n repeat tract. We found that DNA nicks 5' of the (GAA)100 run led to a dramatic increase in both the rate and scale of its expansion in dividing cells. Strikingly, they also promoted large-scale expansions of carrier- and large normal-size (GAA)n repeats, recreating, in a model system, the expansion events that occur in human pedigrees. DNA nicks 3' of the (GAA)100 repeat led to a smaller but significant increase in the expansion rate as well. Our genetic analysis implies that in dividing cells, conversion of nicks into double-strand breaks (DSBs) during DNA replication followed by DSB or fork repair leads to repeat expansions. Finally, we showed that 5' GAA-strand nicks increase expansion frequency in nondividing yeast cells, albeit to a lesser extent than in dividing cells.}, }
@article {pmid39585465, year = {2024}, author = {Qiu, J and Liu, C and Zhu, Y}, title = {Rolling circle amplification cooperating crRNA switch for direct and sensitive methicillin-resistant Staphylococcus aureus (MRSA) analysis.}, journal = {Biotechnology letters}, volume = {47}, number = {1}, pages = {4}, pmid = {39585465}, issn = {1573-6776}, mesh = {*Methicillin-Resistant Staphylococcus aureus/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Staphylococcal Infections/microbiology/diagnosis ; Exodeoxyribonucleases/genetics/metabolism ; RNA, Bacterial/genetics ; Sensitivity and Specificity ; }, abstract = {Evaluating the methicillin resistance of Staphylococcus aureus (S. aureus) is highly important for adapting nursing strategies. Nevertheless, the identification of methicillin-resistant S. aureus (MRSA) that is both sensitive and reliable continues to pose a significant obstacle. This study describes a method for detecting MRSA using a combination of fixed rolling circle amplification (RCA) and the exonuclease-iii (Exo-iii) assisted CRISPR-Cas12a system for signal amplification. When MRSA is present, the interaction between the "b" chain in the capture probe and MRSA allows the "a" chain to be exposed. This "a" chain acts as a primer to initiate the fixed RCA process. The H probe, which includes the crRNA segment, forms a bond with the RCA product and then releases the crRNA segment with the aid of Exo-iii. The Cas12a protein, when combined with the crRNA, generates an activated CRISPR-Cas12a system that cleaves the "Reporter" probe, resulting in the production of fluorescent signals. Furthermore, this fluorescent test has been utilized for the examination of clinical samples with a satisfactory rate of retrieval. Based on the elegant design, the proposed method exhibited a low detection limit of 4.6 cfu/mL, while maintaining a high specificity for MRSA even from a mixture of several interfering bacteria. Due to its cost-effectiveness, simplicity, and adaptability, the sensing system shows potential as a platform for detecting MRSA and evaluating postoperative nursing for stomach cancer patients.}, }
@article {pmid39584421, year = {2024}, author = {Wang, S and Hu, Y and Deng, Z and Liu, R and Lv, Y}, title = {CRISPR/Cas12a-enhanced DNA nanomachine for multiple respiratory pathogens detection.}, journal = {Chemical communications (Cambridge, England)}, volume = {60}, number = {99}, pages = {14814-14817}, doi = {10.1039/d4cc05639f}, pmid = {39584421}, issn = {1364-548X}, mesh = {*CRISPR-Cas Systems/genetics ; *SARS-CoV-2/genetics/isolation &amp; purification ; Humans ; *Influenza A Virus, H1N1 Subtype/isolation &amp; purification/genetics ; *Mycoplasma pneumoniae/genetics/isolation &amp; purification ; DNA/chemistry ; COVID-19/virology/diagnosis ; Limit of Detection ; Nanostructures/chemistry ; Mass Spectrometry ; CRISPR-Associated Proteins/metabolism/genetics ; Respiratory Tract Infections/virology/microbiology/diagnosis ; }, abstract = {Respiratory infection caused by pathogens is among the most prevalent health issues affecting people worldwide. Accurate and rapid screening of respiratory pathogens is crucial for selecting appropriate treatments to control epidemics. However, it is often challenged by two aspects: first, the low concentration of pathogens in the early stages of infection; second, the difficulty of analyzing multiple pathogens. Herein, we report a mass spectrometry strategy combining the CRISPR/Cas12a system with DNA nanomachines for respiratory pathogens detection. Thanks to the high sensitivity of the CRISPR/Cas12a-enhanced DNA nanomachine and the multiple analysis of elemental mass spectrometry, the proposed method was successfully applied for clinical sample analysis with a low detection limit of 28 amol, 30 amol, and 38 amol for SARS-CoV-2, influenza A virus subtype H1N1, and Mycoplasma pneumoniae, respectively.}, }
@article {pmid39584349, year = {2024}, author = {Chen, Z and Wu, Y and Zhang, Y and Gao, Y}, title = {[Knockout of RIG-I in HEK293 cells by CRISPR/Cas9].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {40}, number = {11}, pages = {4254-4265}, doi = {10.13345/j.cjb.240130}, pmid = {39584349}, issn = {1872-2075}, mesh = {Humans ; HEK293 Cells ; *CRISPR-Cas Systems ; *DEAD Box Protein 58/genetics/metabolism ; *Signal Transduction ; *Receptors, Immunologic/genetics/metabolism ; Gene Knockout Techniques ; Transfection ; DEAD-box RNA Helicases/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Interferon-Induced Helicase, IFIH1/genetics/metabolism ; Transcription Factor RelA/genetics/metabolism ; Interferon-beta/genetics/metabolism ; }, abstract = {We knocked out the retinoic acid-inducible gene I (RIG-I) in HEK293 cells via CRISPR/Cas9 to reveal the effects of RIG-I knockout on the key factors in the type I interferon signaling pathway. Three single guide RNAs (sgRNAs) targeting RIG-I were designed, and the recombination vectors were constructed on the basis of the pX459 vector and used to transfect HEK293 cells, which were screened by puromycin subsequently. Furthermore, a mimic of virus, poly I: C, was used to transfect the cells screened out. RIG-I knockout was checked by sequencing, real-time quantitative PCR, Western blotting, and immunofluorescence assay. Meanwhile, the expression levels of key factors of type I interferon signaling pathway such as melanoma differentiation-associated gene 5 (MDA5), interferonβ1 (IFNβ1), and nuclear factor-kappa B p65 [NF-κB(p65)], as well as cell viability, were determined. The results showed that two HEK293 cell lines (S1 and S3) with RIG-I knockout were obtained, which exhibited lower mRNA and protein levels of RIG-I than the wild type HEK293 cells (P < 0.05). The mRNA levels of MDA5 and IFNβ1 in S1 and S3 cells and the protein level of NF-κB(p65) in S3 cells were lower than those in the wild type (P < 0.05). More extranuclear NF-κB(p65) protein was detected in S1 cells than in the wild type after transfection with poly I: C. Plus, the wild-type and S1 cells transfected with poly I: C for 48 h showcased reduced viability (P < 0.05), while S3 cells did not display the reduction in cell viability. In summary, the present study obtained two HEK293 cell lines with RIG-I knockout via CRISPR/Cas9, which provided a stable cell model for exploring the mechanism of type I interferon signaling pathway.}, }
@article {pmid39584322, year = {2024}, author = {Hu, Y and Chen, Z and Zhang, S and Ge, S}, title = {[Advances in visual detection of pathogen nucleic acids by CRISPR-Cas].}, journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology}, volume = {40}, number = {11}, pages = {3872-3887}, doi = {10.13345/j.cjb.240111}, pmid = {39584322}, issn = {1872-2075}, mesh = {*CRISPR-Cas Systems ; Humans ; Nucleic Acids/analysis ; Point-of-Care Testing ; }, abstract = {Visual detection is a technique for evaluating the results through visual judgment without relying on complex optical detection systems. It obtains results quickly based on signals, such as visible light, changes in air pressure, and migration distance, that can be directly observed by naked eyes, being widely used in the in vitro diagnostics industry. The CRISPR-Cas system has the potential to be used in the development of point of care testing (POCT) technologies due to the advantages of mild reaction conditions, no need for thermal cycling or other control measures, and a robust signal amplification capability. In recent years, the combination of visual detection and CRISPR-Cas has significantly reduced the need for laboratory infrastructures, precision instruments, and specialized personnel for nucleic acid detection. This has promoted the development of POCT technology and methods for nucleic acids. This article summarizes the signal output modes and characteristics of the visual detection of nucleic acid by CRISPR-Cas and discusses the issues in the application. Finally, its future clinical translation is envisioned with a view to informing the development of CRISPR-Cas visualization assays.}, }
@article {pmid39582866, year = {2024}, author = {Amiri, M and Moaveni, AK and Majidi Zolbin, M and Shademan, B and Nourazarian, A}, title = {Optimizing cancer treatment: the synergistic potential of CAR-T cell therapy and CRISPR/Cas9.}, journal = {Frontiers in immunology}, volume = {15}, number = {}, pages = {1462697}, pmid = {39582866}, issn = {1664-3224}, mesh = {Humans ; *CRISPR-Cas Systems ; *Immunotherapy, Adoptive/methods ; *Neoplasms/therapy/immunology/genetics ; *Gene Editing/methods ; Animals ; Receptors, Chimeric Antigen/genetics/immunology ; Combined Modality Therapy ; }, abstract = {Optimizing cancer treatment has become a pivotal goal in modern oncology, with advancements in immunotherapy and genetic engineering offering promising avenues. CAR-T cell therapy, a revolutionary approach that harnesses the body's own immune cells to target and destroy cancer cells, has shown remarkable success, particularly in treating acute lymphoblastic leukemia (ALL), and in treating other hematologic malignancies. While CAR-T cell therapy has shown promise, challenges such as high cost and manufacturing complexity remain. However, its efficacy in solid tumors remains limited. The integration of CRISPR/Cas9 technology, a powerful and precise genome-editing tool, also raises safety concerns regarding unintended edits and off-target effects, offers a synergistic potential to overcome these limitations. CRISPR/Cas9 can enhance CAR-T cell therapy by improving the specificity and persistence of CAR-T cells, reducing off-target effects, and engineering resistance to tumor-induced immunosuppression. This combination can also facilitate the knockout of immune checkpoint inhibitors, boosting the anti-tumor activity of CAR-T cells. Recent studies have demonstrated that CRISPR/Cas9-edited CAR-T cells can target previously untreatable cancer types, offering new hope for patients with refractory cancers. This synergistic approach not only enhances the efficacy of cancer treatment but also paves the way for personalized therapies tailored to individual genetic profiles. This review highlights the ongoing research efforts to refine this approach and explores its potential to revolutionize cancer treatment across a broader range of malignancies. As research progresses, the integration of CAR-T cell therapy and CRISPR/Cas9 holds the promise of transforming cancer treatment, making it more effective and accessible. This review explores the current advancements, challenges, and future prospects of this innovative therapeutic strategy.}, }
@article {pmid39581190, year = {2025}, author = {Egawa, M and Uno, N and Komazaki, R and Ohkame, Y and Yamazaki, K and Yoshimatsu, C and Ishizu, Y and Okano, Y and Miyamoto, H and Osaki, M and Suzuki, T and Hosomichi, K and Aizawa, Y and Kazuki, Y and Tomizuka, K}, title = {Generation of Monosomy 21q Human iPS Cells by CRISPR/Cas9-Mediated Interstitial Megabase Deletion.}, journal = {Genes to cells : devoted to molecular &amp; cellular mechanisms}, volume = {30}, number = {1}, pages = {e13184}, pmid = {39581190}, issn = {1365-2443}, support = {21-101//Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences/ ; JP24bm1123038//Japan Agency for Medical Research and Development/ ; JP24ama121046//Japan Agency for Medical Research and Development/ ; JP24gm0010010//Japan Agency for Medical Research and Development/ ; JPMJCR18S4//CREST, Japan Science and Technology Agency/ ; JPMJSP2134//SPRING, Japan Science and Technology Agency/ ; }, mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems ; *Chromosomes, Human, Pair 21/genetics ; *Chromosome Deletion ; Monosomy/genetics ; Cell Line ; }, abstract = {Missing an entire chromosome or chromosome arm in normal diploid cells has a deleterious impact on cell viability, which may contribute to the development of specific birth defects. Nevertheless, the effects of chromosome loss in human cells have remained unexplored due to the lack of suitable model systems. Here, we developed an efficient, selection-free approach to generate partial monosomy in human induced pluripotent stem cells (iPSCs). The introduction of Cas9 proteins and a pair of gRNAs induces over megabase-sized interstitial chromosomal deletions. Using human chromosome 21 (HSA21) as a model, partial monosomy 21q (PM21q) iPSC lines with deletions ranging from 4.5 to 27.9 Mb were isolated. A 33.6 Mb deletion, encompassing all protein-coding genes on 21q, was also achieved, establishing the first 21q monosomy human iPSC line. Transcriptome and proteome analyses revealed that the abundances of mRNA and protein encoded by the majority of genes in the monosomic regions are half of the diploid expression level, indicating an absence of dosage compensation. The ability to generate customized partial monosomy cell lines on an isogenic, karyotypically normal background should facilitate the gain of novel insights into the impact of chromosome loss on cellular fitness.}, }
@article {pmid39580667, year = {2024}, author = {Kim, B and Seo, HW and Lee, K and Yong, D and Park, YK and Lee, Y and Lee, S and Kim, DW and Kim, D and Ryu, CM}, title = {Lipid Nanoparticle-Mediated CRISPR-Cas13a Delivery for the Control of Bacterial Infection.}, journal = {Advanced healthcare materials}, volume = {}, number = {}, pages = {e2403281}, doi = {10.1002/adhm.202403281}, pmid = {39580667}, issn = {2192-2659}, support = {KGM9942421//Korea Research Institute of Bioscience and Biotechnology/ ; RS-2023-00219213//Ministry of Science and ICT, South Korea/ ; }, abstract = {Lipid nanoparticles (LNPs) can assist in the delivery of nucleic acid inside animal cells, as demonstrated by their use in COVID-19 vaccine development. However, LNPs applicable to bacteria have not been reported. Here, the screening of 511 LNPs containing random combinations of different lipid components identified two LNPs, LNP 496 and LNP 470, that efficiently delivered plasmids into Escherichia coli BW25113. Since Gram-negative bacteria have lipid bilayers, the bacteria are pretreated with LNP-helper that weakens the bacterial membrane. The cationic lipid DOTAP improved delivery of LNP-encapsulated plasmid DNA when present at a molar ratio of 10-25 mol% in the LNP. LNP encapsulation of the Cas13a/gRNA expression vector controlled infection by a clinical Escherichia strain in Galleria mellonela larvae and mouse infection models when used in combination with non-cytotoxic concentrations of polymyxin B, a bacterial membrane disruptor. Together, the results show that LNPs can be useful as a delivery platform for agents that counteract pathogenic bacterial infections.}, }
@article {pmid39579910, year = {2025}, author = {Chen, C and Li, YW and Zheng, YY and Li, XJ and Wu, N and Guo, Q and Shi, TQ and Huang, H}, title = {Expanding the frontiers of genome engineering: A comprehensive review of CRISPR-associated transposons.}, journal = {Biotechnology advances}, volume = {78}, number = {}, pages = {108481}, doi = {10.1016/j.biotechadv.2024.108481}, pmid = {39579910}, issn = {1873-1899}, mesh = {Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *DNA Transposable Elements/genetics ; *Gene Editing/methods ; }, abstract = {Genome engineering is extensively utilized in diverse scientific disciplines, advancing human welfare and addressing various challenges. Numerous genome engineering tools have been developed to modify genomic sequences. Among these, the CRISPR-Cas system has transformed the field and remains the most commonly employed genome-editing tool. However, the CRISPR-Cas system relies on induced double-strand breaks, with editing efficiency often limited by factors such as cell type and homologous recombination, impeding further progress. CRISPR-associated transposons (CASTs) represent programmable mobile genetic elements. CASTs identified as active were developed as CAST systems, which can perform RNA-guided DNA integration and are featured by high precision, programmability, and kilobase-level payload capacity. Moreover, CAST system allows for precise genome modifications independent of host DNA repair mechanisms, addressing the constraints of conventional CRISPR-Cas systems. It expands the genome engineering toolkit and is poised to become a representative of next-generation genome editing tools. This review thoroughly examines the research progress on CASTs, highlighting the current challenges faced in genome engineering based on CASTs, and offering insights into the ongoing development of this transformative technology.}, }
@article {pmid39579237, year = {2024}, author = {Kumari, K and Gusain, S and Joshi, R}, title = {Engineering cold resilience: implementing gene editing tools for plant cold stress tolerance.}, journal = {Planta}, volume = {261}, number = {1}, pages = {2}, pmid = {39579237}, issn = {1432-2048}, mesh = {*Gene Editing/methods ; *Cold-Shock Response/genetics ; *CRISPR-Cas Systems ; Crops, Agricultural/genetics ; Cold Temperature ; Acclimatization/genetics ; Plants/genetics ; Genetic Engineering/methods ; Genome, Plant/genetics ; Plant Breeding/methods ; }, abstract = {This paper highlights the need for innovative approaches to enhance cold tolerance. It underscores how genome-editing tools can deepen our understanding of genes involved in cold stress. Cold stress is a significant abiotic factor in high-altitude regions, adversely affecting plant growth and limiting crop productivity. Plants have evolved various mechanisms in response to low temperatures that enable resistance at both physiological and molecular levels during chilling and freezing stress. Several cold-inducible genes have been isolated and characterized, with most playing key roles in providing tolerance against low-temperature stress. However, many plants fail to survive at low temperatures due to the absence of cold acclimatization mechanisms. Conventional breeding techniques, such as inter-specific or inter-genic hybridization, have had limited effectiveness in enhancing the cold resistance of essential crops. Thus, it is crucial to develop crops with improved adaptability, high yields and resistance to cold stress using advanced genomic approaches. The current availability of gene editing tools offers the opportunity to introduce targeted modifications in plant genomes efficiently, thereby developing cold-tolerant varieties. This review discusses advancements in gene editing tools, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)/Cas12a(Cpf1), prime editing (PE) and retron library recombineering (RLR). We focus specifically on the CRISPR/Cas system, which has garnered significant attention in recent years as a groundbreaking tool for genome editing across various species. These techniques will enhance our understanding of molecular interactions under low-temperature stress response and highlight the progress of genome editing in designing future climate-resilient crops.}, }
