Mapping the Genomic Landscape of CRISPR-Cas9 Cleavage

CRISPR RNA-guided Cas9 endonucleases are widely used in genome engineering applications. Despite the development of methods to monitor nuclease specificity, there remains a lack of information on the possible sequences that can be cleaved by Cas9 and under what conditions these sequences accumulate off-target mutations in cells. To address this, we developed a biochemical method, based on the selective enrichment and identification of adapter-tagged DNA ends by sequencing (SITE-Seq), to generate a comprehensive list of sites cleaved by Cas9 within genomic DNA for a given guide RNA. This comprehensive list of sites can then be used to monitor the frequency of off-target mutations in cell-based experiments and to elucidate the factors (Cas9/sgRNA delivery, duration of expression, cell type, etc.) that influence the accumulation of off-target mutations. Importantly, SITE-Seq recovered nearly every off-target site that was identified for specific guide RNAs using alternative methods, as well as additional sites that were not previously identified. Collectively, our results help define a relationship between biochemical and cellular DNA cleavage, and underscore the utility of SITE-Seq as an unbiased approach to map the full spectrum of genomic off-target sites.