Nonsense and missense mutations cause numerous incurable diseases, including Cystic Fibrosis and Duchenne Muscular Dystrophy, that affect millions of people worldwide. Gene therapy is the most promising treatment for these genetic diseases. The majority of current gene therapy techniques focus on correction of DNA sequences; our project focuses on direct editing of RNA sequences. Targeting RNA is advantageous because it permits a tunable and reversible editing system that can repair multiple mutations while circumventing the process of homology-directed repair. We have constructed dCas9 fusions to the editing enzymes APOBEC1 and ADAR1/2 to enable CRISPR-targeted C-to-U and A-to-I edits to RNA, respectively. We have designed and thoroughly characterized a series of GFP-based reporters that enable the detection and quantification of RNA editing by these dCas9 fusions. Our project outlines the theoretical framework for a novel, tunable, and reversible gene editing strategy with myriad applications.
