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.
Highlights
Precise genome editing/correction of DNA double-strand breaks (DSBs) induced by CRISPRCas[9] by homology-dependent repair (HDR) is limited by the competing error-prone nonhomologous end-joining (NHEJ) DNA repair pathway
We designed five different HA-tagged dominant negative (DN) 53BP1 fragments to be delivered by lentiviral vectors: DN1 and DN1S, which contain the focus forming region (FFR); DN2, which excludes the glycine-arginine rich (GAR) motif but retains the oligomerization domain (OD) domain via a linker to the remaining FFR; DN3 and DN4, which exclude both the OD domain and the GAR motif from the FFR
We found that when expressed at modest levels, DN1 or DN1S largely co-localize with endogenous 53BP1 at irradiation induced foci (IRIF) (Fig. 1c)
Summary
Precise genome editing/correction of DNA double-strand breaks (DSBs) induced by CRISPRCas[9] by homology-dependent repair (HDR) is limited by the competing error-prone nonhomologous end-joining (NHEJ) DNA repair pathway. In order to enhance genome editing by HDR, different approaches have been developed, including (i) cell synchronization in S/G2 phases[12] or fusion of Cas[9] to the Geminin degron to regulate Cas[9] expression in the S-phase of cell cycle[13,14] and (ii) overall inhibition of cellular NHEJ repair (global NHEJ inhibition) by inhibiting or depleting NHEJ proteins DNA Ligase IV, DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and 53BP14,15,16 or overexpression of DNA repair proteins such as RAD52 along with mouse dominant negative (mdn) 53BP117,18. We hypothesized that if error-prone NHEJ repair was inhibited only at the DSB generated by Cas[9], HDR efficiency would be enhanced, without compromising genome integrity, thereby promoting cellular homeostasis, and clinical safety
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