Yeast-based screening system to identify and optimize the anti-sense oligos that promote targeted base-editing in mRNA molecules by the ADAR enzymes

Adenosine deaminase acting on RNA (ADAR) proteins are endogenous enzymes catalyzing the deamination of adenosine nucleotides to inosines, which are then read as guanosines during translation. This ability to re-code makes ADAR an attractive therapeutic tool to reprogram genetic information at the mRNA level. Indeed, different studies have reported several site-directed RNA editing approaches for making targeted base changes in RNA molecules. The basic strategy by several groups has been to use an antisense oligo (ASOs) that hybridizes and forms a dsRNA structure with the desired RNA target. These attempts resulted in low editing efficiency implying that more effective guides would be useful for better redirecting endogenous ADAR. However, the determinants that govern such activity are largely unknown. To address this issue, we exogenously expressed the ADAR proteins in the yeast Saccharomyces cerevisiae, an organism whose origins precede the emergence of ADARs but can express the human enzyme. The cells were transformed with a library of plasmids expressing millions of ASOs and engineered to uncover ASOs that are particularly more efficient in the editing of selected targets.

We identified and optimized ASOs sequences that enabled the recruitment of endogenous ADAR for efficient editing of mutations in the genes TRPM1, and USH2A that cause common inherited retinal diseases, a major cause of blindness. The identified ASOs were tested and validated in human cell lines, and currently form the basis for better future designs including applying this genetic therapy to the appropriate knock-in mouse models, retinal organoids, and ultimately in human patients.