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

Rajeswari Jayavaradhan, Devin M. Pillis, Michael Goodman, Fan Zhang, Yue Zhang, Paul R. Andreassen () and Punam Malik ()
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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)
Punam Malik: Cancer and Blood Diseases Institute (CBDI), Cincinnati Children’s Hospital Medical Center (CCHMC)

Nature Communications, 2019, vol. 10, issue 1, 1-13

Abstract: 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.

Date: 2019
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DOI: 10.1038/s41467-019-10735-7

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