@article {pmid39579214, year = {2024}, author = {Karmakar, S and Panda, D and Behera, D and Saha, R and Baig, MJ and Molla, KA}, title = {Adaptation of bacterial natural single guide RNA (tracr-L) for efficient plant genome editing.}, journal = {Plant cell reports}, volume = {43}, number = {12}, pages = {291}, pmid = {39579214}, issn = {1432-203X}, mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; *Streptococcus pyogenes/genetics ; RNA, Bacterial/genetics ; Oryza/genetics/microbiology ; Protoplasts ; DNA Breaks, Double-Stranded ; }, abstract = {A long tracrRNA (tracr-L), which naturally act as single guide RNA, and its truncated version, Δtracr-L, from S. pyogenes, efficiently induce Cas9-mediated double-strand breaks (DSBs) in plant genomic loci, as demonstrated by in vitro cleavage assay and protoplast transfection. CRISPR-Cas system provides a form of immune memory in prokaryotes and archaea, protecting them against viruses and foreign genetic elements. In Streptococcus pyogenes, this system includes the pre-crRNA along with another non-coding RNA, tracrRNA, which aids in CRISPR-based immunity. In S. pyogenes, two distinct tracrRNAs are produced: a long form (tracr-L) and a short form (tracr-S). The tracr-S regulates crRNA biogenesis and Cas9 cleavage, while tracr-L suppresses CRISPR-Cas expression by targeting the Cas9 promoter to prevent autoimmunity. Deleting 79 nucleotides from tracr-L results in Δtracr-L, which retains similar functionality in gene repression. This study investigates, for the first time, the effectiveness of tracr-L, and Δtracr-L in genome editing within plant systems. In vitro cleavage assays using purified Cas9 and synthesized sgRNAs targeting the Cas9 gene, OsPDS, and the OsSWEET11 promoter revealed that across all target sites, tracr-S demonstrated the highest cleavage efficiency compared to tracr-L and Δtracr-L. For in vivo genome editing, we transfected rice protoplasts with tracr-L, Δtracr-L, and tracr-S, targeting three rice genes: OsPDS, OsSPL14, and the promoter of OsSWEET14. Amplicon deep sequencing revealed various types of indels at the target regions across all three tracrRNA versions, indicating comparable levels of efficiency. This study establishes the utility of both the long-form tracrRNA (tracr-L) and its truncated variant (Δtracr-L) in eukaryote genome editing. These two new forms of tracrRNA provide proof of concept and expand the CRISPR-Cas toolkit for plant genome editing applications, and for eukaryotes more broadly.}, }
@article {pmid39578577, year = {2024}, author = {Ingle, H and Molleston, JM and Hall, PD and Bui, D and Wang, L and Bhatt, KD and Foster, L and Antia, A and Ding, S and Lee, S and Fremont, DH and Baldridge, MT}, title = {The neonatal Fc receptor is a cellular receptor for human astrovirus.}, journal = {Nature microbiology}, volume = {9}, number = {12}, pages = {3321-3331}, pmid = {39578577}, issn = {2058-5276}, support = {R01AI127552//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01AI139314//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01AI181955//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01AI141478//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; T32AI106688//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; T32DK077653//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; F30AI181285//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01AI150796//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; U19AI116484//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R00 AI141683/AI/NIAID NIH HHS/United States ; Pathogenesis of Infectious Disease Program//Burroughs Wellcome Fund (BWF)/ ; Pew Biomedical Scholars Program//Pew Charitable Trusts/ ; T32DK007130//U.S. Department of Health &amp; Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes &amp; Digestive &amp; Kidney Diseases)/ ; P20 GM109035/GM/NIGMS NIH HHS/United States ; 75N93022C00035/AI/NIAID NIH HHS/United States ; R00 AI141683/AI/NIAID NIH HHS/United States ; P20 GM109035/GM/NIGMS NIH HHS/United States ; 75N93022C00035/AI/NIAID NIH HHS/United States ; }, mesh = {Humans ; *Receptors, Fc/metabolism/genetics ; *Histocompatibility Antigens Class I/metabolism/genetics ; *Mamastrovirus/metabolism/genetics/physiology ; *Virus Internalization ; *Dipeptidyl Peptidase 4/metabolism/genetics ; Astroviridae Infections/virology/metabolism ; CRISPR-Cas Systems ; Cell Line ; Receptors, Virus/metabolism ; HEK293 Cells ; }, abstract = {Human astroviruses (HAstV) are major causes of gastroenteritis, especially in children, and there are no vaccines or antivirals currently available. Little is known about host factors required for their cellular entry. Here we utilized complementary CRISPR-Cas9-based knockout and activation screens to identify neonatal Fc receptor (FcRn) and dipeptidyl-peptidase IV (DPP4) as entry factors for HAstV infection in vitro. Disruption of FcRn or DPP4 reduced HAstV infection in permissive cells and, reciprocally, overexpression of these factors in non-permissive cells was sufficient to promote infection. We observed direct binding of FcRn, but not DPP4, with HAstV virions and the purified spike protein. This suggests that FcRn is a receptor for HAstVs while DPP4 is a cofactor for entry. Inhibitors for DPP4 and FcRn currently in clinical use prevented HAstV infection in cell lines and human enteroids. Our results reveal mechanisms of HAstV entry as well as druggable targets to limit HAstV infection.}, }
@article {pmid39578074, year = {2025}, author = {Ferdigg, A and Hopp, AK and Wolf, G and Superti-Furga, G}, title = {Membrane transporters modulating the toxicity of arsenic, cadmium, and mercury in human cells.}, journal = {Life science alliance}, volume = {8}, number = {2}, pages = {}, pmid = {39578074}, issn = {2575-1077}, mesh = {Humans ; *Cadmium/toxicity/metabolism ; *Arsenic/toxicity/metabolism ; *Mercury/toxicity/metabolism ; *Multidrug Resistance-Associated Proteins/metabolism/genetics ; Cation Transport Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Membrane Transport Proteins/metabolism/genetics ; Cell Line ; Zinc/metabolism ; HEK293 Cells ; }, abstract = {Non-essential metals are extremely toxic to living organisms, posing significant health risks, particularly in developing nations where they are a major contributor to illness and death. Although their toxicity is widely acknowledged, the mechanisms by which they are regulated within human cells remain incompletely understood. Specifically, the role of membrane transporters in mediating heavy metal toxicity is not well comprehended. Our study demonstrates how specific transporters can modulate the toxicity of cadmium, mercury, and the metalloid arsenic in human cells. Using CRISPR/Cas9 loss-of-function screens, we found that the multidrug resistance protein MRP1/ABCC1 provided protection against toxicity induced by arsenic and mercury. In addition, we found that SLC39A14 and SLC30A1 increased cellular sensitivity to cadmium. Using a reporter cell line to monitor cellular metal accumulation and performing a cDNA gain-of-function screen, we were able to clarify the function of SLC30A1 in controlling cadmium toxicity through the modulation of intracellular zinc levels. This transporter-wide approach provides new insights into the complex roles of membrane transporters in influencing the toxicity of arsenic, cadmium, and mercury in human cell lines.}, }
@article {pmid39577309, year = {2024}, author = {Tran, HD and Denman, CR and Shin, MK and Jeon, D and Kuhn, B and Jo, J}, title = {Establishment of TH-EGFP human embryonic stem cell line for specific labeling of dopaminergic neurons.}, journal = {Stem cell research}, volume = {81}, number = {}, pages = {103613}, doi = {10.1016/j.scr.2024.103613}, pmid = {39577309}, issn = {1876-7753}, mesh = {Humans ; *Dopaminergic Neurons/metabolism/cytology ; *Human Embryonic Stem Cells/metabolism/cytology ; *Green Fluorescent Proteins/metabolism/genetics ; Cell Differentiation ; Cell Line ; Tyrosine 3-Monooxygenase/metabolism/genetics ; CRISPR-Cas Systems ; }, abstract = {Dopaminergic (DA) neurons play critical roles in various neurological processes and disorders, particularly Parkinson's disease. To enable precise visualization and tracking of DA neurons, we generated TH-EGFP, a tyrosine hydroxylase (TH)-driven enhanced green fluorescent protein (EGFP)-expressing knock-in cell line, by employing CRISPR/Cas9 technology. We introduced EGFP into the targeted genomic region of human embryonic stem cells (hESCs) and successfully established a TH-EGFP hESC line. Differentiation of TH-EGFP hESCs into human midbrain organoids confirmed the accurate integration of EGFP into TH-positive cells. The TH-EGFP hESC line serves as a valuable reporter for studying the development, maturation, and function of DA neurons.}, }
@article {pmid39577180, year = {2025}, author = {Dai, F and Zhang, T and Pang, F and Jiao, T and Wang, K and Zhang, Z and Wang, N and Xie, Z and Zhang, Y and Wang, Z and Chen, Z and Yu, M and Wei, H and Song, J}, title = {A compact, palm-sized isothermal fluorescent diagnostic intelligent IoT device for personal health monitoring and beyond via one-tube/one-step LAMP-CRISPR assay.}, journal = {Biosensors &amp; bioelectronics}, volume = {270}, number = {}, pages = {116945}, doi = {10.1016/j.bios.2024.116945}, pmid = {39577180}, issn = {1873-4235}, mesh = {*Biosensing Techniques/instrumentation/methods ; *Nucleic Acid Amplification Techniques/instrumentation/methods ; Humans ; *COVID-19/diagnosis/virology ; *SARS-CoV-2/isolation &amp; purification/genetics ; Molecular Diagnostic Techniques/instrumentation ; CRISPR-Cas Systems ; Smartphone ; Animals ; Equipment Design ; Fluorescence ; }, abstract = {The demand for accurate, user-friendly, and sensitive at-home nucleic acid testing solutions is rising due to occasional outbreaks of various infectious diseases and a growing desire for an improved quality of life. In response, we developed the WeD-mini, a compact, palm-sized isothermal fluorescent diagnostic IoT device that weighs just 61 g. The WeD-mini features a uniquely designed, highly sensitive optical sensing system, ultra-low power consumption, a minimalist industrial design, and an intelligent operating algorithm. It integrates real-time fluorescence detection and automatic result interpretation via a smartphone, with results seamlessly uploaded to the 'EzDx Cloud' for comprehensive health management and spatio-temporal disease mapping. The device supports various assays that operate at different temperatures and with varying fluorescence emission intensities, such as RPA (39 °C, low intensity), LAMP (65 °C, high intensity), and LAMP-PfAgo (65/95 °C, high intensity), while maintaining precise temperature control and exceptional fluorescence detection sensitivity. Additionally, we engineered a more thermostable AapCRISPR-Cas12b variant that operates effectively at 63 °C, enhancing compatibility with LAMP to create a robust One-Tube/One-Step LAMP-CRISPR assay. Adaptable for at-home testing of SARS-CoV-2 and influenza viruses, the WeD-mini achieved 100% sensitivity and specificity with the newly established One-Tube/One-Step LAMP-CRISPR assay. Furthermore, the WeD-mini shows potential applications in detecting meat adulteration, monitoring respiratory diseases in pets, and conducting wastewater surveillance, making it suitable for a wide range of personal and public health use cases.}, }
@article {pmid39577179, year = {2025}, author = {Liu, C and Liu, Q and Chen, X and Guo, M and Chen, Z and Zhao, J and Chen, H and Guo, S and Cen, H and Yao, G and Chen, L and Wang, Y and Yang, PC and Wang, L and Chen, F}, title = {A novel label-free biosensor for myocardial ischemia biomarker detection via CRISPR/12a.}, journal = {Biosensors &amp; bioelectronics}, volume = {270}, number = {}, pages = {116954}, doi = {10.1016/j.bios.2024.116954}, pmid = {39577179}, issn = {1873-4235}, mesh = {*Biosensing Techniques/instrumentation/methods ; *Myocardial Ischemia/diagnosis/genetics ; Animals ; *Biomarkers ; Mice ; Humans ; *CRISPR-Cas Systems ; Adenosine Triphosphate/analysis ; Myocytes, Cardiac ; G-Quadruplexes ; }, abstract = {Myocardial infarction (MI) is the leading cause of death worldwide. Here, we present a novel, label-free biosensor for detecting myocardial ischemia biomarkers via CRISPR/Cas12a. This system utilizes the unique properties of CRISPR/Cas12a and G-quadruplex-ThT-based biosensors, enabling sensitive and specific detection of ATP, a crucial biomarker in cardiovascular diseases, at concentrations as low as 23 nM. Our method demonstrates substantial improvements over traditional ATP detection techniques, such as high-performance liquid chromatography and enzymatic assays, which often require complex sample preparation methods and costly equipment. The feasibility of the biosensor was further demonstrated in various models, including heart failure in mice and hypoxic conditions in cardiomyocytes. This successfully showcased its ability to function as a practical tool for diagnosing and monitoring diseases characterized by ATP dysregulation, highlighting its effectiveness in real-world clinical scenarios. This biosensor is notable for its rapid response, ease of use, and potential for integration into point-of-care diagnostics. These features offer significant advantages for the early diagnosis and management of ischemic heart disease and other conditions where ATP serves as a key metabolic biomarker. This technology also offers significant potential for early diagnosis and monitoring of myocardial ischemia and cardiovascular diagnostics. These findings underscore the biosensor's capacity for real-time ATP monitoring, offering crucial insights into mitochondrial function and disease progression, particularly in cardiovascular and inflammatory diseases.}, }
@article {pmid39577177, year = {2025}, author = {Zhuang, T and Gao, C and Zhao, W and Yu, H and Liu, Y and Zhang, N and Li, N and Ji, M}, title = {A minimal transcription template-based amplification-free CRISPR-Cas13a strategy for DNA detection.}, journal = {Biosensors &amp; bioelectronics}, volume = {270}, number = {}, pages = {116918}, doi = {10.1016/j.bios.2024.116918}, pmid = {39577177}, issn = {1873-4235}, mesh = {*CRISPR-Cas Systems ; Humans ; *Biosensing Techniques/methods ; DNA, Viral/genetics/analysis/isolation &amp; purification ; Adenoviruses, Human/genetics/isolation &amp; purification ; }, abstract = {CRISPR-Cas13a has shown great potential for the rapid and accurate detection of pathogen nucleic acids. However, conventional CRISPR-Cas13a-based assays typically require pre-amplification, which can introduce aerosol contamination and operational complexities. In this study, we developed a Minimalist transcription template-based Amplification-free CRISPR-Cas13a strategy for DNA detection (MAD). This strategy facilitates the release of pathogen DNA and its annealing with primers from nasopharyngeal swab samples in a straightforward manner, followed by T7 transcription and CRISPR-Cas13a detection, completing the entire process within 40 min. MAD eliminates the need for DNA extraction and pre-amplification while maintaining high sensitivity after optimization, allowing for result visualization via lateral flow strips. Furthermore, evaluation of 167 clinical pediatric samples identified 18 positive cases of human adenovirus, demonstrating a 99.4% concordance in detection compared to standard qPCR. We believe that MAD offers new insights into CRISPR-Cas diagnostics and, due to its simplicity, rapidity, and safety, is poised for widespread application in clinical practice.}, }
@article {pmid39576839, year = {2024}, author = {Harrison, EN and Jay, AN and Kent, MR and Sukienik, TP and LaVigne, CA and Kendall, GC}, title = {Engineering an fgfr4 knockout zebrafish to study its role in development and disease.}, journal = {PloS one}, volume = {19}, number = {11}, pages = {e0310100}, pmid = {39576839}, issn = {1932-6203}, support = {R01 CA272872/CA/NCI NIH HHS/United States ; T32 CA269052/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; *Zebrafish/genetics ; *Receptor, Fibroblast Growth Factor, Type 4/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Knockout Techniques ; Zebrafish Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals, Genetically Modified ; }, abstract = {Fibroblast growth factor receptor 4 (FGFR4) has a role in many biological processes, including lipid metabolism, tissue repair, and vertebrate development. In recent years, FGFR4 overexpression and activating mutations have been associated with numerous adult and pediatric cancers. As such, FGFR4 presents an opportunity for therapeutic targeting which is being pursued in clinical trials. To understand the role of FGFR4 signaling in disease and development, we generated and characterized three alleles of fgfr4 knockout zebrafish strains using CRISPR/Cas9. To generate fgfr4 knockout crispants, we injected single-cell wildtype zebrafish embryos with fgfr4 targeting guide RNA and Cas9 proteins, identified adult founders, and outcrossed to wildtype zebrafish to create an F1 generation. The generated mutations introduce a stop codon within the second Ig-like domain of Fgfr4, resulting in a truncated 215, 223, or 228 amino acid Fgfr4 protein compared to 922 amino acids in the full-length protein. All mutant strains exhibited significantly decreased fgfr4 mRNA expression during development, providing evidence for successful knockout of fgfr4 in mutant zebrafish. We found that, consistent with other Fgfr4 knockout animal models, the fgfr4 mutant fish developed normally; however, homozygous fgfr4 mutant zebrafish were significantly smaller than wildtype fish at three months post fertilization. These fgfr4 knockout zebrafish lines are a valuable tool to study the role of FGFR4 in vertebrate development and its viability as a potential therapeutic target in pediatric and adult cancers, as well as other diseases.}, }
