Harnessing type I CRISPR–Cas systems for genome engineering in human cells

Type I CRISPR–Cas systems are the most abundant adaptive immune systems in bacteria and archaea^1,2. Target interference relies on a multi-subunit, RNA-guided complex called Cascade^3,4, which recruits a trans-acting helicase-nuclease, Cas3, for target degradation^5–7. 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 domain^8–11 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.