Comparative analysis of CRISPR off-target discovery tools following ex vivo editing of CD34⁺ hematopoietic stem and progenitor cells.

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 O

SUBMITTER: Cromer MK 

PROVIDER: S-EPMC10124080 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature