Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq

Genome editing using nucleases such as CRISPR-Cas induces programmable DNA damage at a target genomic site but can also affect off-target sites. Here, we develop a powerful, sensitive assay for the unbiased identification of off-target sites that we term DISCOVER-Seq. This approach takes advantage of the recruitment of endogenous DNA repair factors for genome-wide identification of Cas-induced double-strand breaks. One such factor, MRE11, is recruited precisely to double-strand breaks, enabling molecular characterization of nuclease cut sites with single-base resolution. DISCOVER-Seq detects off-targets in cellular models and in vivo upon adenoviral gene editing of mouse livers, paving the way for real-time off-target discovery during therapeutic gene editing. DISCOVER-Seq is furthermore applicable to multiple types of Cas nucleases and provides an unprecedented view of events that precede repair of the affected sites.