@article {pmid39576609, year = {2025}, author = {Fal, K and Carles, CC}, title = {dCas-Based Tools to Visualize Chromatin or Modify Epigenetic Marks at Specific Plant Genomic Loci.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2873}, number = {}, pages = {305-332}, pmid = {39576609}, issn = {1940-6029}, mesh = {*Chromatin/genetics/metabolism ; *Epigenesis, Genetic ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genome, Plant ; Gene Expression Regulation, Plant ; Plants/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genetic Loci ; Plants, Genetically Modified/genetics ; }, abstract = {Development of locus-specific approaches targeting precise regions on chromatin, for locus/transcription visualization or transcription/epigenetic marks editing, is a critical challenge in functional genetics and epigenetics. Systems engineered from the clustered regularly interspaced short palindromic repeats (CRISPR) and its associated endonuclease (Cas) operate through DNA sequence-specific recognition by so-called guide RNAs, which provides high flexibility and modularity for precise chromatin visualization or edition. Here, we provide an overview of the CRISPR/Cas-derived tools developed for visualization of chromatin loci in live imaging or for effective modification of gene expression. These tools make use of effector modules that combine activators, repressors, and epigenetic modifiers with a deactivated Cas protein (dCas). We present how their use in plants brought advances in visualizing or manipulating the expression of loci involved in agronomically interesting traits such as flowering time and response to drought or heat. We also discuss the limitations and future improvements of the dCas-related technologies, such as more compact and combinatorial systems, spatiotemporal targeting for fine-tuning of gene expression, and live visualization of chromatin dynamics.}, }
@article {pmid39576313, year = {2025}, author = {Teng, P and Gao, Z and Quan, Q and He, G and Song, Q and Zhang, X and Xiao, W and Zhao, J and Cao, D and Liang, J and Tang, Y}, title = {SERS-based CRISPR/Cas12a assays for protein biomarker prostate-specific antigen detection.}, journal = {Analytical and bioanalytical chemistry}, volume = {417}, number = {3}, pages = {573-582}, pmid = {39576313}, issn = {1618-2650}, support = {No. 2022B1111020003//Key-Area Research and Development Program of Guangdong Province/ ; No. 32301266//the National Natural Science Foundation of China/ ; 82372099//the National Natural Science Foundation of China/ ; No. 202201011351//the Technology Research Program of Guangzhou City/ ; 2023A03J0283//the Technology Research Program of Guangzhou City/ ; 2024A03J1076//the Technology Research Program of Guangzhou City/ ; No. 2023CXB014//Outstanding Innovative Talents Cultivation Funded Programs for Doctoral Students of Jinan University/ ; No. 2022A1515110772//GuangDong Basic and Applied Basic Research Foundation/ ; 2021A1515220067//GuangDong Basic and Applied Basic Research Foundation/ ; 2021A1515110//GuangDong Basic and Applied Basic Research Foundation/ ; }, mesh = {*Prostate-Specific Antigen/blood/analysis ; *Spectrum Analysis, Raman/methods ; Humans ; *Limit of Detection ; Male ; CRISPR-Cas Systems ; Enzyme-Linked Immunosorbent Assay/methods ; Biomarkers/blood/analysis ; Biosensing Techniques/methods ; Biomarkers, Tumor/blood/analysis ; }, abstract = {Sensitive and accurate detection of protein biomarkers is crucial for disease diagnosis, especially for early diagnosis. Here, we describe surface-enhanced Raman scattering (SERS)-based CRISPR/Cas12a assays (S-CRISPR) for protein biomarker detection. Firstly, an S-CRISPR-driven enzyme-linked immunosorbent assay (S-CasLISA) was developed utilizing a capture antibody coated on a microplate to recognize the target and a detection antibody labeled with active DNA to trigger the activity of CRISPR/Cas12a. With this assay, we achieved detection of prostate-specific antigen (PSA) as models at the picogram level. The limit of detection (LoD) of S-CasLISA was 0.17 pg mL[-1] and in the range of 0.1 pg mL[-1] to 10 ng mL[-1]. Further, we applied aptamer to S-CRISPR (S-Apt-CRISPR), combining the high sensitivity of SERS with the high selectivity of aptamers, while simplifying the operation process of CRISPR detection of protein biomarkers. The proposed S-Apt-CRISPR also could detect picogram-level PSA and without repeated washing steps. The LoD of S-Apt-CRISPR was 0.35 pg mL[-1] and in the range of 0.1 pg mL[-1] to 10 ng mL[-1]. Both SERS-based CRISPR/Cas12a assays were validated with clinical samples and demonstrated accuracy consistent with the chemiluminescence immunoassay. The introduction of the CRISPR/Cas12a system with SERS has the effect of improving the analytical capabilities of the system, thereby broadening and facilitating its application in the analysis of sensitive and accurate protein biomarkers.}, }
@article {pmid39576177, year = {2024}, author = {Samudra, SP and Park, S and Esser, EA and McDonald, TP and Borges, AM and Eggenschwiler, J and Menke, DB}, title = {A new cell culture resource for investigations of reptilian gene function.}, journal = {Development (Cambridge, England)}, volume = {151}, number = {22}, pages = {}, pmid = {39576177}, issn = {1477-9129}, support = {R01 HG013006/HG/NHGRI NIH HHS/United States ; 1827647//National Science Foundation/ ; R01HG013006/NH/NIH HHS/United States ; //University of Georgia/ ; }, mesh = {Animals ; *Hedgehog Proteins/metabolism/genetics ; *Lizards/genetics ; Mice ; *Cilia/metabolism/genetics ; *Signal Transduction/genetics ; CRISPR-Cas Systems/genetics ; Cell Line ; Fibroblasts/metabolism/cytology ; Limb Buds/metabolism/cytology/embryology ; Cell Culture Techniques/methods ; Zinc Finger Protein GLI1/metabolism/genetics ; Gene Editing/methods ; }, abstract = {The establishment of CRISPR/Cas9 gene editing in Anolis sagrei has positioned this species as a powerful model for studies of reptilian gene function. To enhance this model, we developed an immortalized lizard fibroblast cell line (ASEC-1) for the exploration of reptilian gene function in cellular processes. We demonstrate the use of this cell line by scrutinizing the role of primary cilia in lizard Hedgehog (Hh) signaling. Using CRISPR/Cas9 mutagenesis, we disrupted the ift88 gene, which is required for ciliogenesis in diverse organisms. We determined that loss of itf88 from lizard cells leads to an absence of primary cilia, a partial derepression of gli1 transcription, and an inability of the cells to respond to the Smoothened agonist, SAG. Through a cross-species analysis of SAG-induced transcriptional responses in cultured limb bud cells, we further determined that ∼46% of genes induced as a response to Hh pathway activation in A. sagrei are also SAG responsive in Mus musculus limb bud cells. Our results highlight conserved and diverged aspects of Hh signaling in anoles and establish a new resource for investigations of reptilian gene function.}, }
@article {pmid39576014, year = {2024}, author = {Saito, S and Nakamura, Y and Miyashita, S and Sato, T and Hoshina, K and Okada, M and Hasegawa, H and Oishi, M and Fujii, Y and Körbelin, J and Kubota, Y and Tainaka, K and Natsumeda, M and Ueno, M}, title = {CRISPR/CasRx suppresses KRAS-induced brain arteriovenous malformation developed in postnatal brain endothelial cells in mice.}, journal = {JCI insight}, volume = {9}, number = {22}, pages = {}, pmid = {39576014}, issn = {2379-3708}, mesh = {Animals ; Mice ; *Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; *Endothelial Cells/metabolism ; *CRISPR-Cas Systems ; *Intracranial Arteriovenous Malformations/genetics/pathology/metabolism ; *Disease Models, Animal ; Brain/blood supply/pathology/metabolism ; Humans ; Mutation ; }, abstract = {Brain arteriovenous malformations (bAVMs) are anomalies forming vascular tangles connecting the arteries and veins, which cause hemorrhagic stroke in young adults. Current surgical approaches are highly invasive, and alternative therapeutic methods are warranted. Recent genetic studies identified KRAS mutations in endothelial cells of bAVMs; however, the underlying process leading to malformation in the postnatal stage remains unknown. Here we established a mouse model of bAVM developing during the early postnatal stage. Among 4 methods tested, mutant KRAS specifically introduced in brain endothelial cells by brain endothelial cell-directed adeno-associated virus (AAV) and endothelial cell-specific Cdh5-CreERT2 mice successfully induced bAVMs in the postnatal period. Mutant KRAS led to the development of multiple vascular tangles and hemorrhage in the brain with increased MAPK/ERK signaling and growth in endothelial cells. Three-dimensional analyses in cleared tissue revealed dilated vascular networks connecting arteries and veins, similar to human bAVMs. Single-cell RNA-Seq revealed dysregulated gene expressions in endothelial cells and multiple cell types involved in the pathological process. Finally, we employed CRISPR/CasRx to knock down mutant KRAS expression, which efficiently suppressed bAVM development. The present model reveals pathological processes that lead to postnatal bAVMs and demonstrates the efficacy of therapeutic strategies with CRISPR/CasRx.}, }
@article {pmid39575850, year = {2024}, author = {Zhong, J and Wu, X and Guo, C and Liu, C and Zhang, Q and Chen, Y and Liu, Y}, title = {A Single-Tube, Single-Enzyme Clustered Regularly Interspaced Short Palindromic Repeats System (UNISON) with Internal Controls for Accurate Nucleic Acid Detection.}, journal = {Analytical chemistry}, volume = {96}, number = {49}, pages = {19348-19353}, doi = {10.1021/acs.analchem.4c03403}, pmid = {39575850}, issn = {1520-6882}, mesh = {*CRISPR-Cas Systems/genetics ; *Hepatitis B virus/genetics ; Humans ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins have been widely applied in molecular diagnostics. Unlike the Ct value quantification method of PCR, the CRISPR system mainly relies on the rise of the rate of the fluorescence signal to indicate the concentration of the target nucleic acid, which is susceptible to system errors caused by various factors, such as reaction conditions and instrument performance. Therefore, establishing internal controls is essential to improve the accuracy, reliability, and commercial feasibility of the CRISPR system. However, the nonspecific trans-cleavage activity of Cas proteins presents a challenge in establishing internal controls. In this study, we developed unified nucleic acid detection with a single-tube, one-enzyme system (UNISON) for accurate nucleic acid detection with internal controls. By extending the crRNA and modifying it with different fluorophores and quenchers, we achieved that the specific target can only specifically cleave the corresponding folded crRNA and generate a corresponding fluorescence signal. With this design, we established an internal control, achieving accurate and reliable detection of clinical samples of the hepatitis B virus. Integrating internal controls into the CRISPR/Cas system demonstrates significant potential in medical diagnostics and virus monitoring.}, }
@article {pmid39575683, year = {2024}, author = {Siniscalco, AM and Perera, RP and Greenslade, JE and Veeravenkatasubramanian, H and Masters, A and Doll, HM and Raj, B}, title = {Barcoding Notch signaling in the developing brain.}, journal = {Development (Cambridge, England)}, volume = {151}, number = {24}, pages = {}, pmid = {39575683}, issn = {1477-9129}, support = {R00 HD098298/HD/NICHD NIH HHS/United States ; DGE-2236662//National Science Foundation/ ; //University of Pennsylvania/ ; DP2 NS131787/NS/NINDS NIH HHS/United States ; R00HD098298/NH/NIH HHS/United States ; }, mesh = {Animals ; *Zebrafish/genetics/embryology/metabolism ; *Brain/metabolism/embryology/growth &amp; development ; *Signal Transduction/genetics ; *Receptors, Notch/metabolism/genetics ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Neurons/metabolism/cytology ; Gene Expression Regulation, Developmental ; Single-Cell Analysis ; }, abstract = {Developmental signaling inputs are fundamental for shaping cell fates and behavior. However, traditional fluorescent-based signaling reporters have limitations in scalability and molecular resolution of cell types. We present SABER-seq, a CRISPR-Cas molecular recorder that stores transient developmental signaling cues as permanent mutations in cellular genomes for deconstruction at later stages via single-cell transcriptomics. We applied SABER-seq to record Notch signaling in developing zebrafish brains. SABER-seq has two components: a signaling sensor and a barcode recorder. The sensor activates Cas9 in a Notch-dependent manner with inducible control, while the recorder obtains mutations in ancestral cells where Notch is active. We combine SABER-seq with an expanded juvenile brain atlas to identify cell types derived from Notch-active founders. Our data reveal rare examples where differential Notch activities in ancestral progenitors are detected in terminally differentiated neuronal subtypes. SABER-seq is a novel platform for rapid, scalable and high-resolution mapping of signaling activity during development.}, }
@article {pmid39574587, year = {2024}, author = {Kushawah, G and Amaral, DB and Hassan, H and Gogol, M and Nowotarski, SH and Bazzini, AA}, title = {Critical role of Spatio-Temporally Regulated Maternal RNAs in Zebrafish Embryogenesis.}, journal = {bioRxiv : the preprint server for biology}, volume = {}, number = {}, pages = {}, pmid = {39574587}, issn = {2692-8205}, support = {R01 GM136849/GM/NIGMS NIH HHS/United States ; R21 OD034161/OD/NIH HHS/United States ; }, abstract = {The maternal-to-zygotic transition shifts regulatory control from maternal to zygotic messenger RNAs (mRNA) through maternal mRNA degradation. While temporal aspects of maternal mRNA decay are known, spatial mechanisms remain underexplored. Using CRISPR-Cas9 and CRISPR-Cas13d systems, we functionally dissected the contribution of maternal versus zygotic fractions and overcame challenges of studying embryonic lethal genes. We identified differentially distributed maternal mRNAs in specific cells and evidenced the critical role of five maternal mRNAs, cth1, arl4d, abi1b, foxa and lhx1a, in embryogenesis. Further, we focused on the functionally uncharacterized cth1 gene, revealing its essential role in gametogenesis and embryogenesis. Cth1 acts as a spatio-temporal RNA decay factor regulating mRNA stability and accumulation of its targets in a spatio-temporal manner through 3'UTR recognition during early development. Furthermore, Cth1 3'UTR drives its spatio-temporal RNA localization. Our findings provide new insights into spatio-temporal RNA decay mechanisms and highlight dual CRISPR-Cas strategies in studying embryonic development.}, }
@article {pmid39574297, year = {2024}, author = {Zhang, Y and Chen, J and Wu, Z and Zhao, C and Wang, R and Li, Z and Wang, J and Wang, D}, title = {CRISPR/Cas Enzyme Catalysis in Liquid-Liquid Phase-Separated Systems.}, journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)}, volume = {}, number = {}, pages = {e2407194}, doi = {10.1002/advs.202407194}, pmid = {39574297}, issn = {2198-3844}, support = {20230101356JC//Outstanding Youth Foundation of Jilin Province, China/ ; 2024A1515011281//Guangdong Basic and Applied Basic Research/ ; JJKH20241346KJ//Scientific Research project of Education Department of Jilin Province/ ; }, abstract = {The clustered regularly interspaced palindromic repeats (CRISPR) /CRISPR-associated proteins (Cas) system is the immune system in bacteria and archaea and has been extensively applied as a critical tool in bioengineering. Investigation of the mechanisms of catalysis of CRISPR/Cas systems in intracellular environments is essential for understanding the underlying catalytic mechanisms and advancing CRISPR-based technologies. Here, the catalysis mechanisms of CRISPR/Cas systems are investigated in an aqueous two-phase system (ATPS) comprising PEG and dextran, which simulated the intracellular environment. The findings revealed that nucleic acids and proteins tended to be distributed in the dextran-rich phase. The results demonstrated that the cis-cleavage activity of Cas12a is enhanced in the ATPS, while its trans-cleavage activity is suppressed, and this finding is further validated using Cas13a. Further analysis by increasing the concentration of the DNA reporter revealed that this phenomenon is not attributed to the slow diffusion of the reporter, and explained why Cas12a and Cas13a do not randomly cleave nucleic acids in the intracellular compartment. The study provides novel insights into the catalytic mechanisms of CRISPR/Cas systems under physiological conditions and may contribute to the development of CRISPR-based molecular biological tools.}, }
@article {pmid39572140, year = {2024}, author = {Chen, X and Stafford, DW and Tie, JK}, title = {Assessment of gamma-glutamyl carboxylase activity in its native milieu.}, journal = {Methods in enzymology}, volume = {708}, number = {}, pages = {207-236}, doi = {10.1016/bs.mie.2024.10.011}, pmid = {39572140}, issn = {1557-7988}, mesh = {Humans ; *Carbon-Carbon Ligases/metabolism/genetics ; *Vitamin K/metabolism ; Animals ; CRISPR-Cas Systems ; Enzyme Assays/methods ; HEK293 Cells ; Gene Knockout Techniques/methods ; }, abstract = {Gamma-glutamyl carboxylase (GGCX), a polytopic membrane protein found in the endoplasmic reticulum, catalyzes the posttranslational modification of a variety of vitamin K-dependent (VKD) proteins to their functional forms. GGCX uses the free energy from the oxygenation of reduced vitamin K to remove the proton from the glutamate residue to drive VKD carboxylation. During the process of carboxylation, reduced vitamin K is oxidized to vitamin K epoxide. Therefore, GGCX is a dual-function enzyme that possesses both glutamate carboxylation and vitamin K epoxidation activities. Genetic variations in GGCX are mainly associated with bleeding disorders referred to as combined VKD coagulation factors deficiency. Comorbid non-bleeding phenotypes are also observed in patients carrying GGCX mutations. Our current knowledge concerning GGCX's function has been obtained mainly from in vitro experimentation under artificial conditions, which limits its use in interpreting the clinical phenotypes associated with GGCX genotypes. In this chapter, we describe the background, establishment, and application of mammalian cell-based assays for both the carboxylation and epoxidation activities of GGCX. We provide detailed procedures for making the reporter cell lines, creating CRISPR-Cas9-mediated gene-knockout reporter cell lines, and using these cell lines for functional studies of GGCX and its naturally occurring mutations. Combined with different reporter proteins, this cell-based strategy has been successfully used for the functional study of vitamin K-related enzymes, high-throughput screening of VKD carboxylation inhibitors, and genome-wide CRISPR-Cas9 knockout library screening of the unknown enzymes associated with vitamin K reduction.}, }
@article {pmid39571855, year = {2024}, author = {Wan, Y and Huang, C and Feng, D and Wang, L and Lin, X and Zhao, X and Han, L and Zhu, Y and Hao, L and Du, H and Huang, L}, title = {Characterizing the collateral activity of CRISPR/Cas13 in mammalian cells: Implications for RNA editing and therapeutic applications.}, journal = {International journal of biological macromolecules}, volume = {283}, number = {Pt 4}, pages = {137861}, doi = {10.1016/j.ijbiomac.2024.137861}, pmid = {39571855}, issn = {1879-0003}, mesh = {Humans ; *RNA Editing/genetics ; *CRISPR-Cas Systems ; Animals ; Swine ; Cell Proliferation ; HEK293 Cells ; Cell Survival ; Embryonic Development/genetics ; }, abstract = {The CRISPR/Cas13 system has garnered attention as a potential tool for RNA editing. However, the degree of collateral activity among various Cas13 orthologs and their cytotoxic effects in mammalian cells remain contentious, potentially impacting their applications. In this study, we observed differential collateral activities for LwaCas13a and RfxCas13d in 293T and U87 cells by applying both sensitive dual-fluorescence (mRuby/GFP) reporter and quantifiable dual-luciferase (Fluc/Rluc) reporter, with LwaCas13a displaying notable activity contrary to previous reports. However, significant collateral RNA cleavage exerted only a modest impact on cell viability. Furthermore, collateral activity of LwaCas13a mildly impeded, but did not arrest, porcine embryo development. Our findings reveal that distinct collateral RNA cleavage by Cas13 slightly suppresses mammalian cell proliferation and embryo development. This could account for the lack of reported collateral effects in numerous prior studies and offers new insights into the implications of the collateral activity of Cas13 for clinical application.}, }
