CRISPR Journal最新文献_第6页

Benchmark Software and Data for Evaluating CRISPR-Cas9 Experimental Pipelines Through the Assessment of a Calibration Screen. 通过校准筛选评估 CRISPR-Cas9 实验流程的基准软件和数据。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2024-12-01 Epub Date: 2024-01-02 DOI: 10.1089/crispr.2023.0040

Raffaele M Iannuzzi, Ichcha Manipur, Clare Pacini, Fiona M Behan, Mario R Guarracino, Mathew J Garnett, Aurora Savino, Francesco Iorio

{"title":"Benchmark Software and Data for Evaluating CRISPR-Cas9 Experimental Pipelines Through the Assessment of a Calibration Screen.","authors":"Raffaele M Iannuzzi, Ichcha Manipur, Clare Pacini, Fiona M Behan, Mario R Guarracino, Mathew J Garnett, Aurora Savino, Francesco Iorio","doi":"10.1089/crispr.2023.0040","DOIUrl":"10.1089/crispr.2023.0040","url":null,"abstract":"Genome-wide genetic screens using CRISPR-guide RNA libraries are widely performed in mammalian cells to functionally characterize individual genes and for the discovery of new anticancer therapeutic targets. As the effectiveness of such powerful and precise tools for cancer pharmacogenomics is emerging, tools and methods for their quality assessment are becoming increasingly necessary. Here, we provide an R package and a high-quality reference data set for the assessment of novel experimental pipelines through which a single calibration experiment has been executed: a screen of the HT-29 human colorectal cancer cell line with a commercially available genome-wide library of single-guide RNAs. This package and data allow experimental researchers to benchmark their screens and produce a quality-control report, encompassing several quality and validation metrics. The R code used for processing the reference data set, for its quality assessment, as well as to evaluate the quality of a user-provided screen, and to reproduce the figures presented in this article is available at https://github.com/DepMap-Analytics/HT29benchmark. The reference data is publicly available on FigShare.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"355-365"},"PeriodicalIF":3.7,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139075824","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

'Tis the Season: CRISPR Products All Around. 这个季节：CRISPR产品无处不在。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2024-12-01 Epub Date: 2024-12-06 DOI: 10.1089/crispr.2024.0094

Rodolphe Barrangou

引用次数: 0

Early Detection of Wildlife Disease Pathogens Using CRISPR-Cas System Methods. 利用 CRISPR-Cas 系统方法早期检测野生动物疾病病原体。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2024-12-01 Epub Date: 2024-10-31 DOI: 10.1089/crispr.2024.0030

Adam A Pérez, Guelaguetza Vazquez-Meves, Margaret E Hunter

引用次数: 0

Identification of a Guide RNA Targeting an Ultraconserved Element for Evaluation of Cas9 Genome Editors Across Mammalian Species. 鉴定靶向超保守元件的引导核糖核酸，以评估跨哺乳动物物种的 Cas9 基因组编辑器。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2024-12-01 Epub Date: 2024-09-23 DOI: 10.1089/crispr.2024.0053

Benjamin G Gowen, Prachi Khekare, Shannon R McCawley, Kory Melton, Craig Soares, Jean Chan, Vihasi Jani, Pierre Boivin, Ashil Bans, Weng-In Leong, Aaron J Cantor, Jack Walleshauser, Peter B Otoupal, Rina J Mepani, Adam P Silverman, Mary Haak-Frendscho, Spencer C Wei

引用次数: 0

CRISPR-GRIT: Guide RNAs with Integrated Repair Templates Enable Precise Multiplexed Genome Editing in the Diploid Fungal Pathogen Candida albicans. CRISPR-GRIT：带有集成修复模板的引导 RNA 可对二倍体真菌病原体白色念珠菌进行精确的多重基因组编辑。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2024-12-01 Epub Date: 2024-10-22 DOI: 10.1089/crispr.2024.0052

Christopher J Cotter, Cong T Trinh

{"title":"CRISPR-GRIT: Guide RNAs with Integrated Repair Templates Enable Precise Multiplexed Genome Editing in the Diploid Fungal Pathogen Candida albicans.","authors":"Christopher J Cotter, Cong T Trinh","doi":"10.1089/crispr.2024.0052","DOIUrl":"10.1089/crispr.2024.0052","url":null,"abstract":"Candida albicans, an opportunistic fungal pathogen, causes severe infections in immunocompromised individuals. Limited classes and overuse of current antifungals have led to the rapid emergence of antifungal resistance. Thus, there is an urgent need to understand fungal pathogen genetics to develop new antifungal strategies. Genetic manipulation of C. albicans is encumbered by its diploid chromosomes requiring editing both alleles to elucidate gene function. Although the recent development of CRISPR-Cas systems has facilitated genome editing in C. albicans, large-scale and multiplexed functional genomic studies are still hindered by the necessity of cotransforming repair templates for homozygous knockouts. Here, we present CRISPR-GRIT (Guide RNAs with Integrated Repair Templates), a repair template-integrated guide RNA design for expedited gene knockouts and multiplexed gene editing in C. albicans. We envision that this method can be used for high-throughput library screens and identification of synthetic lethal pairs in both C. albicans and other diploid organisms with strong homologous recombination machinery.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"385-394"},"PeriodicalIF":3.7,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142512851","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

