Oligonucleotide-mediated gene editing is underestimated in cells expressing mutated green fluorescent protein and is positively associated with target protein expression.
BACKGROUND
Single-stranded DNA oligonucleotides (ssODNs) can introduce small, specific sequence alterations into genomes. Potential applications include creating disease-associated mutations in cell lines or animals, functional studies of single nucleotide polymorphisms and, ultimately, clinical therapy by correcting genetic point mutations. Here, we report feasibility studies into realizing this potential by targeting a reporter gene, mutated enhanced green fluorescent protein (mEGFP).


METHODS
Three mammalian cell lines, CHO, HEK293T and HepG2, expressing multiple copies of mEGFP were transfected with a 27-mer ssODN capable of restoring fluorescence. Successful cell correction was quantified by flow cytometry.


RESULTS
Gene editing in each isogenic cell line, as measured by percentage of green cells, correlated tightly with target protein levels, and thus gene expression. In the total population, 2.5% of CHO-mEGFP cells were successfully edited, although, remarkably, in the highest decile producing mEGFP protein, over 20% of the cells had restored green fluorescence. Gene-edited clones initially selected for green fluorescence lost EGFP expression during cell passaging, which partly reflected G2-phase cycle arrest and perhaps eventual cell death. The major cause, however, was epigenetic down-regulation; incubation with sodium butyrate or 5-aza-2'-deoxycytidine reactivated fluorescent EGFP expression and hence established that the repaired genotype was stable.


CONCLUSIONS
Our data establish that ssODN-mediated gene editing is underestimated in cultured mammalian cells expressing nonfluorescent mutated EGFP, because of variable expression of this mEGFP target gene in the cell population. This conclusion was endorsed by studies in HEK293T-mEGFP and HepG2-mEGFP cells. We infer that oligonucleotide-directed editing of endogenous genes is feasible, particularly for those that are transcriptionally active.
Division of Medicine, UCL Medical School, London, UK.
Petra
P
Disterer
Ioannis
I
Papaioannou
Vanessa C
VC
Evans
J Paul
JP
Simons
James S
JS
Owen
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D005818:Genetic Engineering / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D056945:Hep G2 Cells; D006801:Humans; D016296:Mutagenesis / Q000235:genetics*; D009841:Oligonucleotides / Q000235:genetics*; D014162:Transfection
D009841:Oligonucleotides; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins