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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. Access through your institution Buy or subscribe This is a preview of subscription content, access via your

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institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS MULTI-PATHWAY DNA-REPAIR REPORTERS REVEAL COMPETITION BETWEEN END-JOINING,

SINGLE-STRAND ANNEALING AND HOMOLOGOUS RECOMBINATION AT CAS9-INDUCED DNA DOUBLE-STRAND BREAKS Article Open access 08 September 2022 PREVALENT INTEGRATION OF GENOMIC REPETITIVE AND REGULATORY

ELEMENTS AND DONOR SEQUENCES AT CRISPR-CAS9-INDUCED BREAKS Article Open access 20 January 2025 TARGETING DOUBLE-STRAND BREAK INDEL BYPRODUCTS WITH SECONDARY GUIDE RNAS IMPROVES CAS9

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Article  CAS  PubMed  PubMed Central  Google Scholar  Download references 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 * Bruna S. Paulsen and Pankaj K. Mandal contributed

equally to this work. AUTHORS AND AFFILIATIONS * 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 * 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 * Department of Pediatrics, Harvard Medical

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

02115, USA Richard L. Frock & Frederick W. Alt * Division of Hematology/Oncology, Boston Children’s Hospital, Boston, MA, 02115, USA Baris Boyraz & Suneet Agarwal * Department of

Basic Oncology, Hacettepe University Cancer Institute, Ankara, Turkey Baris Boyraz * Molecular Neurogenetics Unit, Psychiatric and Neurodevelopmental Genetics Unit, Center for Genomic

Medicine, Massachusetts General Hospital, Boston, MA, 02114, USA Rachita Yadav & Michael E. Talkowski * Broad Institute, Cambridge, MA, 02142, USA Rachita Yadav & Michael E.

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

02115, USA Srigokul Upadhyayula & Tomas Kirchhausen * Department of Pediatrics, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden Anders Fasth * Stem Cell Program,

Boston Children’s Hospital, Boston, MA, 02115, USA Suneet Agarwal * Harvard Stem Cell Institute, Cambridge, MA, 02138, USA Suneet Agarwal & Derrick J. Rossi * The Howard Hughes Medical

Institute, Boston Children’s Hospital, Boston, MA, 02115, USA Frederick W. Alt Authors * Bruna S. Paulsen View author publications You can also search for this author inPubMed Google Scholar

* Pankaj K. Mandal View author publications You can also search for this author inPubMed Google Scholar * Richard L. Frock View author publications You can also search for this author

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publications You can also search for this author inPubMed Google Scholar * Wataru Ebina View author publications You can also search for this author inPubMed Google Scholar * Anders Fasth

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* Michael E. Talkowski View author publications You can also search for this author inPubMed Google Scholar * Suneet Agarwal View author publications You can also search for this author

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search for this author inPubMed Google Scholar 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. CORRESPONDING AUTHOR Correspondence to Derrick J. Rossi. ETHICS DECLARATIONS COMPETING INTERESTS

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

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ARTICLE 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 Download citation * Received: 08 December 2016 * Accepted: 13 September 2017 * Published: 09 October 2017 * Issue Date: November

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