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 l... More
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.
