Abstract

Despite the wide use of the CRISPR/Cas9 system, low efficiency of targeted genome editing still limits its applications. Epigenetics have been suggested before to influence genome editing outcomes. We have tested several small molecular weight compounds, inhibiting epigenetic modifiers, for their effect on double-strand break (DSB) repair pathway choice. An already published traffic light reporter system, expressed in HEK293 cells, was used to detect homology-directed repair (HDR) and non-homologous end-joining (NHEJ) by fluorescent markers. FACS analysis identified one top-candidate inhibitor, which showed repeatedly a trend towards increased HDR efficiency. To test whether standard use of the inhibitor in targeted gene editing applications is advantageous, we tested it for gene-to-gene variation on endogenous gene targets in HEK293T cells. While fluorescent reporters, like the traffic light system, offer a fast and easy readout of HDR and NHEJ efficiencies they limit analysis not only to one cell line but also to one particular transgene. NGS deciphers the exact nucleotide sequence with high preciseness but is laborious and cost- intensive. We therefore tested inhibitor effects for the introduction of 8 base pairs, containing the HindIII restriction site, at an endogenous locus with restriction digest, IDAA, TIDER and NGS. All four methods showed an increase in HDR for inhibitor treated cells. IDAA showed high sensitivity and was hence used to study gene-to-gene variation. Four additional genes, two high and two low expressed, were CRISPR edited with and without inhibitor in HEK293T cells. For all four genes a significant increase in HDR efficiency was detected. We thus identified an epigenetic inhibitor, that successfully increases HDR efficiency in a transgenic reporter cell line as well as at endogenous loci in HEK293T cells. In addition, we validated IDAA as a reliable readout to study CRISPR editing outcomes.