Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR)1,2,3. However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ)4. We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells4,5 and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants6 for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells, blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.
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We are grateful to R. Szilard for critical reading of the manuscript. We thank J. Stark (City of Hope) for the DR- and EJ2-GFP U2OS cell lines, G. Dellaire (Dalhousie University) for the LMNA assay plasmids and Y. Doyon (Université Laval) for the HIST1H2BK-mAG targeting vector. The Lenti-Cas9-2A-Blast construct was a gift from J. Moffat (University of Toronto). Clone 12CA5 was gift from M. Tyers, University of Montréal). Goat anti-GFP was a gift from L. Pelletier, Lunenfeld-Tanenbaum Research Institute. A.F.-T. was a CIHR post-doctoral fellow and A.O. was a recipient of the Terry Fox Foundation Strategic Initiative for Excellence in Radiation Research for the 21st Century at CIHR fellowship. P.A.M.-G. received a Breast Cancer postdoctoral fellowship award from the US Department of Defense (BC134020). M.D.W. held a long-term fellowship from the Human Frontier Science Program. S.M.N. receives a postdoctoral fellowship from the Dutch Cancer Society (KWF). D.D. is the Thomas Kierans Chair in Mechanisms of Cancer Development and a Canada Research Chair (Tier 1) in the Molecular Mechanisms of Genome Integrity. Work was supported by NIH grant U19 HL129902 to PMC, CIHR grants MOP111149 and MOP136956 (to S.S.S.), FDN143277 (to F.S.) and FDN143343 (to D.D.) anda Grant-in-Aid from the Krembil Foundation (to D.D.).
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Scientific Reports (2018)