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SITE-Seq probes Cas9 cleavage sites in vitro and returns a comprehensive list of off-target sites at different Cas9–sgRNA concentrations. RNA-guided CRISPR–Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9–sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.

Type I CRISPR–Cas systems are the most abundant adaptive immune systems in bacteria and archaea1,2. Target interference relies on a multi-subunit, RNA-guided complex called Cascade3,4, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation5–7. Type I systems have rarely been used for eukaryotic genome engineering applications owing to the relative difficulty of heterologous expression of the multicomponent Cascade complex. Here, we fuse Cascade to the dimerization-dependent, non-specific FokI nuclease domain8–11 and achieve RNA-guided gene editing in multiple human cell lines with high specificity and efficiencies of up to ~50%. FokI–Cascade can be reconstituted via an optimized two-component expression system encoding the CRISPR-associated (Cas) proteins on a single polycistronic vector and the guide RNA (gRNA) on a separate plasmid. Expression of the full Cascade–Cas3 complex in human cells resulted in targeted deletions of up to ~200 kb in length. Our work demonstrates that highly abundant, previously untapped type I CRISPR–Cas systems can be harnessed for genome engineering applications in eukaryotic cells.