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.
Bibliographical noteFunding Information:
E.J.A. and W.R.G. designed the experiments, E.J.A. and A.S. performed the experiments and analysis, E.J.A. and K.N.L. designed and constructed fusion constructs, A.S. and R.S. H. designed and created reporter cell line, and E.J.A., K.N.L., and W.R.G. assembled the manuscript with contributions from all authors. Funding: This study was supported by an NIH NIGMS R35 GM119483 grant to W.R.G. and NIGMS R01 GM118000, NIAID R37 AI064046, and NCI R21 CA206309 to R.S.H. E.J.A. received salary support from 3M Graduate Fellowship and A.S. from NSF-GRFP 00039202. W.R.G. is a Pew Biomedical Scholar. R.S.H. is the Margaret Harvey Schering Land Grant Chair for Cancer Research, a Distinguished McKnight University Professor, and an Investigator of the Howard Hughes Medical Institute.
© 2018, The Author(s).
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