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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. A type I CRISPR–Cas system, rather than the conventional type II CRISPR systems, is adapted for gene editing by fusing Cascade to the FokI nuclease domain.

Summary The off-target activity of the CRISPR-associated nuclease Cas9 is a potential concern for therapeutic genome editing applications. Although high-fidelity Cas9 variants have been engineered, they exhibit varying efficiencies and have residual off-target effects, limiting their applicability. Here, we show that CRISPR hybrid RNA-DNA (chRDNA) guides provide an effective approach to increase Cas9 specificity while preserving on-target editing activity. Across multiple genomic targets in primary human T cells, we show that 2′-deoxynucleotide (dnt) positioning affects guide activity and specificity in a target-dependent manner and that this can be used to engineer chRDNA guides with substantially reduced off-target effects. Crystal structures of DNA-bound Cas9-chRDNA complexes reveal distorted guide-target duplex geometry and allosteric modulation of Cas9 conformation. These structural effects increase specificity by perturbing DNA hybridization and modulating Cas9 activation kinetics to disfavor binding and cleavage of off-target substrates. Overall, these results pave the way for utilizing customized chRDNAs in clinical applications.