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

Nature Biomedical Engineering volume 1, pages 878–888 (2017)Cite this article

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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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Fig. 1: Ectopic expression of RAD52 and dn53BP1 increases HDR efficiency.
Fig. 2: Co-transfection of RAD52 and dn53BP1 does not affect NHEJ frequency.
Fig. 3: Co-expression of RAD52 and dn53BP1 improves HDR frequency at multiple loci in human cells.
Fig. 4: Co-expression of RAD52 and dn53BP1 improves precise genomic editing in patient-specific iPS cells.
Fig. 5: Effect of RAD52 and dn53BP1 on microhomology distribution at the target site.
Fig. 6: dn53BP1 competitively antagonizes 53BP1 to improve HDR efficiency.

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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.

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Author notes

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

Authors and 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.

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Correspondence to Derrick J. Rossi.

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Competing interests

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

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Paulsen, B.S., Mandal, P.K., Frock, R.L. et al. Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR–Cas9 genome editing. Nat Biomed Eng 1, 878–888 (2017). https://doi.org/10.1038/s41551-017-0145-2

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