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Commun Biol. 2018 May 31;1:54. doi: 10.1038/s42003-018-0054-2. eCollection 2018.

Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template.

Author information

1Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455 USA.
2Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55108 USA.
3Masonic Cancer Center, Institute for Molecular Virology, University of Minnesota, Minneapolis, MN 55455 USA.
4Howard Hughes Medical Institute, University of Minnesota, Minneapolis, MN 55455 USA.


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). 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 DSB. We present a strategy to enhance HDR efficiency by covalently tethering a single-stranded oligodeoxynucleotide (ssODN) to the Cas9-guide RNA ribonucleoprotein (RNP) complex via a fused HUH endonuclease, thus spatially and temporally co-localizing the DSB machinery and donor DNA. We demonstrate up to a 30-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.

Conflict of interest statement

R.S.H. is a co-founder, shareholder, and consultant of ApoGen Biotechnologies Inc. W.R.G., E.J.A. and K.N.L. have filed a provisional patent on this technology. The remaining authors declare no competing interests.

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