@article {pmid39571575, year = {2024}, author = {Wang, Y and Zhang, Y and Kim, K and Han, J and Okin, D and Jiang, Z and Yang, L and Subramaniam, A and Means, TK and Nestlé, FO and Fitzgerald, KA and Randolph, GJ and Lesser, CF and Kagan, JC and Mathis, D and Benoist, C}, title = {A pan-family screen of nuclear receptors in immunocytes reveals ligand-dependent inflammasome control.}, journal = {Immunity}, volume = {57}, number = {12}, pages = {2737-2754.e12}, pmid = {39571575}, issn = {1097-4180}, support = {R01 AI150686/AI/NIAID NIH HHS/United States ; R37 AI049653/AI/NIAID NIH HHS/United States ; R01 AI169795/AI/NIAID NIH HHS/United States ; R37 AI116550/AI/NIAID NIH HHS/United States ; P30 DK034854/DK/NIDDK NIH HHS/United States ; T32 HL116275/HL/NHLBI NIH HHS/United States ; K00 CA264434/CA/NCI NIH HHS/United States ; R01 AI116550/AI/NIAID NIH HHS/United States ; R01 AI167993/AI/NIAID NIH HHS/United States ; }, mesh = {Animals ; *Inflammasomes/metabolism/immunology ; Mice ; *Receptors, Cytoplasmic and Nuclear/metabolism ; Cell Differentiation ; Mice, Inbred C57BL ; Ligands ; Retinoic Acid Receptor gamma ; Macrophages, Peritoneal/immunology/metabolism ; Mice, Knockout ; Pyroptosis/immunology ; Receptors, Retinoic Acid/metabolism ; CARD Signaling Adaptor Proteins/metabolism ; GATA6 Transcription Factor/metabolism ; Retinoid X Receptor alpha/metabolism ; CRISPR-Cas Systems ; }, abstract = {Ligand-dependent transcription factors of the nuclear receptor (NR) family regulate diverse aspects of metazoan biology, enabling communications between distant organs via small lipophilic molecules. Here, we examined the impact of each of 35 NRs on differentiation and homeostatic maintenance of all major immunological cell types in vivo through a "Rainbow-CRISPR" screen. Receptors for retinoic acid exerted the most frequent cell-specific roles. NR requirements varied for resident macrophages of different tissues. Deletion of either Rxra or Rarg reduced frequencies of GATA6[+] large peritoneal macrophages (LPMs). Retinoid X receptor alpha (RXRα) functioned conventionally by orchestrating LPM differentiation through chromatin and transcriptional regulation, whereas retinoic acid receptor gamma (RARγ) controlled LPM survival by regulating pyroptosis via association with the inflammasome adaptor ASC. RARγ antagonists activated caspases, and RARγ agonists inhibited cell death induced by several inflammasome activators. Our findings provide a broad view of NR function in the immune system and reveal a noncanonical role for a retinoid receptor in modulating inflammasome pathways.}, }
@article {pmid39570677, year = {2024}, author = {Raddaoui, A and Chebbi, Y and Frigui, S and Ammeri, RW and Ben Abdejlil, N and Abbassi, MS and Achour, W}, title = {Deciphering the Resistome and Mobiolme of an Avian-Associated Enterococus faecalis ST249 Clone that Acquired Vancomycin Resistance Isolated from Neutropenic Patient in Tunisia.}, journal = {Microbial drug resistance (Larchmont, N.Y.)}, volume = {30}, number = {12}, pages = {481-488}, doi = {10.1089/mdr.2024.0144}, pmid = {39570677}, issn = {1931-8448}, mesh = {Humans ; *Enterococcus faecalis/drug effects/genetics/isolation &amp; purification ; Tunisia ; *Anti-Bacterial Agents/pharmacology ; *Microbial Sensitivity Tests ; *Neutropenia/microbiology ; Animals ; Child ; *Vancomycin/pharmacology/therapeutic use ; Whole Genome Sequencing ; Vancomycin Resistance/genetics ; Plasmids/genetics ; Gram-Positive Bacterial Infections/drug therapy/microbiology ; DNA Transposable Elements ; Bacterial Proteins/genetics ; Vancomycin-Resistant Enterococci/genetics/drug effects/isolation &amp; purification ; }, abstract = {This study aimed to characterize the first vancomycin-resistant Enterococcus faecalis (VREfs) isolate from patient with neutropenic in Tunisia by whole-genome sequencing (WGS). This strain was detected from routine rectal swab from an 8-year-old child with bone marrow aplasia, residing in a rural area, on September 20, 2021. The strain was isolated after 12 days of hospitalization at the National Bone Marrow Transplant Center. Minimum Inhibitory Concentrations of vancomycin and teicoplanin were >256 and 16 mg/L, respectively. WGS revealed that the strain belonged to the ST249 clone, exclusively reported in avian (poultry and ducks) vancomycin-susceptible E. faecalis isolates in six studies from four countries, primarily Denmark. The vanA gene was carried by the Tn1546 transposon mobilized by a pTW9-like plasmid. The ardA gene, a CRISPR-Cas system neutralization factor, was detected in this strain. In summary, this is the first report of avian-associated E. faecalis ST249 in clinical samples. Initially vancomycin susceptible, the strain acquired a pTW9-like plasmid carrying the classical vanA-Tn1546 transposon. This acquisition was facilitated by the sex pheromone-response mechanisms and the ardA gene and CRISPR-Cas system neutralization.}, }
@article {pmid39570312, year = {2024}, author = {Iturralde, AB and Weller, CA and Giovanetti, SM and Sadhu, MJ}, title = {Comprehensive deletion scan of anti-CRISPR AcrIIA4 reveals essential and dispensable domains for Cas9 inhibition.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {48}, pages = {e2413743121}, pmid = {39570312}, issn = {1091-6490}, support = {ZIA HG200401/ImNIH/Intramural NIH HHS/United States ; 1ZIAHG200401//HHS | NIH | National Human Genome Research Institute (NHGRI)/ ; }, mesh = {*CRISPR-Associated Protein 9/metabolism/genetics ; *CRISPR-Cas Systems ; Sequence Deletion ; Protein Domains ; Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; }, abstract = {Delineating a protein's essential and dispensable domains provides critical insight into how it carries out its function. Here, we developed a high-throughput method to synthesize and test the functionality of all possible in-frame and continuous deletions in a gene of interest, enabling rapid and unbiased determination of protein domain importance. Our approach generates precise deletions using a CRISPR library framework that is free from constraints of gRNA target site availability and efficacy. We applied our method to AcrIIA4, a phage-encoded anti-CRISPR protein that robustly inhibits SpCas9. Extensive structural characterization has shown that AcrIIA4 physically occupies the DNA-binding interfaces of several SpCas9 domains; nonetheless, the importance of each AcrIIA4 interaction for SpCas9 inhibition is unknown. We used our approach to determine the essential and dispensable regions of AcrIIA4. Surprisingly, not all contacts with SpCas9 were required, and in particular, we found that the AcrIIA4 loop that inserts into SpCas9's RuvC catalytic domain can be deleted. Our results show that AcrIIA4 inhibits SpCas9 primarily by blocking PAM binding and that its interaction with the SpCas9 catalytic domain is inessential.}, }
@article {pmid39570287, year = {2025}, author = {Su, KC and Radul, E and Maier, NK and Tsang, MJ and Goul, C and Moodie, B and Marescal, O and Keys, HR and Cheeseman, IM}, title = {Functional genetics reveals modulators of antimicrotubule drug sensitivity.}, journal = {The Journal of cell biology}, volume = {224}, number = {2}, pages = {}, pmid = {39570287}, issn = {1540-8140}, support = {R35 GM126930/GM/NIGMS NIH HHS/United States ; //Massachusetts Institute of Technology Office of Graduate Education/ ; HFCR-18-03-02//Hope Funds for Cancer Research/ ; //G. Harold &amp; Leila Y. Mathers Charitable Foundation/ ; /NH/NIH HHS/United States ; R35GM126930/GM/NIGMS NIH HHS/United States ; }, mesh = {Humans ; *Microtubules/metabolism/drug effects/genetics ; *Paclitaxel/pharmacology ; *Tubulin Modulators/pharmacology ; *Nocodazole/pharmacology ; *CRISPR-Cas Systems ; *Kinesins/genetics/metabolism ; Microtubule-Associated Proteins/metabolism/genetics ; }, abstract = {Microtubules play essential roles in diverse cellular processes and are important pharmacological targets for treating human disease. Here, we sought to identify cellular factors that modulate the sensitivity of cells to antimicrotubule drugs. We conducted a genome-wide CRISPR/Cas9-based functional genetics screen in human cells treated with the microtubule-destabilizing drug nocodazole or the microtubule-stabilizing drug paclitaxel. We further conducted a focused secondary screen to test drug sensitivity for ∼1,400 gene targets across two distinct human cell lines and to additionally test sensitivity to the KIF11 inhibitor, STLC. These screens defined gene targets whose loss enhances or suppresses sensitivity to antimicrotubule drugs. In addition to gene targets whose loss sensitized cells to multiple compounds, we observed cases of differential sensitivity to specific compounds and differing requirements between cell lines. Our downstream molecular analysis further revealed additional roles for established microtubule-associated proteins and identified new players in microtubule function.}, }
@article {pmid39570020, year = {2025}, author = {Yin, L and Zhao, Z and Wang, C and Zhou, C and Wu, X and Gao, B and Wang, L and Man, S and Cheng, X and Wu, Q and Hu, S and Fan, H and Ma, L and Xing, H and Shen, L}, title = {Development and evaluation of a CRISPR/Cas12a-based diagnostic test for rapid detection and genotyping of HR-HPV in clinical specimens.}, journal = {Microbiology spectrum}, volume = {13}, number = {1}, pages = {e0225324}, pmid = {39570020}, issn = {2165-0497}, support = {32302218//MOST | National Natural Science Foundation of China (NSFC)/ ; (2022YL05B,2022YL12A,2022YL07B//Science and Technology Plan (in the field of Medical and health care) of Xiangyang/ ; 82002192//MOST | National Natural Science Foundation of China (NSFC)/ ; 24YDTPJC00800//Tianjin Science and technology planning project/ ; 21JCQNJC01410//Tianjin Municipal Science and Technology Committee (Tianjin Municipal Science and Technology Commission)/ ; 2021M702460//China Postdoctoral Science Foundation (China Postdoctoral Foundation Project)/ ; 2022KF0203//Liquor Making Biological Technology and Application of Key Laboratory of Sichuan Province Microbiology/ ; 2022CFB539,2022CFD107//General Project of Natural Science Foundation of Hubei Province/ ; Q20222605//Young and middle-aged Talents Project of Hubei Provincial Education Department/ ; 2021kpgj06//Scientific Research Ability Cultivation Fund of Hubei University of Arts and Science/ ; }, mesh = {Humans ; *Papillomavirus Infections/diagnosis/virology ; *CRISPR-Cas Systems ; Female ; *Sensitivity and Specificity ; *Genotype ; *Papillomaviridae/genetics/classification/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/methods ; Uterine Cervical Neoplasms/diagnosis/virology ; Molecular Diagnostic Techniques/methods ; Genotyping Techniques/methods ; DNA, Viral/genetics ; Diagnostic Tests, Routine/methods ; }, abstract = {Persistent infection with high-risk human papillomavirus (HR-HPV) is the principal etiological factor of cervical cancer. Considering the gradual progression of cervical cancer, the early, rapid, sensitive, and specific identification of HPV, particularly HR-HPV types, is crucial in halting the advancement of the illness. Here, we established a rapid, highly sensitive, and specific HR-HPV detection platform, leveraging the CRISPR/Cas12a assay in conjunction with multienzyme isothermal rapid amplification. Our platform enables the detection and genotyping of 14 types of HR-HPV by using type-specific crRNAs. The outcomes of the detection can be interpreted either through a fluorescence reader or visually. Furthermore, we achieved one-tube multiplex detection of 14 HR-HPV types through the use of multiple amplifications and a crRNA pool. The detection sensitivity of this method is 2 copies/μL with no cross-reactivity, and the results can be obtained within 30 minutes. This method exhibited 100% clinical sensitivity and 100% clinical specificity when applied to 258 clinical specimens. Based on these findings, our CRISPR/Cas-based HR-HPV detection platform holds promise as a novel clinical detection tool, offering a visually intuitive and expedited alternative to existing HPV infection diagnostics and providing fresh perspectives for clinical cervical cancer screening.IMPORTANCEThis study developed a novel high-risk human papillomavirus (HR-HPV) detection platform based on CRISPR/Cas12a technology. This platform not only enables the rapid, highly sensitive, and specific detection and genotyping of 14 types of HR-HPV but also achieves single-tube multiplex detection of 14 HR-HPV types through ingenious design. The outcomes of the detection can be interpreted either through a fluorescence reader or visually. To the best of our knowledge, this is the first paper to utilize CRISPR/Cas diagnostic technology for the simultaneous detection of 14 types of HPV and to evaluate its feasibility in clinical sample detection using a large number of clinical samples. We hope that this work will facilitate the rapid and accurate detection of HPV and promote the broader application of CRISPR/Cas diagnostic technology.}, }
@article {pmid39569987, year = {2024}, author = {Yamamoto, S and Afifi, OA and Lam, LPY and Takeda-Kimura, Y and Osakabe, Y and Osakabe, K and Bartley, LE and Umezawa, T and Tobimatsu, Y}, title = {Disruption of aldehyde dehydrogenase decreases cell wall-bound p-hydroxycinnamates and improves cell wall digestibility in rice.}, journal = {The Plant journal : for cell and molecular biology}, volume = {120}, number = {6}, pages = {2828-2845}, doi = {10.1111/tpj.17148}, pmid = {39569987}, issn = {1365-313X}, support = {Exploratory Research on Humanosphere Science 2021//Research Institute for Sustainable Humanosphere, Kyoto University/ ; Exploratory Research on Humanosphere Science 2023//Research Institute for Sustainable Humanosphere, Kyoto University/ ; DE-SC0021126//Office of Science/ ; #20H03044//Japan Society for the Promotion of Science/ ; #22J13457//Japan Society for the Promotion of Science/ ; #24K01827//Japan Society for the Promotion of Science/ ; #1015621//National Institute of Food and Agriculture/ ; }, mesh = {*Cell Wall/metabolism ; *Oryza/genetics/metabolism/enzymology ; *Coumaric Acids/metabolism ; *Lignin/metabolism ; *Plant Proteins/genetics/metabolism ; Aldehyde Dehydrogenase/metabolism/genetics ; Xylans/metabolism ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; }, abstract = {In grass cell walls, ferulic acid (FA) serves as an important cross-linker between cell wall polymers, such as arabinoxylan (AX) and lignin, affecting the physicochemical properties of the cell walls as well as the utilization properties of grass lignocellulose for biorefinering. Here, we demonstrate that hydroxycinnamaldehyde dehydrogenase (HCALDH) plays a crucial role in the biosynthesis of the FA used for cell wall feruloylation in rice (Oryza sativa). Bioinformatic and gene expression analyses of aldehyde dehydrogenases (ALDHs) identified two rice ALDH subfamily 2C members, OsHCALDH2 (OsALDH2C2) and OsHCALDH3 (OsALDH2C3), potentially involved in cell wall feruloylation in major vegetative tissues of rice. CRISPR-Cas9 genome editing of OsHCALDH2 and OsHCALDH3 revealed that the contents of AX-bound ferulate were reduced by up to ~45% in the cell walls of the HCALDH-edited mutants, demonstrating their roles in cell wall feruloylation. The abundance of hemicellulosic sugars including arabinosyl units on AX was notably reduced in the cell walls of the HCALDH-edited mutants, whereas cellulose and lignin contents remained unaffected. In addition to reducing cell wall-bound ferulate, the loss of OsHCALDH2 and/or OsHCALDH3 also partially reduced cell wall-bound p-coumarate and sinapate in the vegetative tissues of rice, whereas it did not cause detectable changes in the amount of γ-oryzanol (feruloyl sterols) in rice seeds. Furthermore, the HCALDH-edited mutants exhibited improved cell wall saccharification efficiency, both with and without alkaline pretreatment, plausibly due to the reduction in cell wall cross-linking FA. Overall, HCALDH appears to present a potent bioengineering target for enhancing utilization properties of grass lignocellulose.}, }
@article {pmid39569586, year = {2024}, author = {Kanke, KL and Rayner, RE and Bozik, J and Abel, E and Venugopalan, A and Suu, M and Nouri, R and Stack, JT and Guo, G and Vetter, TA and Cormet-Boyaka, E and Hester, ME and Vaidyanathan, S}, title = {Single-stranded DNA with internal base modifications mediates highly efficient knock-in in primary cells using CRISPR-Cas9.}, journal = {Nucleic acids research}, volume = {52}, number = {22}, pages = {13561-13576}, pmid = {39569586}, issn = {1362-4962}, support = {//Comprehensive Transplant Center Human Tissue Biorepository,&#xa0;OSU&#xa0;Wexner Medical Center/ ; R00 HL151900/HL/NHLBI NIH HHS/United States ; HL151900-03/HL/NHLBI NIH HHS/United States ; DK106829//National Institute of Diabetes, Digestive and Kidney Diseases/ ; UL1TR002733//Center for Clinical and Translational Science/ ; //Ohio State University/ ; MCCOY19Ro//Nationwide Children's Hospital/ ; VAIDYA22A0-KB//Cystic Fibrosis Foundation/ ; UL1 TR002733/TR/NCATS NIH HHS/United States ; U54 DK106829/DK/NIDDK NIH HHS/United States ; K99 HL151900/HL/NHLBI NIH HHS/United States ; }, mesh = {*CRISPR-Cas Systems ; Humans ; *DNA, Single-Stranded/genetics/metabolism ; *Gene Knock-In Techniques ; *Induced Pluripotent Stem Cells/metabolism ; Receptors, CCR5/genetics/metabolism ; Gene Editing/methods ; Cells, Cultured ; Hematopoietic Stem Cells/metabolism ; }, abstract = {Single-stranded DNA (ssDNA) templates along with Cas9 have been used for knocking-in exogenous sequences in the genome but suffer from low efficiency. Here, we show that ssDNA with chemical modifications in 12-19% of internal bases, which we denote as enhanced ssDNA (esDNA), improve knock-in (KI) by 2-3-fold compared to end-modified ssDNA in airway basal stem cells (ABCs), CD34 + hematopoietic cells (CD34 + cells), T-cells and endothelial cells. Over 50% of alleles showed KI in three clinically relevant loci (CFTR, HBB and CCR5) in ABCs using esDNA and up to 70% of alleles showed KI in the HBB locus in CD34 + cells in the presence of a DNA-PKcs inhibitor. This level of correction is therapeutically relevant and is comparable to adeno-associated virus-based templates. The esDNA templates did not improve KI in induced pluripotent stem cells (iPSCs). This may be due to the absence of the nuclease TREX1 in iPSCs. Indeed, knocking out TREX1 in other cells improved KI using unmodified ssDNA. esDNA can be used to modify 20-30 bp regions in primary cells for therapeutic applications and biological modeling. The use of this approach for gene length insertions will require new methods to produce long chemically modified ssDNA in scalable quantities.}, }
