Letter

Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR–Cas9 genome-editing efficiency

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Abstract

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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Change history

  • Corrected online 14 December 2017

    In the version of this article initially published, the affiliation footnotes of Andrew Vorobyov and Sachdev S Sidhu were erroneously given as number 4, the Skirball Institute of Biomolecular Medicine, instead of number 5, The Donnelly Centre for Cellular and Biomolecular Research. The errors have been corrected for the print, PDF and HTML versions of this article.

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Primary accessions

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Acknowledgements

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.).

Author information

Author notes

    • Amélie Fradet-Turcotte
    • , Alexandre Orthwein
    •  & Andreas Ernst

    Present addresses: St-Patrick Research Group in Basic Oncology, Université Laval Cancer Research Center, and Oncology Axis-CHU de Québec Research Center – Université Laval, Quebec City, Quebec, Canada (A.F.-T.); Department of Oncology, McGill University, Montreal, Quebec, Canada and Segal Cancer Center, Jewish General Hospital, Lady Davis Institute for Medical Research, Montreal, Quebec, Canada (A.O.); and Institute of Biochemistry II, Goethe University School of Medicine, Frankfurt am Main, Germany (A.E.).

    • Marella D Canny
    •  & Nathalie Moatti

    These authors contributed equally to this work.

Affiliations

  1. The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.

    • Marella D Canny
    • , Nathalie Moatti
    • , Leo C K Wan
    • , Amélie Fradet-Turcotte
    • , Michal Zimmermann
    • , Alexandre Orthwein
    • , Yu-Chi Juang
    • , Sylvie M Noordermeer
    • , Marcus D Wilson
    • , Meagan Munro
    • , Timothy F Ng
    • , Tiffany Cho
    • , Frank Sicheri
    •  & Daniel Durocher
  2. Department of Molecular Genetics, University of Toronto, Ontario, Canada.

    • Leo C K Wan
    • , Timothy F Ng
    • , Tiffany Cho
    • , Sachdev S Sidhu
    • , Frank Sicheri
    •  & Daniel Durocher
  3. Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.

    • Danielle Krasner
    • , Eduardo Seclen
    •  & Paula M Cannon
  4. Skirball Institute of Biomolecular Medicine, Department of Cell Biology, NYU Langone Medical Center, New York, New York, USA.

    • Pedro A Mateos-Gomez
  5. The Donnelly Centre for Cellular and Biomolecular Research, Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.

    • Wei Zhang
    • , Andrew Vorobyov
    • , Andreas Ernst
    •  & Sachdev S Sidhu

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Contributions

M.D.C. generated the mutations for the analysis by pull-down and carried out most of the pull-down experiments, generated the protein crystals, conducted the ITC experiments and carried out some of the initial assays demonstrating efficacy of i53. N.M. carried out the DR- and EJ2-GFP assays, all assays with AAV-i53 in human cells along with all ssODN assays and the determination of 53BP1 foci in G1 cells. L.C.K.W. refined and analyzed the crystal structure and prepared the figures describing the structure. A.F.-T. produced the GST-53BP1 protein for Ubv selection and the GST-fusion Ubv proteins for the initial pull-down assays she carried out. Y.-C.J. supervised the crystallization and collected the diffraction data. A.O. carried out the LMNA gene targeting assays with the help of T.F.N. W.Z. carried out the Ubv selections with A.V. and A.E. D.K. and E.S. conducted HDR analyses in K562 cells. T.C. assessed the impact of i53 on p53 to answer a reviewer comment whereas M.Z. tested the impact of i53 of SCE rates. P.M.C. supervised D.K. and E.S. P.A.M.-G. performed mouse gene targeting assay. S.M.N. carried out the PARP inhibitor assays using cells generated by M.Z. M.D.W. helped with biochemical experiments. M.M. did the immunoprecipitation mass spectrometry and helped M.D.C., A.F.-T. and A.O. S.S.S. supervised W.Z., A.V. and A.E. F.S. supervised L.C.K.W. and Y.-C.J. D.D. conceived and supervised the project, obtained funding and wrote the manuscript with L.C.K.W., M.D.C. and F.S., with input from the other authors.

Competing interests

The use of 53BP1-blocking Ubvs for stimulating genome editing are the subject of a patent application and a license agreement.

Corresponding author

Correspondence to Daniel Durocher.

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