Simultaneous inhibition of DNA-PK and Polϴ improves integration efficiency and precision of genome editing

Genome editing tools, especially CRISPR/Cas9-based strategies, have transformed biomedical research and opened opportunities for developing curative treatments for genetic diseases. Despite rapid progress, low efficiency of targeted DNA integration and generation of undesired mutations represent major limitations for genome editing applications. Both issues arise from the interplay between the main DNA Double-Strand Break (DSB) repair pathways, Homology-Directed Repair (HDR), Non-Homologous End Joining (NHEJ), and Microhomology-Mediated End Joining (MMEJ). To improve efficiencies of targeted CRISPR-Cas9 genome editing, we screened a large compound library. This led to the discovery of AZD7648, a DNA-dependent protein kinase (DNA-PK) inhibitor and potent enhancer of CRISPR-Cas9-mediated integration. We demonstrated that AZD7648 increased HDR and decreased mutagenic NHEJ repair, thus resulting in improved performance of precise gene editing. Furthermore, we observed additional improvement of integration efficiency by impairing MMEJ repair through DNA polymerase ϴ (Polϴ) inhibition. Combined treatment with AZD7648 and Polϴ inhibitors (which we named 2iHDR) substantially increased precision of templated insertions, with efficiencies of up to 80%, and nearly no formation of undesired Insertion-Deletions (InDels). Importantly, 2iHDR also decreased Cas9-associated off-target activity, dramatically improving the performance and fidelity of CRISPR-Cas9 gene editing. ### Competing Interest Statement S.W., N.A., M.F., J.B., S.E., A.L., P.H., S.L., S.C., J.S., B.B., B.S., B.M., S.DC., P.I., M.B., T.M., S.R., O.E., E.C., J.V.F., S.S., P.A., A.T.G. and M.M. are presently or were previously employed by AstraZeneca and may be AstraZeneca shareholders. M.K.S., M.R.S., and TM are presently employed by Promega Corporation.