Discovery and Tuning of RNA Editing Guides via High‐Throughput Screening and Chemical Modification

Adenosine deaminases acting on RNA (ADAR) catalyze the deamination of adenosine to inosine in double‐stranded RNA. Because inosine is read as guanosine during translation, this process enables programmable A‐to‐G recoding at the transcript level. ADARs can be harnessed for therapeutic correction of pathogenic mutations through site‐directed RNA editing with guide RNAs. To expand the design space of editing‐enabling guides, we applied EMERGe, a high‐throughput screening platform, to identify motifs targeting a premature termination codon in the MeCP2 transcript associated with Rett syndrome. This uncovered a guide RNA motif that supported efficient ADAR2‐mediated editing in vitro , featuring a 5′‐GUG‐3' sequence predicted to form an asymmetric loop. To enable therapeutic application, structure–activity relationship studies and chemical optimization were performed, yielding a fully modified guide RNA with 2′‐O‐methyl, 2′‐fluoro, and phosphorothioate linkages. This stabilized guide retained the activity of unmodified RNA and showed enhanced nuclease resistance. The optimized guide induces dose‐dependent editing at two MECP2 loci in reporter assays in HEK293T cells, demonstrating that EMERGe‐selected motifs can be rendered viable in cells through targeted chemical modification. These findings highlight the utility of EMERGe as a discovery platform and establish a pipeline for identifying and optimizing editing‐enabling guide RNA features beyond traditional design rules.