Article

Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR–Cas9 genome editing

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Abstract

Gene disruption by clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) is highly efficient and relies on the error-prone non-homologous end-joining pathway. Conversely, precise gene editing requires homology-directed repair (HDR), which occurs at a lower frequency than non-homologous end-joining in mammalian cells. Here, by testing whether manipulation of DNA repair factors improves HDR efficacy, we show that transient ectopic co-expression of RAD52 and a dominant-negative form of tumour protein p53-binding protein 1 (dn53BP1) synergize to enable efficient HDR using a single-stranded oligonucleotide DNA donor template at multiple loci in human cells, including patient-derived induced pluripotent stem cells. Co-expression of RAD52 and dn53BP1 improves multiplexed HDR-mediated editing, whereas expression of RAD52 alone enhances HDR with Cas9 nickase. Our data show that the frequency of non-homologous end-joining-mediated double-strand break repair in the presence of these two factors is not suppressed and suggest that dn53BP1 competitively antagonizes 53BP1 to augment HDR in combination with RAD52. Importantly, co-expression of RAD52 and dn53BP1 does not alter Cas9 off-target activity. These findings support the use of RAD52 and dn53BP1 co-expression to overcome bottlenecks that limit HDR in precision genome editing.

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Acknowledgements

This work was supported in part by National Institutes of Health grants R01AI020047 and R01AI077595 (to F.W.A.) and RO1HL107630, HL107440, UC4DK104218 and U19HL129903 (to D.J.R.), the Translational Research Program (Boston Children’s Hospital), Pedals for Pediatrics (Dana-Farber Cancer Institute) awards (to S.A. and B.B.), The Leona M. and Harry B. Helmsley Charitable Trust (to D.J.R.) and the New York Stem Cell Foundation (to D.J.R.). The HEK293 broken-GFP reporter cell line was kindly provided by G. Church. The gRNA constructs targeting B2M were provided by C. Cowan.

Author information

Author notes

  1. Bruna S. Paulsen and Pankaj K. Mandal contributed equally to this work.

Affiliations

  1. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, 02138, USA

    • Bruna S. Paulsen
    • , Pankaj K. Mandal
    • , Paula Gutierrez-Martinez
    • , Wataru Ebina
    •  & Derrick J. Rossi
  2. Program in Cellular and Molecular Medicine at Boston Children’s Hospital, Boston, MA, 02115, USA

    • Bruna S. Paulsen
    • , Pankaj K. Mandal
    • , Richard L. Frock
    • , Srigokul Upadhyayula
    • , Paula Gutierrez-Martinez
    • , Wataru Ebina
    • , Tomas Kirchhausen
    • , Frederick W. Alt
    •  & Derrick J. Rossi
  3. Department of Pediatrics, Harvard Medical School, Boston, MA, 02115, USA

    • Pankaj K. Mandal
    • , Srigokul Upadhyayula
    • , Tomas Kirchhausen
    • , Suneet Agarwal
    •  & Derrick J. Rossi
  4. Department of Genetics, Harvard Medical School, Boston, MA, 02115, USA

    • Richard L. Frock
    •  & Frederick W. Alt
  5. Division of Hematology/Oncology, Boston Children’s Hospital, Boston, MA, 02115, USA

    • Baris Boyraz
    •  & Suneet Agarwal
  6. Department of Basic Oncology, Hacettepe University Cancer Institute, Ankara, Turkey

    • Baris Boyraz
  7. Molecular Neurogenetics Unit, Psychiatric and Neurodevelopmental Genetics Unit, Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA

    • Rachita Yadav
    •  & Michael E. Talkowski
  8. Broad Institute, Cambridge, MA, 02142, USA

    • Rachita Yadav
    •  & Michael E. Talkowski
  9. Department of Neurology, Harvard Medical School, Boston, MA, 02115, USA

    • Rachita Yadav
    •  & Michael E. Talkowski
  10. Department of Cell Biology, Harvard Medical School, Boston, MA, 02115, USA

    • Srigokul Upadhyayula
    •  & Tomas Kirchhausen
  11. Department of Pediatrics, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden

    • Anders Fasth
  12. Stem Cell Program, Boston Children’s Hospital, Boston, MA, 02115, USA

    • Suneet Agarwal
  13. Harvard Stem Cell Institute, Cambridge, MA, 02138, USA

    • Suneet Agarwal
    •  & Derrick J. Rossi
  14. The Howard Hughes Medical Institute, Boston Children’s Hospital, Boston, MA, 02115, USA

    • Frederick W. Alt

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Contributions

B.S.P., P.K.M. and D.J.R. designed the experiments. B.S.P. and P.K.M. performed the experiments. P.K.M., R.L.F. and F.W.A. designed and performed the HTGTS experiments. B.S.P., B.B., A.F. and S.A. designed and performed the human DKC1 iPS cell line experiments. B.S.P., P.G.-M. and W.E. designed and performed the experiments for the selection of the candidate factors. P.K.M., R.Y. and M.E.T. designed and performed the capture deep sequencing experiments. S.U. and T.K. performed the image analyses. All authors were involved in data analysis. B.S.P., P.K.M and D.J.R. wrote the manuscript.

Competing interests

D.J.R. is an academic co-founder of Intellia Therapeutics (Cambridge, MA), a biotechnology company focused on developing CRISPR–Cas9 therapies.

Corresponding author

Correspondence to Derrick J. Rossi.

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