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

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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.

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Steven Lin

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

Competing interests
The authors declare that no competing interests exist.
Brett Staahl

Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, United States

Competing interests
The authors declare that no competing interests exist.
Ravi K Alla

Computational Genomics Resource Lab, QB3, University of California, Berkeley, Berkeley, United States

Competing interests
The authors declare that no competing interests exist.
Jennifer A Doudna

Department of Molecular and Cell Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States

For correspondence
doudna@berkeley.edu

Competing interests
The authors declare that no competing interests exist.

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This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.