@article {pmid39569582, year = {2024}, author = {Arantes, PR and Chen, X and Sinha, S and Saha, A and Patel, AC and Sample, M and Nierzwicki, &#x141; and Lapinaite, A and Palermo, G}, title = {Dimerization of the deaminase domain and locking interactions with Cas9 boost base editing efficiency in ABE8e.}, journal = {Nucleic acids research}, volume = {52}, number = {22}, pages = {13931-13944}, pmid = {39569582}, issn = {1362-4962}, support = {//Advanced Cyberinfrastructure Coordination Ecosystem: Services &amp; Support (ACCESS) program/ ; CHE-2144823//National Science Foundation/ ; R01GM141329/NH/NIH HHS/United States ; BIO230007//Pittsburgh Supercomputer Center/ ; DP2 GM149550/GM/NIGMS NIH HHS/United States ; TC-24-063//Camille and Henry Dreyfus Foundation/ ; MCB160059//San Diego Supercomputing Center/ ; FG-2023-20431//Sloan Foundation/ ; 2138259//National Science Foundation/ ; R01GM141329/NH/NIH HHS/United States ; }, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism/chemistry ; *DNA/metabolism/genetics/chemistry ; Protein Multimerization ; Humans ; Adenosine Deaminase/metabolism/genetics/chemistry ; Protein Domains ; Deamination ; Molecular Dynamics Simulation ; Adenine/metabolism/chemistry ; }, abstract = {CRISPR-based DNA adenine base editors (ABEs) hold remarkable promises to address human genetic diseases caused by point mutations. ABEs were developed by combining CRISPR-Cas9 with a transfer RNA (tRNA) adenosine deaminase enzyme and through directed evolution, conferring the ability to deaminate DNA. However, the molecular mechanisms driving the efficient DNA deamination in the evolved ABEs remain unresolved. Here, extensive molecular simulations and biochemical experiments reveal the biophysical basis behind the astonishing base editing efficiency of ABE8e, the most efficient ABE to date. We demonstrate that the ABE8e's DNA deaminase domain, TadA8e, forms remarkably stable dimers compared to its tRNA-deaminating progenitor and that the strength of TadA dimerization is crucial for DNA deamination. The TadA8e dimer forms robust interactions involving its R98 and R129 residues, the RuvC domain of Cas9 and the DNA. These locking interactions are exclusive to ABE8e, distinguishing it from its predecessor, ABE7.10, and are indispensable to boost DNA deamination. Additionally, we identify three critical residues that drive the evolution of ABE8e toward improved base editing by balancing the enzyme's activity and stability, reinforcing the TadA8e dimer and improving the ABE8e's functionality. These insights offer new directions to engineer superior ABEs, advancing the design of safer precision genome editing tools.}, }
@article {pmid39567138, year = {2025}, author = {Lu, X and Wang, Y and Zhen, X and Che, Y and Yu, H and Ge, Y and Wang, X and Li, R and Geng, M and Zhou, B and Liu, J and Guo, J and Yao, Y}, title = {Editing of the soluble starch synthase gene MeSSIII-1 enhanced the amylose and resistant starch contents in cassava.}, journal = {Carbohydrate polymers}, volume = {348}, number = {Pt B}, pages = {122903}, doi = {10.1016/j.carbpol.2024.122903}, pmid = {39567138}, issn = {1879-1344}, mesh = {*Manihot/genetics/chemistry/metabolism ; *Starch Synthase/genetics/metabolism ; *Amylose/metabolism/chemistry ; *Resistant Starch/metabolism ; Gene Editing/methods ; Mutation ; CRISPR-Cas Systems/genetics ; Starch/metabolism/chemistry ; Plant Proteins/genetics/metabolism/chemistry ; Amylopectin/chemistry/metabolism ; Viscosity ; }, abstract = {Foods with high amylose and resistant starch (RS) contents have great potential to enhance human health. In this study, cassava soluble starch synthase MeSSIII-1 gene mutants were generated using CRISPR/Cas9 system. The results showed that the storage roots of messiii-1 mutants had higher contents of amylose, RS, and total starch than those in CK. The rates of small and large-sized starch granules were increased. Additionally, amylopectin starch in messiii-1 mutants had a higher proportion of medium- and long- chains, and a lower proportion of short-chains than those in CK. The onset, peak, and conclusion temperatures of starch gelatinization in messiii-1 mutants were significantly lower than those in CK, and the peak viscosity, trough viscosity and final viscosity all increased. MeSSIII-1 mutation could increase the contents of sucrose, glucose, and fructose in cassava storage roots. We hypothesize that these soluble sugars serve a dual role: they provide the necessary carbon source for starch synthesis and act as sugar signals to trigger the transcriptional reprogramming of genes involved in starch biosynthesis. This process results in a collective enhancement of amylose, RS, and total starch contents, accompanied by changes in starch granule morphology, fine structure, and physicochemical properties.}, }
@article {pmid39566816, year = {2025}, author = {Baek, M and Kim, CL and Kim, SH and la Cour Karottki, KJ and Hefzi, H and Grav, LM and Pedersen, LE and Lewis, NE and Lee, JS and Lee, GM}, title = {Unraveling productivity-enhancing genes in Chinese hamster ovary cells via CRISPR activation screening using recombinase-mediated cassette exchange system.}, journal = {Metabolic engineering}, volume = {87}, number = {}, pages = {11-20}, doi = {10.1016/j.ymben.2024.11.009}, pmid = {39566816}, issn = {1096-7184}, mesh = {CHO Cells ; *Cricetulus ; Animals ; *CRISPR-Cas Systems ; Antibodies, Bispecific/genetics/metabolism ; Recombinases/genetics/metabolism ; Cricetinae ; }, abstract = {Chinese hamster ovary (CHO) cells, which are widely used for therapeutic protein production, have been genetically manipulated to enhance productivity. Nearly half of the genes in CHO cells are silenced, which are promising targets for CHO cell engineering. To identify novel gene targets among the silenced genes that can enhance productivity, we established a genome-wide clustered regularly interspaced short palindromic repeats activation (CRISPRa) screening platform for bispecific antibody (bsAb)-producing CHO (CHO-bsAb) cells with 110,979 guide RNAs (gRNAs) targeting 13,812 silenced genes using a virus-free recombinase-mediated cassette exchange-based gRNA integration method. Using this platform, we performed a fluorescence-activated cell sorting-based cold-capture assay to isolate cells with high fluorescence intensity, which is indicative of high specific bsAb productivity (qbsAb), and identified 90 significantly enriched genes. To verify the screening results, 14 high-scoring candidate genes were individually activated in CHO-bsAb cells via CRISPRa. Among these, 10 genes demonstrated enhanced fluorescence intensity of CHO-bsAb cells in the cold-capture assay when activated. Furthermore, the overexpression of the identified novel gene target Syce3 in CHO-bsAb cells resulted in a 1.4- to 1.9-fold increase in the maximum bsAb concentration, owing to improved qbsAb and specific growth rate. Thus, this virus-free CRISPRa screening platform is a potent tool for identifying novel engineering targets in CHO cells to improve bsAb production.}, }
@article {pmid39566812, year = {2025}, author = {Delshad, M and Davoodi-Moghaddam, Z and Khademi, M and Pourbagheri-Sigaroodi, A and Zali, MR and Bashash, D}, title = {Advancements in gene therapy for human diseases: Trend of current clinical trials.}, journal = {European journal of pharmacology}, volume = {986}, number = {}, pages = {177143}, doi = {10.1016/j.ejphar.2024.177143}, pmid = {39566812}, issn = {1879-0712}, mesh = {Humans ; *Genetic Therapy/methods ; *Clinical Trials as Topic ; Genetic Vectors ; }, abstract = {In an era of rapid scientific advancement, gene therapy has emerged as a groundbreaking approach with the potential to revolutionize the treatment of a myriad of diseases and medical conditions. The trend of current clinical trials suggests that there is growing interest and investment in exploring gene therapy as a viable treatment option. In 2023, a significant milestone was achieved with the approval of seven gene therapies by the Food and Drug Administration (FDA). Projections indicate that between 10 and 20 gene therapies could receive annual FDA approval by 2025. In this review, we conducted a comprehensive analysis of registered clinical trials on Clinicaltrials.gov to determine the progression status of gene therapies. Upon extraction of the data, we conducted a comprehensive analysis of the 2809 included studies. This involved a systematic approach, commencing with an overview, followed by a detailed examination of gene therapy strategies employed in various malignant and non-malignant disorders. Additionally, the study will cover the types of vectors utilized in current trials. Lastly, a meticulous review of 105 phase III-IV clinical trials was conducted to identify potential therapies demonstrating promise. We trust that the comprehensive overview provided will serve as a solid foundation for forthcoming research and study designs, ultimately contributing to the progression of gene therapy and its practical application within healthcare settings. Also, we anticipate that such inquiries will bolster the formulation of practical policies and guidelines for pharmaceutical companies engaged in gene therapy research and development.}, }
@article {pmid39566771, year = {2024}, author = {Panda, G and Ray, A}, title = {Deciphering Cas9 specificity: Role of domain dynamics and RNA:DNA hybrid interactions revealed through machine learning and accelerated molecular simulations.}, journal = {International journal of biological macromolecules}, volume = {283}, number = {Pt 4}, pages = {137835}, doi = {10.1016/j.ijbiomac.2024.137835}, pmid = {39566771}, issn = {1879-0003}, mesh = {*Molecular Dynamics Simulation ; *CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; *Machine Learning ; *DNA/chemistry/metabolism ; *RNA/chemistry/metabolism ; Francisella/enzymology ; Protein Domains ; CRISPR-Cas Systems ; Substrate Specificity ; Bacterial Proteins/chemistry/metabolism/genetics ; }, abstract = {CRISPR/Cas9 technology is widely used for gene editing, but off-targeting still remains a major concern in therapeutic applications. Although Cas9 variants with better mismatch discrimination have been developed, they have significantly lower rates of on-target DNA cleavage. This study compares the dynamics of the highly specific Cas9 from Francisella novicida (FnCas9) to the commonly used SpCas9. Using long-scale atomistic Gaussian accelerated molecular dynamic simulations and machine learning techniques, we deciphered the structural factors behind FnCas9's higher specificity in native and off-target forms. Our analysis revealed that Cas9's cleavage specificity relies more on its domain rearrangement than on RNA:DNA heteroduplex shape, with significant conformational variations in Cas9 domains among off-target forms, while the RNA:DNA hybrid showed minimal changes, especially in FnCas9 compared to SpCas9. REC1-REC3 domains contacts with the RNA:DNA hybrid in FnCas9 acted as critical discriminator of off-target effects playing a pivotal role in influencing specificity. In FnCas9, allosteric signal transmission involves the REC3 and HNH domain, bypassing REC2, leading to a superior efficiency in information transmission. This study offers a quantitative framework for understanding the structural basis of elevated specificity, paving the way for the rational design of Cas9 variants with improved precision and specificity in genome editing applications.}, }
@article {pmid39566123, year = {2024}, author = {Dara, M and Dianatpour, M and Azarpira, N and Tanideh, N and Tanideh, R}, title = {Integrating CRISPR technology with exosomes: Revolutionizing gene delivery systems.}, journal = {Biochemical and biophysical research communications}, volume = {740}, number = {}, pages = {151002}, doi = {10.1016/j.bbrc.2024.151002}, pmid = {39566123}, issn = {1090-2104}, mesh = {*Exosomes/genetics ; Humans ; *Gene Transfer Techniques ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genetic Therapy/methods ; }, abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) serves as an adaptive immune system in bacteria and archaea, offering a defense mechanism against invading genetic elements such as viruses (bacteriophages) and plasmids. Today, CRISPR has evolved into a powerful gene-editing technology that enables highly specific and rapid modifications of DNA within a genome. It has a broad range of applications across various fields, including medicine, agriculture, and fundamental research. One of the significant challenges facing this technology is the efficient transfer of CRISPR constructs into target cells for gene editing. There are several methods to deliver this system into target cells, which can be classified as viral and non-viral methods. Each of these approaches has its own advantages and disadvantages. Recently, the use of extracellular vesicles for delivery has garnered particular attention. Exosomes are nano-sized extracellular vesicles that have emerged as promising carriers for drug delivery due to their unique properties. These naturally occurring vesicles, typically ranging from 30 to 150 nm in diameter, facilitate intercellular communication by transferring bioactive molecules such as proteins, lipids, and nucleic acids between cells. Exosome therapy has surfaced as a promising strategy in regenerative medicine, utilizing small extracellular vesicles to deliver therapeutic molecules to target cells. One of the emerging options for transferring the CRISPR system is exosomes. The integration of these two advanced technologies holds significant potential for developing efficient and targeted gene editing and advancing precision medicine. In contemporary medicine, there is an increasing focus on personalized and targeted treatments that cater to the distinct genetic and molecular profiles of individual patients. The synergy of CRISPR technology and exosome therapy presents a remarkable opportunity to develop highly targeted and effective therapeutic strategies customized to individual patient requirements. This review article examines the potential of incorporating CRISPR technology within exosomes for precision therapeutic applications.}, }
@article {pmid39565688, year = {2024}, author = {Asano, K and Yoshimi, K and Takeshita, K and Mitsuhashi, S and Kochi, Y and Hirano, R and Tingyu, Z and Ishida, S and Mashimo, T}, title = {CRISPR Diagnostics for Quantification and Rapid Diagnosis of Myotonic Dystrophy Type 1 Repeat Expansion Disorders.}, journal = {ACS synthetic biology}, volume = {13}, number = {12}, pages = {3926-3935}, pmid = {39565688}, issn = {2161-5063}, mesh = {*Myotonic Dystrophy/genetics/diagnosis ; Humans ; *CRISPR-Cas Systems/genetics ; *Trinucleotide Repeat Expansion/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Sensitivity and Specificity ; }, abstract = {Repeat expansion disorders, exemplified by myotonic dystrophy type 1 (DM1), present challenges in diagnostic quantification because of the variability and complexity of repeat lengths. Traditional diagnostic methods, including PCR and Southern blotting, exhibit limitations in sensitivity and specificity, necessitating the development of innovative approaches for precise and rapid diagnosis. Here, we introduce a CRISPR-based diagnostic method, REPLICA (repeat-primed locating of inherited disease by Cas3), for the quantification and rapid diagnosis of DM1. This method, using in vitro-assembled CRISPR-Cas3, demonstrates superior sensitivity and specificity in quantifying CTG repeat expansion lengths, correlated with disease severity. We also validate the robustness and accuracy of CRISPR diagnostics in quantitatively diagnosing DM1 using patient genomes. Furthermore, we optimize a REPLICA-based assay for point-of-care-testing using lateral flow test strips, facilitating rapid screening and detection. In summary, REPLICA-based CRISPR diagnostics offer precise and rapid detection of repeat expansion disorders, promising personalized treatment strategies.}, }
@article {pmid39565578, year = {2025}, author = {Hwang, I and Nikoli, H and Paik, J}, title = {Functional Motif Discovery in FOXO1 Through CRISPR/Cas9 Exon Tiling Scan.}, journal = {Methods in molecular biology (Clifton, N.J.)}, volume = {2871}, number = {}, pages = {57-68}, pmid = {39565578}, issn = {1940-6029}, mesh = {*Forkhead Box Protein O1/metabolism/genetics ; *CRISPR-Cas Systems ; *Exons/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; }, abstract = {The study of FOXO1, a pivotal transcription factor, has garnered significant attention due to its critical role in diverse cellular processes, including lineage differentiation, apoptosis, cell cycle regulation, and metabolism. To comprehensively understand the functional intricacies of FOXO1, an innovative approach is essential. This chapter highlights employing CRISPR exon scanning as a strategic tool to dissect the functional domains of FOXO1 and unravel its diverse regulatory functions. CRISPR exon scan allows for the identification of functionally important domains based on the levels of sgRNA depletion or enrichment within the FOXO1 gene, providing a unique opportunity to investigate the domain function under relevant biological contexts. This approach enables the systematic exploration of FOXO1's structural domains, shedding light on how distinct regions contribute to its overall function. The comprehensive exon scan analysis using CRISPR technology allows gaining a nuanced understanding of FOXO1's functional diversity and regulatory mechanisms.}, }
