RNA editing activity can be exploited for the restoration of disease-causing nonsense and missense mutations and as a tool to manipulate the transcriptome in a simple and programmable way. The general concept is called site-directed RNA editing and has high potential for translation into the clinics. Due to its different mode of action RNA editing may well complement gene editing and other gene therapy options. In this method chapter, we particularly highlight RNA editing strategies that harness endogenous ADARs. Such strategies circumvent the delivery and expression of engineered editases and are notably precise and simple. This is particularly true if endogenous ADARs are recruited with chemically modified antisense oligonucleotides, an approach we call RESTORE (recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNA editing). To foster the research and development of RESTORE we now report a detailed protocol for the procedure of editing reactions, and a protocol for the generation of partly chemically modified RESTORE ASOs with a combination of in vitro transcription and ligation.
Keywords: ADAR; Antisense oligonucleotide; In vitro transcription—therapeutic RNA editing; RESTORE; RNA editing; RNA ligation; Site-directed RNA editing.