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

Eric J. Aird, Klaus N. Lovendahl, Amber St Martin, Reuben Harris, Wendy R Gordon

Research output: Contribution to journalArticle

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.
Original languageEnglish (US)
Article number54
JournalCommunications Biology- Nature
Volume1
DOIs
StatePublished - 2018

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Clustered Regularly Interspaced Short Palindromic Repeats
Guide RNA
Ribonucleoproteins
DNA Repair
Genome
Oligodeoxyribonucleotides
Endonucleases
DNA
Proteins
Gene Editing

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Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template. / Aird, Eric J.; Lovendahl, Klaus N.; St Martin, Amber; Harris, Reuben; Gordon, Wendy R.

In: Communications Biology- Nature, Vol. 1, 54, 2018.

Research output: Contribution to journalArticle

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