@article {pmid39565132, year = {2025}, author = {Khongthongdam, M and Phetruen, T and Chanarat, S}, title = {Development of ptxD/Phi as a new dominant selection system for genetic manipulation in Cryptococcus neoformans.}, journal = {Microbiology spectrum}, volume = {13}, number = {1}, pages = {e0161824}, pmid = {39565132}, issn = {2165-0497}, mesh = {*Cryptococcus neoformans/genetics/pathogenicity ; *Virulence Factors/genetics ; CRISPR-Cas Systems ; Fungal Proteins/genetics/metabolism ; Cryptococcosis/microbiology ; Oxidoreductases/genetics/metabolism ; }, abstract = {Cryptococcus neoformans is a globally distributed pathogenic fungus posing a significant threat to immunocompromised individuals, particularly those with HIV/AIDS. Effective genetic manipulation tools are essential for understanding its biology and developing new therapies. However, current genetic tools, including the variation of versatile selectable markers, are limited. This study develops and validates the phosphite dehydrogenase gene (ptxD)/phosphite (Phi) selection system as a non-antibiotic selectable marker for genetic manipulation in C. neoformans. A codon-optimized ptxD gene from Pseudomonas stutzeri was cloned under the TEF promoter. Using the transient CRISPR-Cas9 coupled with electroporation system, we integrated the ptxD gene into the C. neoformans genome and assessed the impact of ptxD integration on cell growth and virulence factors. The ptxD/Phi system effectively selected transformed cells on Phi-containing media. Growth assays showed that ptxD integration did not adversely affect cell growth or key virulence factors, including pleomorphism, capsule size, and melanin production. Additionally, we successfully disrupted the ADE2 gene using this system, confirming its applicability for gene deletion. Taken together, the ptxD/Phi system provides a robust and versatile tool for genetic manipulation in C. neoformans, facilitating further research into its biology and pathogenicity.IMPORTANCECryptococcus neoformans is a type of fungus that can cause serious illnesses in people who have weakened immune systems, like those with HIV/AIDS. To better study this fungus and find new treatments, scientists need tools to change its genes in precise ways. However, the current tools available for this are somewhat limited. This research introduces a new tool called the phosphite dehydrogenase gene/phosphite system, which does not rely on antibiotics to work. It uses a gene from a different bacterium that helps select and grow only the fungus cells that have successfully incorporated new genetic information. This is particularly useful because it does not interfere with the normal growth of the fungus or the features that make it harmful (like its ability to change shape or produce protective coatings). By making it easier and more effective to manipulate the genetics of C. neoformans, this tool opens up new possibilities for understanding how this fungus operates and for developing therapies to combat its infections. This is crucial for improving the treatment of infections in vulnerable populations.}, }
@article {pmid39563182, year = {2024}, author = {Wang, Y and Chen, P and Wen, H and Gui, Y and Yan, D and Huang, D and Wang, D and Tang, BZ and Tan, H}, title = {Advanced Nanoplatform Mediated by CRISPR-Cas9 and Aggregation-Induced Emission Photosensitizers to Boost Cancer Theranostics.}, journal = {ACS nano}, volume = {18}, number = {48}, pages = {33168-33180}, doi = {10.1021/acsnano.4c11757}, pmid = {39563182}, issn = {1936-086X}, mesh = {*Photosensitizing Agents/chemistry/pharmacology ; *Theranostic Nanomedicine ; Humans ; *CRISPR-Cas Systems/genetics ; Mice ; Animals ; *Reactive Oxygen Species/metabolism ; Nanoparticles/chemistry ; Neoplasms/therapy/genetics/diagnostic imaging/pathology ; Cell Line, Tumor ; Antineoplastic Agents/pharmacology/chemistry ; Immunotherapy ; Mice, Inbred BALB C ; Cell Proliferation/drug effects ; Drug Screening Assays, Antitumor ; }, abstract = {Immunotherapy combined with phototherapy is emerging as a promising strategy to treat omnipotent cancers. In this study, a clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system, aggregation-induced emission (AIE) photosensitizer (PS) and surface coating of polyethylene imine/hyaluronic acid were combined to construct a multifunctional nanoplatform, denoted as TCPH nanoparticles (NPs), for comprehensive cancer theranostics. TCPH NPs are featured by intrinsic functions including efficient reactive oxygen species (ROS) production, good photothermal conversion, programmed death-ligand 1 (PD-L1)-eliminating capability, and effective intracellular transport. The generated ROS and hyperthermia do not only achieve primary tumor elimination but also regulate the tumor immune microenvironment. Genomic disruption of PD-L1 conspicuously augments its therapeutic efficacy, especially in tumor metastasis and recurrence. Exceptional multimodal imaging navigation has also been developed. Excellent theranostics performance was substantiated in diverse tumor models, implying that this synergistic strategy of phototheranostics and immunotherapy provides a paradigm shift in emerging CRISPR-mediated nanomedicines.}, }
@article {pmid39563029, year = {2025}, author = {Broksø, AD and Bendixen, L and Fammé, S and Mikkelsen, K and Jensen, TI and Bak, RO}, title = {Orthogonal transcriptional modulation and gene editing using multiple CRISPR-Cas systems.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {1}, pages = {71-89}, doi = {10.1016/j.ymthe.2024.11.024}, pmid = {39563029}, issn = {1525-0024}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Streptococcus pyogenes/genetics ; Staphylococcus aureus/genetics ; Gene Knockout Techniques ; CRISPR-Associated Protein 9/metabolism/genetics ; T-Lymphocytes/metabolism ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation ; Transcriptional Activation ; Endonucleases/metabolism/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; }, abstract = {CRISPR-Cas-based transcriptional activation (CRISPRa) and interference (CRISPRi) enable transient programmable gene regulation by recruitment or fusion of transcriptional regulators to nuclease-deficient Cas (dCas). Here, we expand on the emerging area of transcriptional engineering and RNA delivery by benchmarking combinations of RNA-delivered dCas and transcriptional modulators. We utilize dCas9 from Staphylococcus aureus and Streptococcus pyogenes for orthogonal transcriptional modulation to upregulate one set of genes while downregulating another. We also establish trimodal genetic engineering by combining orthogonal transcriptional regulation with gene knockout by Cas12a (Acidaminococcus; AsCas12a) ribonucleoprotein delivery. To simplify trimodal engineering, we explore optimal parameters for implementing truncated single guide RNAs (sgRNAs) to make use of SpCas9 for knockout and CRISPRa. We find the Cas9 protein/sgRNA ratio to be crucial for avoiding sgRNA cross-complexation and for balancing knockout and activation efficiencies. We demonstrate high efficiencies of trimodal genetic engineering in primary human T cells while preserving basic T cell health and functionality. This study highlights the versatility and potential of complex genetic engineering using multiple CRISPR-Cas systems in a simple one-step process yielding transient transcriptome modulation and permanent DNA changes. We believe such elaborate engineering can be implemented in regenerative medicine and therapies to facilitate more sophisticated treatments.}, }
@article {pmid39563028, year = {2025}, author = {Nieland, L and Vrijmoet, AB and Jetten, IW and Rufino-Ramos, D and de Reus, AJEM and Breyne, K and Kleinstiver, BP and Maguire, CA and Broekman, MLD and Breakefield, XO and Abels, ER}, title = {CRISPR targeting of mmu-miR-21a through a single adeno-associated virus vector prolongs survival of glioblastoma-bearing mice.}, journal = {Molecular therapy : the journal of the American Society of Gene Therapy}, volume = {33}, number = {1}, pages = {133-151}, doi = {10.1016/j.ymthe.2024.11.023}, pmid = {39563028}, issn = {1525-0024}, support = {P01 CA069246/CA/NCI NIH HHS/United States ; U19 CA179563/CA/NCI NIH HHS/United States ; }, mesh = {Animals ; *Glioblastoma/therapy/genetics/pathology ; *Dependovirus/genetics ; *MicroRNAs/genetics ; Mice ; *Genetic Vectors/genetics/administration &amp; dosage ; Humans ; *CRISPR-Cas Systems ; Gene Editing/methods ; Cell Line, Tumor ; Genetic Therapy/methods ; Disease Models, Animal ; Brain Neoplasms/therapy/genetics/pathology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Xenograft Model Antitumor Assays ; Gene Targeting ; }, abstract = {Glioblastoma (GB), the most aggressive tumor of the central nervous system (CNS), has poor patient outcomes with limited effective treatments available. MicroRNA-21 (miR-21(a)) is a known oncogene, abundantly expressed in many cancer types. miR-21(a) promotes GB progression, and lack of miR-21(a) reduces the tumorigenic potential. Here, we propose a single adeno-associated virus (AAV) vector strategy targeting mmu-miR-21a using the Staphylococcus aureus Cas9 ortholog (SaCas9) guided by a single-guide RNA (sgRNA). Our results demonstrate that AAV8 is a well-suited AAV serotype to express SaCas9-KKH/sgRNA at the tumor site in an orthotopic GB model. The SaCas9-KKH induced a genomic deletion, resulting in lowered mmu-miR-21a levels in the brain, leading to reduced tumor growth and improved overall survival. In this study, we demonstrated that disruption of genomic mmu-miR-21a with a single AAV vector influenced glioma development, resulting in beneficial anti-tumor outcomes in GB-bearing mice.}, }
@article {pmid39562573, year = {2024}, author = {Kunitake, K and Mizuno, T and Hattori, K and Oneyama, C and Kamiya, M and Ota, S and Urano, Y and Kojima, R}, title = {Barcoding of small extracellular vesicles with CRISPR-gRNA enables comprehensive, subpopulation-specific analysis of their biogenesis and release regulators.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {9777}, pmid = {39562573}, issn = {2041-1723}, support = {24H00868//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JPMJPR17H5//MEXT | JST | Precursory Research for Embryonic Science and Technology (PRESTO)/ ; CDA-00008/2019-C//Human Frontier Science Program (HFSP)/ ; K05 DA000008/DA/NIDA NIH HHS/United States ; JPMJCR19H1, JPMJCR23B7//MEXT | JST | Core Research for Evolutional Science and Technology (CREST)/ ; }, mesh = {*Extracellular Vesicles/metabolism/genetics ; Humans ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Tetraspanin 29/metabolism/genetics ; *Tetraspanin 30/metabolism/genetics ; HEK293 Cells ; Exosomes/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; }, abstract = {Small extracellular vesicles (sEVs) are important intercellular information transmitters in various biological contexts, but their release processes remain poorly understood. Herein, we describe a high-throughput assay platform, CRISPR-assisted individually barcoded sEV-based release regulator (CIBER) screening, for identifying key players in sEV release. CIBER screening employs sEVs barcoded with CRISPR-gRNA through the interaction of gRNA and dead Cas9 fused with an sEV marker. Barcode quantification enables the estimation of the sEV amount released from each cell in a massively parallel manner. Barcoding sEVs with different sEV markers in a CRISPR pooled-screening format allows genome-wide exploration of sEV release regulators in a subpopulation-specific manner, successfully identifying previously unknown sEV release regulators and uncovering the exosomal/ectosomal nature of CD63[+]/CD9[+] sEVs, respectively, as well as the synchronization of CD9[+] sEV release with the cell cycle. CIBER should be a valuable tool for detailed studies on the biogenesis, release, and heterogeneity of sEVs.}, }
@article {pmid39562558, year = {2024}, 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 = {Discovering CRISPR-Cas system with self-processing pre-crRNA capability by foundation models.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {10024}, pmid = {39562558}, issn = {2041-1723}, mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; DNA Cleavage ; CRISPR-Associated Proteins/metabolism/genetics ; }, abstract = {The discovery of CRISPR-Cas systems has paved the way for advanced gene editing tools. However, traditional Cas discovery methods relying on sequence similarity may miss distant homologs and aren't suitable for functional recognition. With protein large language models (LLMs) evolving, there is potential for Cas system modeling without extensive training data. Here, we introduce CHOOSER (Cas HOmlog Observing and SElf-processing scReening), an AI framework for alignment-free discovery of CRISPR-Cas systems with self-processing pre-crRNA capability using protein foundation models. By using CHOOSER, we identify 11 Casλ homologs, nearly doubling the known catalog. Notably, one homolog, EphcCasλ, is experimentally validated for self-processing pre-crRNA, DNA cleavage, and trans-cleavage, showing promise for CRISPR-based pathogen detection. This study highlights an innovative approach for discovering CRISPR-Cas systems with specific functions, emphasizing their potential in gene editing.}, }
@article {pmid39561776, year = {2025}, author = {Xu, Y and Morrow, CA and Laksir, Y and Holt, OM and Taylor, K and Tsiappourdhi, C and Collins, P and Jia, S and Andreadis, C and Whitby, MC}, title = {DNA nicks in both leading and lagging strand templates can trigger break-induced replication.}, journal = {Molecular cell}, volume = {85}, number = {1}, pages = {91-106.e5}, doi = {10.1016/j.molcel.2024.10.026}, pmid = {39561776}, issn = {1097-4164}, mesh = {*Schizosaccharomyces/genetics/metabolism ; *DNA Replication ; *Schizosaccharomyces pombe Proteins/metabolism/genetics ; *DNA Breaks, Double-Stranded ; *DNA Breaks, Single-Stranded ; *DNA, Fungal/metabolism/genetics ; CRISPR-Cas Systems ; DNA End-Joining Repair ; DNA-Binding Proteins/metabolism/genetics ; DNA Repair ; Recombinational DNA Repair ; DNA Helicases/metabolism/genetics ; Homologous Recombination ; }, abstract = {Encounters between replication forks and unrepaired DNA single-strand breaks (SSBs) can generate both single-ended and double-ended double-strand breaks (seDSBs and deDSBs). seDSBs can be repaired by break-induced replication (BIR), which is a highly mutagenic pathway that is thought to be responsible for many of the mutations and genome rearrangements that drive cancer development. However, the frequency of BIR's deployment and its ability to be triggered by both leading and lagging template strand SSBs were unclear. Using site- and strand-specific SSBs generated by nicking enzymes, including CRISPR-Cas9 nickase (Cas9n), we demonstrate that leading and lagging template strand SSBs in fission yeast are typically converted into deDSBs that are repaired by homologous recombination. However, both types of SSBs can also trigger BIR, and the frequency of these events increases when fork convergence is delayed and the non-homologous end joining protein Ku70 is deleted.}, }
@article {pmid39561528, year = {2024}, author = {Zhang, K and Wang, P and Li, S and Xie, X and Wang, Z and Li, Y and Jiao, X and Li, Q}, title = {Type I-E CRISPR-Cas system regulates fimZY and T3SS1 genes expression in Salmonella enterica serovar Pullorum.}, journal = {Veterinary microbiology}, volume = {299}, number = {}, pages = {110301}, doi = {10.1016/j.vetmic.2024.110301}, pmid = {39561528}, issn = {1873-2542}, mesh = {*CRISPR-Cas Systems ; *Salmonella enterica/genetics ; Animals ; *Chickens ; *Gene Expression Regulation, Bacterial ; Bacterial Proteins/genetics/metabolism ; Salmonella Infections, Animal/microbiology ; Poultry Diseases/microbiology ; Virulence/genetics ; Type III Secretion Systems/genetics ; Plasmids/genetics ; Cell Line, Tumor ; Serogroup ; }, abstract = {Clustered regularly interspaced short palindromic repeats and associated Cas proteins (CRISPR-Cas) provide prokaryotes with adaptive immunity against invasion by plasmids or phages. In Salmonella, the type I-E CRISPR-Cas system is typically considered silent in immunity against foreign genetic elements. To elucidate the role of the CRISPR-Cas system, we chose Salmonella enterica serovar Pullorum S06004 as a model organism due to its four spacers and well-defined biological characteristics observed in previous studies. Western blot analysis revealed expression of Cas3 in S06004 cultured in vitro, but plasmid transformation assays demonstrated that both wild-type (WT) and S06004 strains overexpressing LeuO (a positive regulator of CRISPR-Cas) showed no immunity against the target plasmid. RNA-Seq analysis detected significant downregulation of the fim cluster, encoding type I fimbriae, and T3SS1-related genes in the cas cluster mutant compared to the WT. This downregulation was further confirmed in mutants of CR1 and individual cas genes by qRT-PCR. Consequently, mutants of CR1 and cas clusters exhibited decreased invasion of chicken hepatocellular carcinoma cells. The consistent regulation of T3SS1 genes by the CRISPR-Cas system in S. Pullorum, S. Enteritidis, and S. Typhimurium indicates a common role for the type I-E CRISPR-Cas system in promoting bacterial virulence. However, the specific molecular mechanisms underlying this regulation require further investigation.}, }
@article {pmid39561207, year = {2024}, author = {Osia, B and Merkell, A and Lopezcolorado, FW and Ping, X and Stark, JM}, title = {RAD52 and ERCC6L/PICH have a compensatory relationship for genome stability in mitosis.}, journal = {PLoS genetics}, volume = {20}, number = {11}, pages = {e1011479}, pmid = {39561207}, issn = {1553-7404}, support = {R01 CA256989/CA/NCI NIH HHS/United States ; R01 CA240392/CA/NCI NIH HHS/United States ; T32 CA186895/CA/NCI NIH HHS/United States ; F32 CA275207/CA/NCI NIH HHS/United States ; P30 CA033572/CA/NCI NIH HHS/United States ; }, mesh = {*Rad52 DNA Repair and Recombination Protein/genetics/metabolism ; *Genomic Instability ; *Mitosis/genetics ; Humans ; *DNA Helicases/genetics/metabolism ; Poly-ADP-Ribose Binding Proteins/genetics/metabolism ; DNA Repair/genetics ; Anaphase/genetics ; CRISPR-Cas Systems ; HeLa Cells ; Synthetic Lethal Mutations/genetics ; DNA Damage/genetics ; }, abstract = {Mammalian RAD52 is a DNA repair factor with strand annealing and recombination mediator activities that appear important in both interphase and mitotic cells. Nonetheless, RAD52 is dispensable for cell viability. To query RAD52 synthetic lethal relationships, we performed genome-wide CRISPR knock-out screens and identified hundreds of candidate synthetic lethal interactions. We then performed secondary screening and identified genes for which depletion causes reduced viability and elevated genome instability (increased 53BP1 nuclear foci) in RAD52-deficient cells. One such factor was ERCC6L, which marks DNA bridges during anaphase, and hence is important for genome stability in mitosis. Thus, we investigated the functional interrelationship between RAD52 and ERCC6L. We found that RAD52 deficiency increases ERCC6L-coated anaphase ultrafine bridges, and that ERCC6L depletion causes elevated RAD52 foci in prometaphase and interphase cells. These effects were enhanced with replication stress (i.e. hydroxyurea) and topoisomerase IIα inhibition (ICRF-193), where post-treatment effect timings were consistent with defects in addressing stress in mitosis. Altogether, we suggest that RAD52 and ERCC6L co-compensate to protect genome stability in mitosis.}, }
