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• W4320921285 abstract "While a number of methods exist to investigate CRISPR off-target (OT) editing, few have been compared head-to-head in primary cells after clinically relevant editing processes. Therefore, we compared in silico tools (COSMID, CCTop, and Cas-OFFinder) and empirical methods (CHANGE-Seq, CIRCLE-Seq, DISCOVER-Seq, GUIDE-Seq, and SITE-Seq) after ex vivo hematopoietic stem and progenitor cell (HSPC) editing. We performed editing using 11 different gRNAs complexed with Cas9 protein (high-fidelity [HiFi] or wild-type versions), then performed targeted next-generation sequencing of nominated OT sites identified by in silico and empirical methods. We identified an average of less than one OT site per guide RNA (gRNA) and all OT sites generated using HiFi Cas9 and a 20-nt gRNA were identified by all OT detection methods with the exception of SITE-seq. This resulted in high sensitivity for the majority of OT nomination tools and COSMID, DISCOVER-Seq, and GUIDE-Seq attained the highest positive predictive value (PPV). We found that empirical methods did not identify OT sites that were not also identified by bioinformatic methods. This study supports that refined bioinformatic algorithms could be developed that maintain both high sensitivity and PPV, thereby enabling more efficient identification of potential OT sites without compromising a thorough examination for any given gRNA." @default.
• W4320921285 created "2023-02-16" @default.
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• W4320921285 date "2023-04-01" @default.
• W4320921285 modified "2023-10-16" @default.
• W4320921285 title "Comparative analysis of CRISPR off-target discovery tools following ex vivo editing of CD34+ hematopoietic stem and progenitor cells" @default.
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• W4320921285 doi "https://doi.org/10.1016/j.ymthe.2023.02.011" @default.
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