Harnessing type I CRISPR-Cas systems for genome engineering in human cells.
Nat Biotechnol
; 37(12): 1471-1477, 2019 12.
Article
en En
| MEDLINE
| ID: mdl-31740839
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Sistemas CRISPR-Cas
/
Edición Génica
Límite:
Humans
Idioma:
En
Revista:
Nat Biotechnol
Asunto de la revista:
BIOTECNOLOGIA
Año:
2019
Tipo del documento:
Article
País de afiliación:
Estados Unidos