CRISPR-Cas9-Mediated Targeting of Multidrug Resistance Genes in Methicillin-Resistant Staphylococcus aureus. CRISPR-Cas9 介导的耐甲氧西林金黄色葡萄球菌多药耐药性基因靶向。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2024-12-01 Epub Date: 2024-11-08 DOI: 10.1089/crispr.2024.0001

Aysegul Ates, Cihan Tastan, Safak Ermertcan

{"title":"CRISPR-Cas9-Mediated Targeting of Multidrug Resistance Genes in Methicillin-Resistant Staphylococcus aureus.","authors":"Aysegul Ates, Cihan Tastan, Safak Ermertcan","doi":"10.1089/crispr.2024.0001","DOIUrl":"10.1089/crispr.2024.0001","url":null,"abstract":"Antibiotic resistance poses a global health crisis limiting the efficacy of available therapeutic agents. We explored CRISPR-Cas-based antimicrobials to combat multidrug resistance in methicillin-resistant Staphylococcus aureus (MRSA), targeting methicillin (mecA), gentamicin (aacA), and ciprofloxacin (grlA, grlB) resistance genes. Engineered CRISPR plasmids with specific single-guide RNAs were electroporated into MRSA strains. Real-time polymerase chain reaction assessed gene expression changes, while antibiotic susceptibility tests (ASTs) evaluated resistance status. Results showed a 1.5-fold decrease in mecA, a 5.5-fold decrease in grlA, a 6-fold decrease in grlB, and a 4-fold decrease in aacA expression. ASTs demonstrated the reversal of resistance to beta-lactam, quinolone, and aminoglycoside antibiotics. Western blot analysis revealed a 70% decrease in penicillin-binding protein 2a expression. Sanger sequencing confirmed point mutations in the grlB and aacA genes. Our findings highlight the potential of CRISPR-Cas9 technology to restore antibiotic efficacy against multidrug-resistant pathogens.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"374-384"},"PeriodicalIF":3.7,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142607408","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Engineering CjCas9 for Efficient Base Editing and Prime Editing. 对 CjCas9 进行工程改造，以实现高效的碱基编辑和基序编辑。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2024-12-01 Epub Date: 2024-11-18 DOI: 10.1089/crispr.2024.0018

Siyuan Liu, Yingdi Zhao, Qiqin Mo, Yadong Sun, Hanhui Ma

{"title":"Engineering CjCas9 for Efficient Base Editing and Prime Editing.","authors":"Siyuan Liu, Yingdi Zhao, Qiqin Mo, Yadong Sun, Hanhui Ma","doi":"10.1089/crispr.2024.0018","DOIUrl":"10.1089/crispr.2024.0018","url":null,"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 CjCas9P47K. 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 CjCas9P47K 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.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"395-405"},"PeriodicalIF":3.7,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142649684","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Repurposing an Endogenous CRISPR-Cas System to Generate and Study Subtle Mutations in Bacteriophages. 重新利用内源性 CRISPR-Cas 系统，生成并研究噬菌体中的微妙突变。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2024-12-01 Epub Date: 2024-09-30 DOI: 10.1089/crispr.2024.0047

Kotaro Kamata, Nils Birkholz, Marijn Ceelen, Robert D Fagerlund, Simon A Jackson, Peter C Fineran

{"title":"Repurposing an Endogenous CRISPR-Cas System to Generate and Study Subtle Mutations in Bacteriophages.","authors":"Kotaro Kamata, Nils Birkholz, Marijn Ceelen, Robert D Fagerlund, Simon A Jackson, Peter C Fineran","doi":"10.1089/crispr.2024.0047","DOIUrl":"10.1089/crispr.2024.0047","url":null,"abstract":"While bacteriophage applications benefit from effective phage engineering, selecting the desired genotype after subtle modifications remains challenging. Here, we describe a two-phase endogenous CRISPR-Cas-based phage engineering approach that enables selection of small defined edits in Pectobacterium carotovorum phage ZF40. We designed plasmids containing sequences homologous to ZF40 and a mini-CRISPR array. The plasmids allowed genome editing through homologous recombination and counter-selection against non-recombinant phage genomes using an endogenous type I-E CRISPR-Cas system. With this technique, we first deleted target genes and subsequently restored loci with modifications. This two-phase approach circumvented major challenges in subtle phage modifications, including inadequate sequence distinction for CRISPR-Cas counter-selection and the requirement of a protospacer-adjacent motif, limiting sequences that can be modified. Distinct 20-bp barcodes were incorporated through engineering as differential target sites for programmed CRISPR-Cas activity, which allowed quantification of phage variants in mixed populations. This method aids studies and applications that require mixtures of similar phages.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"343-354"},"PeriodicalIF":3.7,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142332259","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Mutation-Specific CRISPR Targeting with SaCas9 and AsCas12a Restores Therapeutic Sensitivity in Treatment-Resistant Melanoma. 用 SaCas9 和 AsCas12a 进行突变特异性 CRISPR 靶向可恢复耐药黑色素瘤的治疗敏感性。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2024-12-01 Epub Date: 2024-10-10 DOI: 10.1089/crispr.2024.0003

