689. Defining Genome-Wide Off-Target Cleavage Profiles of CRISPR-Cas RNA-Guided Nucleases Using GUIDE-Seq

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) RNA-guided nucleases (RGNs) have been broadly adopted by the scientific community for use as robust genome editing tools. However, CRISPR-Cas RGNs have been demonstrated to exhibit high-frequency off-targets at sites with up to 5 mismatches from the intended target site, raising questions about their global specificity. We have developed a novel method called GUIDE-seq (for Genome-wide Unbiased Identification of DSBs enabled by Sequencing), based on the efficient integration of a double-stranded oligodeoxynucleotide (dsODN) tag followed by tag-specific amplification and high-throughput sequencing. We performed GUIDE-seq on 10 RGNs in 2 human cell lines and identified all known off-target cleavage sites of these RGNs as well as hundreds of additional novel sites. The number of off-target cleavage sites identified varied widely among these RGNs, and the majority of identified sites were not predicted by existing computational methods or ChIP-seq. GUIDE-seq provides a robust method for evaluating the genome-wide specificities of RGNs that will be useful for the clinical translation of these important and widely applicable genome editing reagents.