@article {pmid39560384, year = {2024}, author = {Steiner, S and Roy, CR}, title = {CRISPR-Cas9-based approaches for genetic analysis and epistatic interaction studies in Coxiella burnetii.}, journal = {mSphere}, volume = {9}, number = {12}, pages = {e0052324}, pmid = {39560384}, issn = {2379-5042}, support = {R21 AI171333/AI/NIAID NIH HHS/United States ; R56 AI182383/AI/NIAID NIH HHS/United States ; S10 OD030363/OD/NIH HHS/United States ; R21AI171333,R56AI182383//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; }, mesh = {*Coxiella burnetii/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Epistasis, Genetic ; Bacterial Proteins/genetics ; Humans ; Animals ; Phenotype ; }, abstract = {UNLABELLED: Coxiella burnetii is an obligate intracellular bacterial pathogen that replicates to high numbers in an acidified lysosome-derived vacuole. Intracellular replication requires the Dot/Icm type IVB secretion system, which translocates over 100 different effector proteins into the host cell. Screens employing random transposon mutagenesis have identified several C. burnetii effectors that play an important role in intracellular replication; however, the difficulty in conducting directed mutagenesis has been a barrier to the systematic analysis of effector mutants and to the construction of double mutants to assess epistatic interactions between effectors. Here, two CRISPR-Cas9 technology-based approaches were developed to study C. burnetii phenotypes resulting from targeted gene disruptions. CRISPRi was used to silence gene expression and demonstrated that silencing of effectors or Dot/Icm system components resulted in phenotypes similar to those of transposon insertion mutants. A CRISPR-Cas9-mediated cytosine base editing protocol was developed to generate targeted loss-of-function mutants through the introduction of premature stop codons into C. burnetii genes. Cytosine base editing successfully generated double mutants in a single step. A double mutant deficient in both cig57 and cig2 had a robust and additive intracellular replication defect when compared to either single mutant, which is consistent with Cig57 and Cig2 functioning in independent pathways that both contribute to a vacuole that supports C. burnetii replication. Thus, CRISPR-Cas9-based technologies expand the genetic toolbox for C. burnetii and will facilitate genetic studies aimed at investigating the mechanisms this pathogen uses to replicate inside host cells.<br><br>IMPORTANCE: Understanding the genetic mechanisms that enable C. burnetii to replicate in mammalian host cells has been hampered by the difficulty in making directed mutations. Here, a reliable and efficient system for generating targeted loss-of-function mutations in C. burnetii using a CRISPR-Cas9-assisted base editing approach is described. This technology was applied to make double mutants in C. burnetii that enabled the genetic analysis of two genes that play independent roles in promoting the formation of vacuoles that support intracellular replication. This advance will accelerate the discovery of mechanisms important for C. burnetii host infection and disease.}, }
@article {pmid39560197, year = {2024}, author = {Wang, Y and He, D and Li, W and Dong, Y and Fang, L and Liu, D and Tang, Y and Xiao, S}, title = {Field-deployable porcine epidemic diarrhea virus diagnostics utilizing CRISPR-Cas13a.}, journal = {Virulence}, volume = {15}, number = {1}, pages = {2429022}, pmid = {39560197}, issn = {2150-5608}, mesh = {*Porcine epidemic diarrhea virus/genetics/isolation &amp; purification ; Animals ; Swine ; *CRISPR-Cas Systems ; *Swine Diseases/diagnosis/virology ; *Coronavirus Infections/diagnosis/veterinary/virology ; Sensitivity and Specificity ; CRISPR-Associated Proteins/genetics ; }, abstract = {Porcine epidemic diarrhoea virus (PEDV), a pathogenic microorganism that induces epidemic diarrhoea in swine, causes substantial economic damage to swine-farming nations. To prevent and control PEDV infections, the availability of upgraded and rapid virus detection techniques is crucial. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas)13a system, namely, programmability of CRISPR RNA (crRNA) and "collateral" promiscuous RNase activity of Cas13a after target RNA identification. In this study, we aimed to develop a recombinase polymerase amplification (RPA)-based CRISPR-Cas13a approach for PEDV diagnosis for the first time. The results showed that up to 10 copies of the target PEDV DNA standard/µL were detected after 40 min at 37 °C. PEDV detection exhibited remarkable specificity compared to that of other selected pathogens. Additionally, this RPA-based CRISPR-Cas13a approach could be used to clinical samples, with similar performance to that of reverse transcription-quantitative polymerase chain reaction (RT - qPCR). The results of our proposed approach were visualized using either lateral flow strips or fluorescence for field-deployable viral diagnostics, thereby facilitating its use in endemic regions. Overall, our proposed approach showed good reliability, sensitivity, and specificity, suggesting that it is applicable for detecting other viruses in diagnosing diseases and inspecting food safety.}, }
@article {pmid39558904, year = {2024}, author = {Chen, Y and Kang, R and Jiang, Y and Zheng, Q and Yang, Y and Liu, J and Wu, G and Zhao, W and Li, Z and Peng, C and Zhang, P and Peng, F and Liu, Q and Hu, S and Luo, X and Wu, G and Cui, K and Huang, J and Wang, Y and Songyang, Z and Liang, P}, title = {Rationally designed Campylobacter jejuni Cas9 enables efficient gene activation and base editing.}, journal = {Molecular therapy. Nucleic acids}, volume = {35}, number = {4}, pages = {102366}, pmid = {39558904}, issn = {2162-2531}, abstract = {Compact and adaptable CRISPR-Cas systems enable genome engineering applications in various contexts via high-efficiency delivery. The adeno-associated virus (AAV) is a widely used delivery system. One of the most compact type II-C Cas9 orthologs-CjCas9, derived from Campylobacter jejuni, is particularly appealing for AAV delivery. However, the editing efficiency of CjCas9 limits its applications. In this study, we used structure-guided protein engineering to improve the editing efficiency of CjCas9. Subsequently, we developed a miniature transcriptional activator (LDE-CjCas9-VPR) and base editors engineered from CjCas9 (LDE-CjABE and LDE-CjCBE). LDE-CjABE effectively induced genome editing in human and mouse cells. Through AAV delivery, LDE-CjABE enhanced the on-target editing efficiency, and off-target editing was not detected in the mouse retina. Therefore, the compact size and high editing efficiency of LDE-CjCas9 broadens the target scope of transcription activation and base editing toolsets for therapeutic applications.}, }
@article {pmid39558417, year = {2024}, author = {Santos, IFM and Moreira, DS and Costa, KF and Ribeiro, JM and Murta, SMF and Santi, AMM}, title = {Ascorbate peroxidase modulation confirms key role in Leishmania infantum oxidative defence.}, journal = {Parasites &amp; vectors}, volume = {17}, number = {1}, pages = {472}, pmid = {39558417}, issn = {1756-3305}, support = {Finance Code 001//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior/ ; CNPq 309994-2023-3//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; APQ-02816-21; BDP-00657; RED-00104-22//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de Minas Gerais/ ; Chamada de Redes Colaborativas de Pesquisa do Instituto Ren&#xe9; Rachou-Fiocruz Minas (Rede FarVac)//Funda&#xe7;&#xe3;o Oswaldo Cruz/ ; }, mesh = {*Leishmania infantum/genetics/drug effects/enzymology ; *Ascorbate Peroxidases/genetics/metabolism ; *Oxidative Stress ; *Macrophages/parasitology ; Hydrogen Peroxide/pharmacology ; Animals ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Protozoan Proteins/genetics/metabolism ; Antiprotozoal Agents/pharmacology ; Mice ; Drug Resistance/genetics ; }, abstract = {BACKGROUND: Ascorbate peroxidase (APX) has emerged as a promising target for chemotherapy because of its absence in humans and crucial role in the antioxidant defence of trypanosomatids. APXs, which are class I haeme-containing enzymes, reduces hydrogen peroxide using ascorbate to produce water and monodehydroascorbate, thereby preventing cell damage caused by H2O2.<br><br>METHODS: We aimed to create an APX-knockout L. infantum line using CRISPR/Cas9. Despite unsuccessful attempts at full knockouts, we achieved deletion of chromosomal copies post-APX episomal insertion, yielding Li&#x394;chrAPX::LbAPX parasites. We performed phenotypic characterization to assess the impact of these genetic modifications, which included the determination of APX transcript expression levels using quantitative PCR, drug sensitivity, infectivity, and parasite survival in macrophages.<br><br>RESULTS: Quantitative polymerase chain reaction (PCR) analysis revealed a 10- to 13-fold reduction in APX transcript expression in Li&#x394;chrAPX::LbAPX compared with wild-type (LiWT) and APX-overexpressing (Li::Cas9::LbAPX) parasites, respectively. The episomes in those knockdown parasites remained stable even after 20 drug-free passages in vitro. Li::Cas9::LbAPX parasites showed increased resistance to trivalent antimony (Sb[III]) and isoniazid, reduced tolerance to H2O2, and unchanged macrophage infectivity compared with LiWT. In contrast, Li&#x394;chrAPX::LbAPX parasites were more sensitive to Sb[III] and isoniazid, exhibited greater susceptibility to H2O2-induced oxidative stress, and 72&#xa0;h post-infection, showed fewer infected macrophages and intracellular amastigotes compared with LiWT parasites.<br><br>CONCLUSIONS: Our findings hint at the indispensability of APX in L. infantum and raise the possibility of its potential as a therapeutic target for leishmaniasis.}, }
@article {pmid39558180, year = {2024}, author = {Wang, K and Liu, S and Zhou, S and Qileng, A and Wang, D and Liu, Y and Chen, C and Lei, C and Nie, Z}, title = {Ligand-Responsive Artificial Protein-Protein Communication for Field-Deployable Cell-Free Biosensing.}, journal = {Angewandte Chemie (International ed. in English)}, volume = {}, number = {}, pages = {e202416671}, doi = {10.1002/anie.202416671}, pmid = {39558180}, issn = {1521-3773}, support = {2020YFA0907500//Chongqing Municipal Key Research and Development Program of China/ ; 22034002//National Natural Science Foundation of China/ ; 22374036//National Natural Science Foundation of China/ ; 92253304//National Natural Science Foundation of China/ ; 22061160466//National Natural Science Foundation of China/ ; 22277063//National Natural Science Foundation of China/ ; 2022JJ20004//Natural Science Foundation of Hunan Province/ ; }, abstract = {Natural protein-protein communications, such as those between transcription factors (TFs) and RNA polymerases/ribosomes, underpin cell-free biosensing systems operating on the transcription/translation (TXTL) paradigm. However, their deployment in field analysis is hampered by the delayed response (hour-level) and the complex composition of in vitro TXTL systems. For this purpose, we present a de novo-designed ligand-responsive artificial protein-protein communication (LIRAC) by redefining the connection between TFs and non-interacting CRISPR/Cas enzymes. By rationally designing a chimeric DNA adaptor and precisely regulating its binding affinities to both proteins, LIRAC immediately transduces target-induced TF allostery into rapid CRISPR/Cas enzyme activation within a homogeneous system. Consequently, LIRAC obviates the need for RNA/protein biosynthesis inherent to conventional TXTL-based cell-free systems, substantially reducing reaction complexity and time (from hours to 10 minutes) with improved sensitivity and tunable dynamic range. Moreover, LIRAC exhibits excellent versatility and programmability for rapidly and sensitively detecting diverse contaminants, including antibiotics, heavy metal ions, and preservatives. It also enables the creation of a multi-protein communication-based tristate logic for the intelligent detection of multiple contaminants. Integrated with portable devices, LIRAC has been proven effective in the field analysis of environmental samples and personal care products, showcasing its potential for environmental and health monitoring.}, }
@article {pmid39558174, year = {2024}, author = {Stadelmann, T and Heid, D and Jendrusch, M and Mathony, J and Aschenbrenner, S and Rosset, S and Correia, BE and Niopek, D}, title = {A deep mutational scanning platform to characterize the fitness landscape of anti-CRISPR proteins.}, journal = {Nucleic acids research}, volume = {52}, number = {22}, pages = {e103}, pmid = {39558174}, issn = {1362-4962}, support = {453202693//German Research Foundation/ ; //Aventis Foundation/ ; //German Academic Scholarship Foundation/ ; //Biltema Foundation/ ; //National Center of Competence in Research Chemical Biology/ ; 716058/ERC_/European Research Council/International ; /SNSF_/Swiss National Science Foundation/Switzerland ; }, mesh = {*CRISPR-Cas Systems ; *Escherichia coli/genetics ; *Gene Editing/methods ; Mutation ; CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Associated Proteins/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; }, abstract = {Deep mutational scanning is a powerful method for exploring the mutational fitness landscape of proteins. Its adaptation to anti-CRISPR proteins, which are natural CRISPR-Cas inhibitors and key players in the co-evolution of microbes and phages, facilitates their characterization and optimization. Here, we developed a robust anti-CRISPR deep mutational scanning pipeline in Escherichia coli that combines synthetic gene circuits based on CRISPR interference with flow cytometry coupled sequencing and mathematical modeling. Using this pipeline, we characterized comprehensive single point mutation libraries for AcrIIA4 and AcrIIA5, two potent inhibitors of CRISPR-Cas9. The resulting mutational fitness landscapes revealed considerable mutational tolerance for both Acrs, suggesting an intrinsic redundancy with respect to Cas9 inhibitory features, and - for AcrIIA5 - indicated mutations that boost Cas9 inhibition. Subsequent in vitro characterization suggested that the observed differences in inhibitory potency between mutant inhibitors were mostly due to changes in binding affinity rather than protein expression levels. Finally, to demonstrate that our pipeline can inform Acrs-based genome editing applications, we employed a selected subset of mutant inhibitors to increase CRISPR-Cas9 target specificity by modulating Cas9 activity. Taken together, our work establishes deep mutational scanning as a powerful method for anti-CRISPR protein characterization and optimization.}, }
@article {pmid39557836, year = {2024}, author = {Tsui, CK and Twells, N and Durieux, J and Doan, E and Woo, J and Khosrojerdi, N and Brooks, J and Kulepa, A and Webster, B and Mahal, LK and Dillin, A}, title = {CRISPR screens and lectin microarrays identify high mannose N-glycan regulators.}, journal = {Nature communications}, volume = {15}, number = {1}, pages = {9970}, pmid = {39557836}, issn = {2041-1723}, support = {F32 AG069388/AG/NIA NIH HHS/United States ; 5F32AG069388-03//U.S. Department of Health &amp; Human Services | NIH | National Institute on Aging (U.S. National Institute on Aging)/ ; }, mesh = {*Polysaccharides/metabolism ; *Mannose/metabolism/chemistry ; Humans ; Glycosylation ; *Golgi Apparatus/metabolism ; *Lectins/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; HEK293 Cells ; }, abstract = {Glycans play critical roles in cellular signaling and function. Unlike proteins, glycan structures are not templated from genetic sequences but synthesized by the concerted activity of many genes, making them historically challenging to study. Here, we present a strategy that utilizes CRISPR screens and lectin microarrays to uncover and characterize regulators of glycosylation. We applied this approach to study the regulation of high mannose glycans - the starting structure of all asparagine(N)-linked-glycans. We used CRISPR screens to uncover the expanded network of genes controlling high mannose levels, followed by lectin microarrays to fully measure the complex effect of select regulators on glycosylation globally. Through this, we elucidated how two high mannose regulators - TM9SF3 and the CCC complex - control complex N-glycosylation via regulating Golgi morphology and function. Notably, this allows us to interrogate Golgi function in-depth and reveals that similar disruption to Golgi morphology can lead to drastically different glycosylation outcomes. Collectively, this work demonstrates a generalizable approach for systematically dissecting the regulatory network underlying glycosylation.}, }
@article {pmid39557480, year = {2024}, author = {Shi, Y and Yu, Y and Lü, YL and Lü, H}, title = {Design and practice of educational experiments on genetic epistasis.}, journal = {Yi chuan = Hereditas}, volume = {46}, number = {11}, pages = {958-970}, doi = {10.16288/j.yczz.24-248}, pmid = {39557480}, issn = {0253-9772}, mesh = {*Epistasis, Genetic ; *Saccharomyces cerevisiae/genetics ; CRISPR-Cas Systems ; Mutation ; Phenotype ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; Adenine/metabolism ; }, abstract = {Genetic epistasis is a fundamental concept in genetics that describes how interactions between genes determine phenotypic traits. To enhance students' understanding and practical application of genetic epistasis, this experiment is designed and conducted using gene mutations in the adenine biosynthesis pathway of Saccharomyces cerevisiae (baker's yeast). S. cerevisiae is a classic model organism for genetic teaching experiments. In its adenine biosynthesis pathway, a mutation in the ADE2 gene leads to the accumulation of the intermediate 5'-phosphoribosylaminoimidazole (AIR), causing the cells to appear red. However, if a gene upstream of ADE2 in the adenine biosynthesis pathway (such as ADE8) is defective, the red phenotype of yeast will disappear. Conversely, a defect in a gene downstream of ADE2 (such as ADE1) does not alter the red phenotype. Therefore, ADE8 is epistatic to ADE2. In this experiment, the CRISPR-Cas9 genome editing technology is employed, allowing students to perform single knockout of ade2Δ, as well as double knockouts of ade2Δade8Δ and ade2Δade1Δ in S. cerevisiae. By observing the phenotypic changes in yeast mutants from white to red and back to white, students gain a profound understanding of the basic genetic theory of how genes determine phenotypes and the concept of epistasis in gene interactions. This experiment also enables students to master fundamental yeast genetic techniques, significantly enhancing their ability to design and conduct experiments in real research environments. This is of great significance for their future research work and academic development.}, }
@article {pmid39557225, year = {2025}, author = {Zuo, M and Du, J and Liu, Y and Chen, M and Liu, B and Li, G and Li, M and Huang, S and Yu, G}, title = {Deletion of the gsk-3β (Glycogen synthase kinase-3β) in zebrafish results in decreased susceptibility to Aeromonas hydrophila.}, journal = {Microbial pathogenesis}, volume = {198}, number = {}, pages = {107129}, doi = {10.1016/j.micpath.2024.107129}, pmid = {39557225}, issn = {1096-1208}, mesh = {Animals ; *Aeromonas hydrophila/pathogenicity ; *Zebrafish/microbiology/immunology ; *Glycogen Synthase Kinase 3 beta/genetics/metabolism ; *Gram-Negative Bacterial Infections/immunology/microbiology ; *Fish Diseases/microbiology/immunology ; *Immunity, Innate/genetics ; *CRISPR-Cas Systems ; *Gene Editing ; Gene Knockout Techniques ; Autophagy/genetics ; Disease Models, Animal ; Gene Deletion ; Aquaculture ; Zebrafish Proteins/genetics/metabolism ; Disease Susceptibility ; }, abstract = {Aeromonas hydrophila is a significant pathogen in the field of fish farming, resulting in substantial financial losses for the aquaculture industry. As the pathogen's resistance to commercially available antibiotics continues to rise, the identification of novel antimicrobial strategies becomes increasingly crucial. This study aims to explore the modulatory impact of gsk-3β (Glycogen synthase kinase-3β) on the intrinsic immunity against Aeromonas hydrophila in zebrafish, with the objective of uncovering a new avenue for enhancing fish antimicrobial activity through gene editing. Our investigation involved an analysis of the evolutionary patterns and protein sequence of gsk-3β, elucidating its conserved characteristics in zebrafish and fish species of economic importance. In this research, CRISPR-Cas9 technology was employed to generate a zebrafish model with a knockout of gsk-3β, resulting in a decreased resistance of zebrafish to Aeromonas hydrophila (ATCC 7966) infection. Furthermore, we conducted preliminary investigations into the potential mechanisms through which gsk-3β governs antimicrobial immunity. Our findings revealed that knockout of gsk-3β resulted in diminished activation of innate immunity, antioxidant capacity, and autophagy. Hence, the findings of this study are highly significant in improving the economic benefits of aquaculture and in effectively preventing and controlling infection caused by Aeromonas hydrophila.}, }
