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

Peter Cameron, Mary M. Coons, Sanne E. Klompe, Alexandra M. Lied, Stephen C. Smith, Bastien Vidal, Rachel Kennedy, Stan J.J. Brouns, More Authors

Research output: Contribution to journalLetterScientificpeer-review

11 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)1471-1477
Number of pages7
JournalNature Biotechnology
Volume37
Issue number12
DOIs
Publication statusPublished - 2019

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