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, Cas(3), 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 similar 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 similar 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.