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

Eric J Aird; Klaus N Lovendahl; Amber St Martin; Reuben S Harris; Wendy R Gordon

doi:10.1038/s42003-018-0054-2

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

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

Authors

Eric J Aird^{1

2}, Klaus N Lovendahl^{1

2}, Amber St Martin^{1

2

3}, Reuben S Harris^{1

2

3

4}, Wendy R Gordon^{5

6}

Affiliations

¹ Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, 55455, USA.
² Center for Genome Engineering, University of Minnesota, Minneapolis, MN, 55108, USA.
³ Masonic Cancer Center, Institute for Molecular Virology, University of Minnesota, Minneapolis, MN, 55455, USA.
⁴ Howard Hughes Medical Institute, University of Minnesota, Minneapolis, MN, 55455, USA.
⁵ Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, 55455, USA. [email protected].
⁶ Center for Genome Engineering, University of Minnesota, Minneapolis, MN, 55108, USA. [email protected].

Abstract

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.

Abstract

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