Recruitment of DNA repair MRN complex by fusion of intrinsically disordered protein domain to Cas9 improves genome editing

CRISPR-Cas9 is a powerful tool for genome engineering. Cas9 makes a specific double-strand break in the genome, directed by a guide RNA, which is then repaired by the cellular DNA repair machinery. In order to make specific template-directed changes in the genome, homology-directed repair (HDR) is needed, but this pathway is not the predominant one in most cells. To improve the success rate of genome editing, we fused a recruitment domain for the MRN complex (Mre11/Rad50/Nbs1) to Cas9. The MRN complex is needed for HDR, and by targeting it to the break site, we can increase HDR locally, and avoid potential complications that could arise from global alteration of DNA repair pathways. We found that the MRN-recruiting chimeric Cas9 constructs increased editing two-fold, and were effective at much lower concentrations than wild-type Cas9 in mediating editing of endogenous loci. These constructs were better at mediating a wide range of genomic edits, from small insertions or point mutations encoded by ssODN donors, to larger insertions of fluorescent proteins or other cassettes encoded by plasmid donors. Notably, correction of a genetic defect to restore wild type activity was more efficient with the chimeric constructs, suggesting that these constructs could be relevant in clinical applications. These Cas9 chimeric constructs provide a simple means of achieving higher efficiency and fidelity of genome editing.