Homo sapiens Raw sequence reads
While CRISPR-based editing most often occurs at DNA sequences with perfect homology to the guide RNA (gRNA), unintended editing can occur at highly homologous regions (i.e., off-target (OT) sites). Due to the pace at which genome editing therapies are approaching clinical applications, there is an emerging needto define effective workflows for investigating OT editing effects. A number of homology-dependent, in silico-based prediction methods and wet lab-based empirical methods exist to investigate OT editing, but few have been subjected to analytical assessment or head-to-head comparison in human primary cellsusing an ex vivo editing process optimized for high-fidelity gene editing. Therefore, we sought to compare publicly available in silico tools (COSMID, CCTop, and Cas-OFFinder) as well as empirical methods (CHANGE-Seq, CIRCLE-Seq, DISCOVER-Seq, GUIDE-Seq, and SITE-Seq) in the context of ex vivo hematopoietic stem and progenitor cell (HSPC) editing. To do so, we edited CD34+ HSPCs using 11 differentguide RNAs (gRNAs) complexed with HiFi Cas9, then performed targeted next-generation sequencing of ~200-site panels containing a range of nominated OT sites identified by in silico and empirical methods.