Enhanced proofreading governs CRISPR-Cas9 targeting accuracy.
Nature
; 550(7676): 407-410, 2017 10 19.
Article
en En
| MEDLINE
| ID: mdl-28931002
ABSTRACT
The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity are unknown. Here, using single-molecule Förster resonance energy transfer experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we design a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Streptococcus pyogenes
/
Mutagénesis
/
Proteínas Asociadas a CRISPR
/
Sistemas CRISPR-Cas
/
Edición Génica
Límite:
Humans
Idioma:
En
Revista:
Nature
Año:
2017
Tipo del documento:
Article
País de afiliación:
Estados Unidos