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Elife. 2014 Dec 15;3:e04766. doi: 10.7554/eLife.04766.

Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery.

Author information

1
Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States.
2
Computational Genomics Resource Laboratory, QB3, University of California, Berkeley, Berkeley, United States.

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.

KEYWORDS:

CRISPR/Cas9; cell biology; cell cycle synchronization; chromosomes; genes; genome engineering; homologous recombination; human; nocodazole; non-homologous end joining

PMID:
25497837
PMCID:
PMC4383097
DOI:
10.7554/eLife.04766
[Indexed for MEDLINE]
Free PMC Article

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