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

• Published in: Nature communications 2019-06, Vol.10 (1), p.2866-13, Article 2866
• Main Authors: Jayavaradhan, Rajeswari, Pillis, Devin M., Goodman, Michael, Zhang, Fan, Zhang, Yue, Andreassen, Paul R., Malik, Punam
• Format: Article
• Language: English
• Subjects: 13/1; 13/106; 13/31; 13/51; 14/19; 14/35; 38/22; 38/77; 42/41; 631/1647/1511; 631/337; 631/337/4041/3196; 631/61; Cell Line; Clustered Regularly Interspaced Short Palindromic Repeats; CRISPR; CRISPR-Associated Protein 9 - metabolism; CRISPR-Cas Systems; Deoxyribonucleic acid; DNA; DNA Breaks, Double-Stranded; DNA damage; DNA End-Joining Repair; DNA Repair; Editing; Error correction; Error reduction; Gene Editing - methods; Genome editing; Genomes; Homology; Humanities and Social Sciences; Humans; Leukocytes; Lymphocytes; Lymphocytes B; multidisciplinary; Mutagenesis, Insertional; Non-homologous end joining; Nuclease; Proteins; Recombinational DNA Repair - physiology; Repair; Science; Science (multidisciplinary); Tumor Suppressor p53-Binding Protein 1 - genetics; Tumor Suppressor p53-Binding Protein 1 - metabolism
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-019-10735-7

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. Global inhibition of NHEJ factors has been one strategy to improve CRISPR-Cas9 mediated HDR. Here the authors fuse a dominant-negative mutant of 53BP1 to Cas9 to enhance HDR frequency, reduce NHEJ specifically at the Cas9 cut sites, and reduce the toxicity associated with global NHEJ inhibition.