|| Checking for direct PDF access through Ovid
Site-directed RNA editing is an important technique for correcting gene sequences and ultimately tuning protein function. In this study, we engineered the deaminase domain of adenosine deaminase acting on RNA (ADAR1) and the MS2 system to targetspecific adenosines, with the goal of correcting G-to-A mutations at the RNA level. For this purpose, the ADAR1 deaminase domain was fused downstream of the RNA-binding protein MS2, which has affinity for the MS2 RNA. To direct editing to specific targets, we designed guide RNAs complementary to target RNAs. The guide RNAs directed the ADAR1 deaminase to the desired editing site, where it converted adenosine to inosine. To provide proof of principle, we used an allele of enhanced green fluorescent protein (EGFP) bearing a mutation at the 58th amino acid (TGG), encoding Trp, into an amber (TAG) or ochre (TAA) stop codon. In HEK-293 cells, our system could convert stop codons to read-through codons, thereby turning on fluorescence. We confirmed the specificity of editing at the DNA level by restriction fragment length polymorphism analysis and sequencing, and at the protein level by western blotting. The editing efficiency of this enzyme system was ˜ 5%. We believe that this system could be used to treat genetic diseases resulting from G-to-A point mutations.