@article {pmid39556729, year = {2024}, author = {Du, SW and Newby, GA and Salom, D and Gao, F and Menezes, CR and Suh, S and Choi, EH and Chen, PZ and Liu, DR and Palczewski, K}, title = {In vivo photoreceptor base editing ameliorates rhodopsin-E150K autosomal-recessive retinitis pigmentosa in mice.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {121}, number = {48}, pages = {e2416827121}, pmid = {39556729}, issn = {1091-6490}, support = {UG3 AI150551/AI/NIAID NIH HHS/United States ; F30 EY033642/EY/NEI NIH HHS/United States ; F30EY029136 T32GM007250 T32AR080622//HHS | NIH (NIH)/ ; F30 EY029136/EY/NEI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; UG3AI150551 U01AI142756 R35GM118062 RM1HG009490//HHS | NIH (NIH)/ ; R35 GM118062/GM/NIGMS NIH HHS/United States ; R00HL163805//HHS | NIH (NIH)/ ; U01 AI142756/AI/NIAID NIH HHS/United States ; R01 EY034501/EY/NEI NIH HHS/United States ; T32 GM007250/GM/NIGMS NIH HHS/United States ; R01 EY009339/EY/NEI NIH HHS/United States ; P30 EY034070/EY/NEI NIH HHS/United States ; T32 GM008620/GM/NIGMS NIH HHS/United States ; R00 HL163805/HL/NHLBI NIH HHS/United States ; R01EY009339 R01EY034501 P30EY034070//HHS | NIH (NIH)/ ; T32 AR080622/AR/NIAMS NIH HHS/United States ; T32GM008620 F30EY033642//HHS | NIH (NIH)/ ; }, mesh = {Animals ; *Rhodopsin/genetics/metabolism ; *Retinitis Pigmentosa/genetics/therapy/metabolism/pathology ; Mice ; *Gene Editing/methods ; Disease Models, Animal ; CRISPR-Cas Systems ; Retinal Rod Photoreceptor Cells/metabolism/pathology ; Mutation ; }, abstract = {Rhodopsin, the prototypical class-A G-protein coupled receptor, is a highly sensitive receptor for light that enables phototransduction in rod photoreceptors. Rhodopsin plays not only a sensory role but also a structural role as a major component of the rod outer segment disc, comprising over 90% of the protein content of the disc membrane. Mutations in RHO which lead to structural or functional abnormalities, including the autosomal recessive E150K mutation, result in rod dysfunction and death. Therefore, correction of deleterious rhodopsin mutations could rescue inherited retinal degeneration, as demonstrated for other visual genes such as RPE65 and PDE6B. In this study, we describe a CRISPR/Cas9 adenine base editing strategy to correct the E150K mutation and demonstrate precise in vivo editing in a Rho-E150K mouse model of autosomal recessive retinitis pigmentosa (RP). Using ultraviolet-visible spectroscopy, mass spectrometry, and the G-protein activation assay, we characterized wild-type rhodopsin and rhodopsin variants containing bystander base edits. Subretinal injection of dual-adeno-associated viruses delivering our base editing strategy yielded up to 44% Rho correction in homozygous Rho-E150K mice. Injection at postnatal day 15, but not later time points, restored rhodopsin expression, partially rescued retinal function, and partially preserved retinal structure. These findings demonstrate that in vivo base editing can restore the function of mutated structural and functional proteins in animal models of disease, including rhodopsin-associated RP and suggest that the timing of gene-editing is a crucial determinant of successful treatment outcomes for degenerative genetic diseases.}, }
@article {pmid39556419, year = {2024}, author = {Ipoutcha, T and Tsarmpopoulos, I and Gourgues, G and Baby, V and Dubos, P and Hill, GE and Arfi, Y and Lartigue, C and Thébault, P and Bonneaud, C and Sirand-Pugnet, P}, title = {Evolution of the CRISPR-Cas9 defence system in Mycoplasma gallisepticum following colonization of a novel bird host.}, journal = {Microbial genomics}, volume = {10}, number = {11}, pages = {}, doi = {10.1099/mgen.0.001320}, pmid = {39556419}, issn = {2057-5858}, mesh = {*CRISPR-Cas Systems ; *Mycoplasma gallisepticum/genetics/pathogenicity ; Animals ; *Evolution, Molecular ; Bacteriophages/genetics ; Phylogeny ; }, abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are bacterial defences that target bacteriophages and mobile genetic elements. How these defences evolve in novel host environments remains largely unknown. We studied the evolution of the CRISPR-Cas system in Mycoplasma gallisepticum (also named Mycoplasmoides gallisepticum), a bacterial pathogen of poultry that jumped into a passerine host ~30 years ago. Over the decade following the host shift, all isolates displaying a functional CRISPR-Cas system were found not only to harbour completely new sets of spacers, but the DNA protospacer adjacent motif recognized by the main effector M. gallisepticum Cas9 (MgCas9) was also different. These changes in CRISPR-Cas diversity and specificity are consistent with a change in the community of phages and mobile elements infecting M. gallisepticum as it colonized the novel host. In the years following the host shift, we also detected a gradual rise in isolates displaying non-functional MgCas9. After 12 years, all circulating isolates harboured inactive forms only. This loss of CRISPR-Cas function comes at a time when the passerine host is known to have evolved widespread resistance, which in turn drove the evolution of increasing M. gallisepticum virulence through antagonistic coevolution. Such striking concordance in the rise of inactivated forms of CRISPR-Cas and the evolution of host resistance suggests that the inactivation of the CRISPR-Cas system was necessary for enabling adaptive bacterial responses to host-driven selection. We highlight the need to consider both host and pathogen selection pressures on bacteria for understanding the evolution of CRISPR-Cas systems and the key factors driving the emergence of a pathogenic bacterium in a novel host.}, }
@article {pmid39556313, year = {2024}, author = {Liu, S and Zhao, Y and Mo, Q and Sun, Y and Ma, H}, title = {Engineering CjCas9 for Efficient Base Editing and Prime Editing.}, journal = {The CRISPR journal}, volume = {7}, number = {6}, pages = {395-405}, doi = {10.1089/crispr.2024.0018}, pmid = {39556313}, issn = {2573-1602}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; Humans ; Campylobacter jejuni/genetics ; Cytosine/metabolism ; Genetic Therapy/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Adenine/metabolism ; Bacterial Proteins/genetics ; Streptococcus pyogenes/genetics/enzymology ; }, abstract = {The CRISPR-Cas9 system has been applied for clinical applications of gene therapy. Most CRISPR-based gene therapies are derived from Streptococcus pyogenes Cas9, which is challenging to package into a single adeno-associated virus vector and limits its clinical applications. Campylobacter jejuni Cas9 (CjCas9) is one of the smallest Cas9 proteins. CjCas9-mediated base editing (CjBE) efficiency varies across genomic sites, while CjCas9-mediated prime editing (CjPE) efficiency is less than 5% on average. Here we developed enhanced cytosine base editors (enCjCBEs) and adenine base editors (enCjABEs) by engineered CjCas9[P47K]. We demonstrated the robust C-to-T conversion (70% on average) by enCjCBE or A-to-G conversion (76% on average) by enCjABE. Meanwhile, we applied the CjCas9[P47K] variant to generate enhanced CjPE (enCjPE), which increases the editing efficiency 17-fold at the PRNP site over wild-type CjPE. Fusing nonspecific DNA binding protein Sso7d to enCjCas9 and MS2 stem-loop RNA aptamer to the 3-terminal of cognate pegRNA resulted in 12% editing efficiency on average with a 24-fold increase over wild-type CjPE, and we termed it SsenCjPE. The SsenCjPE can also be combined with hMLH1dn to further increase the editing efficiency and MMLV RTaseΔRnH to reduce size. Finally, we introduced an additional mutation D829R into SsenCjPE and generated SsenCjPE-M2 with a 61-fold increase of PE efficiency over wild-type at the PRNP site. In summary, enCjBEs, SsenCjPEs, or SsenCjPE-M2 are compact Cas9-derived BE or prime editors in biological research or biomedical applications.}, }
@article {pmid39556245, year = {2024}, author = {Poth, T and Schirmacher, P}, title = {[Centers for experimental animal pathology still occupy a small but valuable niche in translational medicine-time for change?].}, journal = {Pathologie (Heidelberg, Germany)}, volume = {45}, number = {Suppl 1}, pages = {31-36}, pmid = {39556245}, issn = {2731-7196}, mesh = {Animals ; *Translational Research, Biomedical/methods ; Humans ; Disease Models, Animal ; Mice ; Pathology/methods ; Germany ; }, abstract = {Since CRISPR/Cas systems can be easily used to establish new genetically engineered mouse models, application of these models in the translational research field to explore predictive and therapeutic approaches for human diseases is rising.Integrative centers for experimental animal pathology, such as the CMCP in Heidelberg and the CEP in Munich, link the veterinary and human pathology discipline and contribute substantially to meaningful study results by combining the technical, research, and diagnostic expertise for phenotyping, evaluating, and interpretating complex animal models in the context of human pathology. They provide high-quality tissue processing in a broad spectrum of standard and specialized tissue-based technologies. The histopathology platform enables animal model evaluation including phenotyping, scoring, and detecting species-specific "background lesions." By providing essential prerequisites for high-quality, reproducible, and sustainable research in translational medicine, facilities for experimental animal pathology optimally fulfill the requirements for publications in top-class professional journals, contribute to the implementation of the 3R principle for animal welfare, and help to reduce costs in the preclinical research.The importance of animal models reflecting human diseases will increase in the future but the requirements for high-quality tissue processing and comparative pathology/morphological phenotyping cannot be met by single facilities in a niche position. To cover the existing gap and expected rising demand in the German and European translational research landscape, further facilities for experimental animal pathology that are integrated in the human pathology environment should be established.}, }
@article {pmid39555930, year = {2025}, author = {Krausfeldt, LE and Samuel, PS and Smith, RP and Urakawa, H and Rosen, BH and Colwell, RR and Lopez, JV}, title = {Transcriptional profiles of Microcystis reveal gene expression shifts that promote bloom persistence in in situ mesocosms.}, journal = {Microbiology spectrum}, volume = {13}, number = {1}, pages = {e0136924}, pmid = {39555930}, issn = {2165-0497}, support = {W912HZ-19-SOI-0022//DOD | USA | U.S. Army Corps of Engineers (USACE)/ ; }, mesh = {*Microcystis/genetics/metabolism/growth &amp; development ; *Harmful Algal Bloom ; *Nitrogen/metabolism ; Gene Expression Regulation, Bacterial ; Ecosystem ; Transcriptome ; Microcystins/genetics/metabolism ; Urea/metabolism ; Photosynthesis ; Gene Expression Profiling ; }, abstract = {Harmful algal blooms caused by cyanobacteria threaten aquatic ecosystems, the economy, and human health. Previous work has tried to identify the mechanisms that allow blooms to form, focusing on the role of nutrients. However, little is known about how introduced nutrients influence gene expression in situ. To address this knowledge gap, we used in situ mesocosms initiated with water experiencing a Microcystis bloom. We added pulses of nutrients that are commonly associated with anthropogenic sources to the mesocosms for 72 hours and collected samples for metatranscriptomics to examine how the physiological function of Microcystis and bloom status changed. The addition of nitrogen (N) as urea, but not the addition of PO4, resulted in conspicuous bloom persistence for at least 9 days after the final introduction of nutrients. The addition of urea initially resulted in the upregulation of photosynthesis machinery, as well as phosphate, carbon, and N transport and metabolism. Once Microcystis presumably became N-replete, upregulation of amino acid metabolism, microcystin biosynthesis, and other processes associated with biomass generation occurred. These capacities coincided with the upregulation of toxin-antitoxin systems, CRISPR-cas genes, and transposases suggesting that phage defense and genome rearrangement are critical in bloom persistence. Overall, our results show the stepwise transcriptional response of a Microcystis bloom to the introduction of nutrients, specifically urea, as it is sustained in a natural setting. The transcriptomic shifts observed herein may serve as markers of the longevity of blooms while providing insight into why Microcystis blooms over other cyanobacteria.IMPORTANCEHarmful algal blooms represent a threat to human health and ecosystems. Understanding why blooms persist may help us develop warning indicators of bloom persistence and create novel mitigation strategies. Using mesocosm experiments initiated with water with an active bloom, we measured the stepwise transcription changes of the toxin-producing cyanobacterium Microcystis in response to the addition of nutrients that are important in causing blooms. We found that nitrogen (N), but not phosphorus, promoted bloom longevity. The initial introduction of N resulted in the upregulation of genes involved in photosynthesis and N import. At later times in the bloom, upregulation of genes involved in biomass generation, phage protection, genomic rearrangement, and toxin production was observed. Our results suggest that Microcystis first fulfills nutritional requirements before investing energy in pathways associated with growth and protection against competitors, which allowed bloom persistence more than a week after the final addition of nutrients.}, }
@article {pmid39555828, year = {2024}, author = {Fontana, M and Solomon, SD and Kachadourian, J and Walsh, L and Rocha, R and Lebwohl, D and Smith, D and Täubel, J and Gane, EJ and Pilebro, B and Adams, D and Razvi, Y and Olbertz, J and Haagensen, A and Zhu, P and Xu, Y and Leung, A and Sonderfan, A and Gutstein, DE and Gillmore, JD}, title = {CRISPR-Cas9 Gene Editing with Nexiguran Ziclumeran for ATTR Cardiomyopathy.}, journal = {The New England journal of medicine}, volume = {391}, number = {23}, pages = {2231-2241}, doi = {10.1056/NEJMoa2412309}, pmid = {39555828}, issn = {1533-4406}, mesh = {Aged ; Aged, 80 and over ; Female ; Humans ; Male ; Middle Aged ; *Amyloid Neuropathies, Familial/blood/complications/genetics/therapy ; *Cardiomyopathies/blood/genetics/therapy ; CRISPR-Cas Systems ; Gene Editing/methods ; *Genetic Therapy/adverse effects/methods ; Infusions, Intravenous/adverse effects ; Natriuretic Peptide, Brain/blood ; Peptide Fragments/blood ; *Prealbumin/genetics ; Troponin T/blood ; *Liposomes/administration &amp; dosage/adverse effects ; *RNA, Guide, Kinetoplastida/administration &amp; dosage/adverse effects/genetics ; *Nanoparticles/administration &amp; dosage/adverse effects ; }, abstract = {BACKGROUND: Transthyretin amyloidosis with cardiomyopathy (ATTR-CM) is a progressive, often fatal disease. Nexiguran ziclumeran (nex-z) is an investigational therapy based on CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease) targeting the gene encoding transthyretin (TTR).<br><br>METHODS: In this phase 1, open-label trial, we administered a single intravenous infusion of nex-z to patients with ATTR-CM. Primary objectives included assessment of the effect of nex-z on safety and pharmacodynamics, including the serum TTR level. Secondary end points included changes in N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels, high-sensitivity cardiac troponin T levels, the 6-minute walk distance, and the New York Heart Association (NYHA) class.<br><br>RESULTS: A total of 36 patients received nex-z and completed at least 12 months of follow-up. Of these patients, 50% were in NYHA class III and 31% had variant ATTR-CM. The mean percent change from baseline in the serum TTR level was -89% (95% confidence interval [CI], -92 to -87) at 28 days and -90% (95% CI, -93 to -87) at 12 months. Adverse events were reported in 34 patients. Five had transient infusion-related reactions, and two had transient liver-enzyme elevations that were assessed as treatment-related. Serious adverse events, most of which were consistent with ATTR-CM, were reported in 14 patients. The geometric mean factor change from baseline to month 12 was 1.02 (95% CI, 0.88 to 1.17) in the NT-proBNP level and 0.95 (95% CI, 0.89 to 1.01) in the high-sensitivity cardiac troponin T level. The median change from baseline to month 12 in the 6-minute walk distance was 5 m (interquartile range, -33 to 49). A total of 92% of the patients had either improvement or no change in their NYHA class.<br><br>CONCLUSIONS: In this phase 1 study involving patients with ATTR-CM, treatment with a single dose of nex-z was associated with transient infusion-related reactions and consistent, rapid, and durable reductions in serum TTR levels. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.).}, }
@article {pmid39555799, year = {2024}, author = {Inen, J and Han, CM and Farrel, DM and Bilousova, G and Kogut, I}, title = {CIRCLE-Seq for Interrogation of Off-Target Gene Editing.}, journal = {Journal of visualized experiments : JoVE}, volume = {}, number = {213}, pages = {}, doi = {10.3791/67069}, pmid = {39555799}, issn = {1940-087X}, mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; DNA/genetics ; Sequence Analysis, DNA/methods ; }, abstract = {Circularization for In Vitro Reporting of Cleavage Effects by Sequencing (CIRCLE-seq) is a novel technique developed for the impartial identification of unintended cleavage sites of CRISPR-Cas9 through targeted sequencing of CRISPR-Cas9 cleaved DNA. The protocol involves circularizing genomic DNA (gDNA), which is subsequently treated with the Cas9 protein and a guide RNA (gRNA) of interest. Following treatment, the cleaved DNA is purified and prepared as a library for Illumina sequencing. The sequencing process generates paired-end reads, offering comprehensive data on each cleavage site. CIRCLE-seq provides several advantages over o