Brett M Sansbury, Sophia B Masciarelli, Salma Kaouser, Olivia M Tharp, Kelly H Banas, Eric B Kmiec

{"title":"Mutation-Specific CRISPR Targeting with SaCas9 and AsCas12a Restores Therapeutic Sensitivity in Treatment-Resistant Melanoma.","authors":"Brett M Sansbury, Sophia B Masciarelli, Salma Kaouser, Olivia M Tharp, Kelly H Banas, Eric B Kmiec","doi":"10.1089/crispr.2024.0003","DOIUrl":"10.1089/crispr.2024.0003","url":null,"abstract":"Background: Melanoma remains one of the most challenging cancers to treat effectively with drug resistant remaining a constant concern, primarily with activating BRAF mutations. Mutations in the BRAF gene appear in approximately 50% of patients, 90% of which are V600E. Two frontline BRAF inhibitors (BRAFi), vemurafenib and dabrafenib, are frequently used to treat unresectable or metastatic BRAF V600E melanoma. Initial response rates are high, but soon thereafter, 70-80% of patients develop resistance to treatment within a year. A major mechanism of resistance is the generation of a secondary Q61K mutation in the NRAS gene. Methods: We have developed an approach in which a CRISPR-Cas complex can be designed to distinguish between mutant genes enabling resistance to standard care in tumor cells and normal genomes of healthy cells. For the first time, we demonstrated the utility of two CRISPR-directed mutation-specific editing approaches to restore BRAFi sensitivity in BRAFV600E/NRASQ61K resistant A375 cells. Results: We utilize an AsCas12a protospacer adjacent motif site created by the NRAS Q61K mutation and the Q61K mutation in the critical seed region of an SaCas9 sgRNA for Q61K-selective targeting. We show here that both approaches allow for effective NRAS targeting of only mutated-Q61K and after CRISPR-directed Q61K-targeting, previously resistant A375 cells are re-sensitized to BRAFi treatment. Conclusion: Our data support the feasibility of the development of CRISPR-Cas therapeutic approaches to the treatment of melanoma. Successful therapeutic CRISPR-directed gene editing would enable both specific and efficient editing of a mutation-specific targeting approach eliminate concern for on- and off-target damage to the genomes of healthy cells.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":" ","pages":"366-373"},"PeriodicalIF":3.7,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142480624","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Engineering of HEK293T Cell Factory for Lentiviral Production by High-Throughput Selected Genes. 通过高通量选择基因对 HEK293T 细胞工厂进行工程改造，以生产慢病毒。

IF 3.7 4区生物学

CRISPR Journal Pub Date : 2024-10-01 Epub Date: 2024-10-16 DOI: 10.1089/crispr.2024.0016

Zhang Xinyue, Siwei Li, Wang Yujie, Dai Yingcai, Bi Changhao, Zhang Xueli

{"title":"Engineering of HEK293T Cell Factory for Lentiviral Production by High-Throughput Selected Genes.","authors":"Zhang Xinyue, Siwei Li, Wang Yujie, Dai Yingcai, Bi Changhao, Zhang Xueli","doi":"10.1089/crispr.2024.0016","DOIUrl":"10.1089/crispr.2024.0016","url":null,"abstract":"Lentiviral vectors (LVs) are crucial tools in gene therapy and bioproduction, but high-yield LV production systems are urgently needed. Using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 high-throughput screening, we identified nine critical genes (LDAH, GBP3, BPIFC, NHLRC1, NHLRC3, ZNF425, TTC37, LRRC4B, and SPINK6) from 17,501 genes that limit LV packaging and formation. Knocking out these genes in HEK293T cells significantly increased virus production, with LDAH knockout exhibiting a 6.63-fold increase. Studies on multigene knockouts demonstrated that the cumulative effects of different gene knockouts can significantly enhance lentivirus production in HEK293T cells. Triple knockout of GBP3, BPIFC, and LDAH increased LV titer by ∼8.33-fold, and knockout (or knockdown) of GBP3, NHLRC1, and NHLRC3 increased LV titer by ∼6.53-fold. This study established HEK293T cell lines with multiple genes knockout for efficient LV production, providing reliable technical support for LV production and application and offering new perspectives for studying LV packaging mechanisms and related virus research.","PeriodicalId":54232,"journal":{"name":"CRISPR Journal","volume":"7 5","pages":"272-282"},"PeriodicalIF":3.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142480626","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0