CRISPR/Cas9 is a powerful tool for genome editing in cells and organisms. Nevertheless,\nintroducing directed templated changes by homology-directed repair (HDR) requires the cellular\nDNA repair machinery, such as the MRN complex (Mre11/Rad50/Nbs1). To improve the process,\nwe tailored chimeric constructs of Cas9, in which SpCas9 was fused at its N- or C-terminus to a\n126aa intrinsically disordered domain from HSV-1 alkaline nuclease (UL12) that recruits the MRN\ncomplex. The chimeric Cas9 constructs were two times more efficient in homology-directed editing of\nendogenous loci in tissue culture cells. This effect was dependent upon the MRN-recruiting activity\nof the domain and required lower amounts of the chimeric Cas9 in comparison with unmodified Cas9.\nThe new constructs improved the yield of edited cells when making endogenous point mutations\nor inserting small tags encoded by oligonucleotide donor DNA (ssODN), and also with larger\ninsertions encoded by plasmid DNA donor templates. Improved editing was achieved with both\ntransfected plasmid-encoded Cas9 constructs as well as recombinant Cas9 protein transfected as\nribonucleoprotein complexes. Our strategy was highly efficient in restoring a genetic defect in a cell\nline, exemplifying the possible implementation of our strategy in gene therapy. These constructs\nprovide a simple approach to improve directed editing.
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