RT Journal Article
SR Electronic
T1 Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 231035
DO 10.1101/231035
A1 Eric J. Aird
A1 Klaus N. Lovendahl
A1 Amber St. Martin
A1 Reuben S. Harris
A1 Wendy R. Gordon
YR 2017
UL http://biorxiv.org/content/early/2017/12/08/231035.abstract
AB The CRISPR-Cas9 system is a powerful genome-editing tool in which a guide RNA targets Cas9 to a site in the genome where the Cas9 nuclease then induces a double stranded break (DSB)1,2. The potential of CRISPR-Cas9 to deliver precise genome editing is hindered by the low efficiency of homology-directed repair (HDR), which is required to incorporate a donor DNA template encoding desired genome edits near the DSB3,4. We present a strategy to enhance HDR efficiency by covalently tethering a single-stranded donor oligonucleotide (ssODN) to the Cas9/guide RNA ribonucleoprotein (RNP) complex via a fused HUH endonuclease5, thus spatially and temporally co-localizing the DSB machinery and donor DNA. We demonstrate up to an 8-fold enhancement of HDR using several editing assays, including repair of a frameshift and in-frame insertions of protein tags. The improved HDR efficiency is observed in multiple cell types and target loci, and is more pronounced at low RNP concentrations.