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CRISPR-Cas9 fusion to dominant-negative 53BP1 enhances HDR and inhibits NHEJ specifically at Cas9 target sites

Author

Listed:
  • Rajeswari Jayavaradhan

    (Cancer and Blood Diseases Institute (CBDI), Cincinnati Children’s Hospital Medical Center (CCHMC))

  • Devin M. Pillis

    (Cancer and Blood Diseases Institute (CBDI), Cincinnati Children’s Hospital Medical Center (CCHMC))

  • Michael Goodman

    (TriHealth)

  • Fan Zhang

    (Cancer and Blood Diseases Institute (CBDI), Cincinnati Children’s Hospital Medical Center (CCHMC))

  • Yue Zhang

    (Cancer and Blood Diseases Institute (CBDI), Cincinnati Children’s Hospital Medical Center (CCHMC))

  • Paul R. Andreassen

    (Cancer and Blood Diseases Institute (CBDI), Cincinnati Children’s Hospital Medical Center (CCHMC)
    University of Cincinnati College of Medicine)

  • Punam Malik

    (Cancer and Blood Diseases Institute (CBDI), Cincinnati Children’s Hospital Medical Center (CCHMC)
    CBDI, CCHMC
    University of Cincinnati College of Medicine)

Abstract

Precise genome editing/correction of DNA double-strand breaks (DSBs) induced by CRISPR-Cas9 by homology-dependent repair (HDR) is limited by the competing error-prone non-homologous end-joining (NHEJ) DNA repair pathway. Here, we define a safer and efficient system that promotes HDR-based precise genome editing, while reducing NHEJ locally, only at CRISPR-Cas9-induced DSBs. We fused a dominant-negative mutant of 53BP1, DN1S, to Cas9 nucleases, and the resulting Cas9-DN1S fusion proteins significantly block NHEJ events specifically at Cas9 cut sites and improve HDR frequency; HDR frequency reached 86% in K562 cells. Cas9-DN1S protein maintains this effect in different human cell types, including leukocyte adhesion deficiency (LAD) patient-derived immortalized B lymphocytes, where nearly 70% of alleles were repaired by HDR and 7% by NHEJ. Our CRISPR-Cas9-DN1S system is clinically relevant to improve the efficiencies of precise gene correction/insertion, significantly reducing error-prone NHEJ events at the nuclease cleavage site, while avoiding the unwanted effects of global NHEJ inhibition.

Suggested Citation

  • Rajeswari Jayavaradhan & Devin M. Pillis & Michael Goodman & Fan Zhang & Yue Zhang & Paul R. Andreassen & Punam Malik, 2019. "CRISPR-Cas9 fusion to dominant-negative 53BP1 enhances HDR and inhibits NHEJ specifically at Cas9 target sites," Nature Communications, Nature, vol. 10(1), pages 1-13, December.
    • Handle: RePEc:nat:natcom:v:10:y:2019:i:1:d:10.1038_s41467-019-10735-7
    DOI: 10.1038/s41467-019-10735-7
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    Cited by:

    1. Arianna Moiani & Gil Letort & Sabrina Lizot & Anne Chalumeau & Chloe Foray & Tristan Felix & Diane Clerre & Sonal Temburni-Blake & Patrick Hong & Sophie Leduc & Noemie Pinard & Alan Marechal & Eduardo, 2024. "Non-viral DNA delivery and TALEN editing correct the sickle cell mutation in hematopoietic stem cells," Nature Communications, Nature, vol. 15(1), pages 1-21, December.
    2. Zhiqian Li & Lang You & Anita Hermann & Ethan Bier, 2024. "Developmental progression of DNA double-strand break repair deciphered by a single-allele resolution mutation classifier," Nature Communications, Nature, vol. 15(1), pages 1-19, December.

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