Engineered circular ADAR-recruiting RNAs increase the efficiency and fidelity of RNA editing in vitro and in vivo.

Current methods for programmed RNA editing using endogenous ADAR enzymes and engineered ADAR-recruiting RNAs (arRNAs) suffer from low efficiency and bystander off-target editing. Here, we describe LEAPER 2.0, an updated version of LEAPER that uses covalently closed circular arRNAs, termed circ-arRNAs. We demonstrate on average ~3.1-fold higher editing efficiency than their linear counterparts when expressed in cells or delivered as in vitro-transcribed circular RNA oligonucleotides. To lower off-target editing we deleted pairings of uridines with off-target adenosines, which almost completely eliminated bystander off-target adenosine editing. Engineered circ-arRNAs enhanced the efficiency and fidelity of editing endogenous CTNNB1 and mutant TP53 transcripts in cell culture. Delivery of circ-arRNAs using adeno-associated virus in a mouse model of Hurler syndrome corrected the pathogenic point mutation and restored α-L-iduronidase catalytic activity, lowering glycosaminoglycan accumulation in the liver. LEAPER 2.0 provides a new design of arRNA that enables more precise, efficient RNA editing with broad applicability for therapy and basic research.