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

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.
Competing Interests: Declaration of interests The authors of this study also wish to declare the following conflicts of interest: M.H.P. is on the Board of Directors of Graphite Bio. M.H.P. serves on the SAB of Allogene Tx and is an advisor to Versant Ventures. M.H.P. and M.K.C. have equity in Graphite Bio. M.H.P. has equity in CRISPR Tx. G.R.R., K.M., G.K., C.A.V., and M.A.B .are employees of IDT, Inc.
(Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)