Low Mutagenicity of Phosphate and Sugar Modifications Used in Donor DNA for Genome Editing

Genome editing with combinations of an artificial nuclease and donor nucleic acid is expected to cure genetic diseases, including cancers. Chemically modified oligodeoxyribonucleotides constitute a class of donor nucleic acids, and phosphorothioate (P-S) and locked nucleic acid (LNA/2',4'-BNA) modifications to the donor nucleic acid have been successfully employed. However, the genotoxicities of the P-S and LNA in DNA have not been evaluated. In this study, the two modifications were separately introduced into the supF reporter gene, and their mutagenicities were examined in human cells. The ethyl phosphotriester (P-OEt) modification was also examined for comparison. These modified supF plasmid DNAs were introduced into human U2OS cells, and the replicated DNAs were electroporated into indicator bacterial cells. The supF mutant frequencies of these plasmids were examined by next-generation sequencing (NGS). No difference was observed in the supF mutant frequencies and mutation spectra, suggesting that these modifications are safe for genome editing therapies.