Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery.
Elife
; 3: e04766, 2014 Dec 15.
Article
en En
| MEDLINE
| ID: mdl-25497837
ABSTRACT
The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ADN
/
Genoma Humano
/
ARN Guía de Kinetoplastida
/
Endonucleasas
/
Reparación del ADN por Recombinación
/
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas
Límite:
Humans
/
Newborn
Idioma:
En
Revista:
Elife
Año:
2014
Tipo del documento:
Article
País de afiliación:
Estados Unidos