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

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.