Oligonucleotide-mediated gene editing is underestimated in cells expressing mutated GFP, and correlates with target protein expression

Short single-stranded DNA oligonucleotides (ssODNs) can create small, specific genomic sequence alterations in eukaryotic cells. Potential applications include introducing disease-associated mutations into cell lines or animals, functional studies of SNPs and, ultimately, clinical therapy by correcting genetic point mutations. Here, we show that ssODN-mediated gene editing is underestimated in three cultured mammalian cell lines, CHO, HEK293T and HepG2, expressing a non-fluorescent mutant version of enhanced GFP (mEGFP), because of variable expression of the mEGFP target gene in the cell population. In each isogenic cell line, gene editing efficiency as measured by recovery of green fluorescence using flow cytometry correlated tightly with levels of the target protein, and thus gene expression. Remarkably, for CHO-mEGFP cells over 20% of cells in the highest expressing decile showed restored green fluorescence, whereas for the overall population it was 2.5%. Moreover, while EGFP expression was lost during passsaging of gene-edited clones initially selected for restored green fluorescence, this merely reflected variable expression characteristics of the parental cells. Reactivation of fluorescent EGFP expression, by treatment with sodium butyrate or 5-aza-2’-deoxycytidine, demonstrated that although the phenotype was labile, the edited genotype was stable. We infer that oligonucleotide-directed editing of endogenous genes is feasible, particularly when highly transcribed