Construction of a guide-RNA for site-directed RNA mutagenesis utilising intracellular A-To-I RNA editing

Abstract

As an alternative to DNA mutagenesis, RNA mutagenesis can potentially become a powerful gene-regulation method for fundamental research and applied life sciences. Adenosine-To-inosine (A-To-I) RNA editing alters genetic information at the transcript level and is an important biological process that is commonly conserved in metazoans. Therefore, a versatile RNA-mutagenesis method can be achieved by utilising the intracellular RNA-editing mechanism. Here, we report novel guide RNAs capable of inducing A-To-I mutations by guiding the editing enzyme, human adenosine deaminase acting on RNA (ADAR). These guide RNAs successfully introduced A-To-I mutations into the target-site, which was determined by the reprogrammable antisense region. In ADAR2-over expressing cells, site-directed RNA editing could also be performed by simply introducing the guide RNA. Our guide RNA framework provides basic insights into establishing a generally applicable RNA-mutagenesis method.