Incorporation of bridged nucleic acids into CRISPR RNAs improves Cas9 endonuclease specificity.
Nat Commun
; 9(1): 1448, 2018 04 13.
Article
en En
| MEDLINE
| ID: mdl-29654299
ABSTRACT
Off-target DNA cleavage is a paramount concern when applying CRISPR-Cas9 gene-editing technology to functional genetics and human therapeutic applications. Here, we show that incorporation of next-generation bridged nucleic acids (2',4'-BNANC[N-Me]) as well as locked nucleic acids (LNA) at specific locations in CRISPR-RNAs (crRNAs) broadly reduces off-target DNA cleavage by Cas9 in vitro and in cells by several orders of magnitude. Using single-molecule FRET experiments we show that BNANC incorporation slows Cas9 kinetics and improves specificity by inducing a highly dynamic crRNA-DNA duplex for off-target sequences, which shortens dwell time in the cleavage-competent, "zipped" conformation. In addition to describing a robust technique for improving the precision of CRISPR/Cas9-based gene editing, this study illuminates an application of synthetic nucleic acids.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ADN
/
ARN
/
ARN Guía de Kinetoplastida
/
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas
/
Sistemas CRISPR-Cas
Tipo de estudio:
Diagnostic_studies
Límite:
Humans
Idioma:
En
Revista:
Nat Commun
Asunto de la revista:
BIOLOGIA
/
CIENCIA
Año:
2018
Tipo del documento:
Article
País de afiliación:
Canadá