Correction of monomeric enhanced green fluorescent protein (mEGFP) gene by short 5'-tailed duplexes.

Mutations of important genes elicit various disorders, including cancer. Recently, a new version of a 5'-tailed duplex (short TD), consisting of a ∼100-base editor strand containing the wild-type sequence and a ∼35-base assistant strand, was shown to correct a base substitution mutation in a target gene in human cells. In that previous study, the target was the copepod green fluorescent protein (copGFP) gene. To examine the usefulness of the short TD, we performed gene correction experiments using a mutant form of the monomeric enhanced Aequorea victoria green fluorescent protein (mEGFP) gene containing a TAC to CAC mutation in codon 75 (corresponding to the tyrosine to histidine substitution in the chromophore). The short TDs with the wild-type sequence efficiently corrected the inactivated gene in human U2OS cells. These results indicated that the short TDs are effective for gene editing.

Gene correction by 5'-tailed duplexes with short editor oligodeoxyribonucleotides.

Various diseases, including cancer, are caused by genetic mutations. A 5'-tailed duplex (TD) DNA, consisting of a long single-stranded (ss) editor DNA and a short (∼35-base) ss assistant oligodeoxyribonucleotide, can introduce a base-substitution in living cells and thus correct mutated genes. Previously, several hundred-base DNAs were employed as the editor DNAs. In this study, 5'-TDs were prepared from various editor DNAs with different lengths and examined for their gene correction abilities, using plasmid DNA bearing a mutated copepod green fluorescent protein (copGFP) gene, in human cells. High-throughput analysis was performed by the reactivated fluorescence of the wild-type protein encoded by the corrected gene as the indicator. The analysis revealed that 5'-TDs with ∼100-base ss editor DNAs enabled gene editing at least as efficiently as those with longer editor DNAs. Moreover, the antisense strand was more effective as the editor than the sense strand, in contrast to the 5'-TDs with longer editor strands. These results indicated that the 5'-TD fragments with shorter editor strands than those used in previous studies are useful nucleic acids for gene correction.