Aptazyme-directed A-to-I RNA editing.

As a promising therapeutic approach, the RNA editing process can correct pathogenic mutations and is reversible and tunable, without permanently altering the genome. RNA editing mediated by human ADAR proteins offers unique advantages, including high specificity and low immunogenicity. Compared to CRISPR-based gene editing techniques, RNA editing events are temporary, which can reduce the risk of long-term unintended side effects, making off-target edits less concerning than DNA-targeting methods. Moreover, ADAR-based RNA editing tools are less likely to elicit immune reactions because ADAR proteins are of human origin, and their small size makes them relatively easy to incorporate into gene therapy vectors, such as adeno-associated virus vectors (AAVs), which have limited space. Despite the promise of RNA editing as a therapeutic approach, precise temporal and spatial control of RNA editing is still lacking. Therefore, we have developed a small molecule-inducible RNA editing strategy by incorporating aptazymes into the guide RNA of the BoxB-λN-ADAR system. This chapter provides detailed protocols for targeted RNA editing by ADAR deaminases using aptazyme-based guide RNAs controlled by exogenous small molecules, marking the earliest use of aptazymes to regulate RNA editing strategies. Once small molecules are added or removed, aptazymes trigger self-cleavage to release the guide RNA, thus achieving small molecule-controlled RNA editing. To satisfy different RNA editing applications, we have realized the conditional activation and deactivation of A-to-I RNA editing of target mRNA using switch aptazymes. We provide step-by-step protocols for constructing guide RNA plasmids for regulatory purposes and conducting small molecule-induced RNA regulatory editing experiments in cells.