Precise in-vivo RNA base editing with a wobble-enhanced circular CLUSTER guide RNA

Adenosine-to-inosine RNA base editing is a strategy developed to safely manipulate genetic information at the RNA level. Particularly promising for clinical implementation is the use of the ubiquitously expressed endogenous editing enzyme ADAR (adenosine deaminase acting on RNA) with tailored guide RNAs. However, the precision and efficiency of editing is often compromised by off-target events. While global off-target events can occur throughout the transcriptome, bystander editing events happen at the guide RNAs binding site on the target transcript. In this study, we introduce a novel circular CLUSTER guide RNA design that recruits endogenous ADAR and investigate the strategic placement of GU wobble base pairs to suppress bystander editing in vivo. The goals of the deep amplicon sequencing experiment were to precisely quantify the on-target editing yield and to verify the suppression of bystander off-target events. The goals of the whole transcriptome sequencing experiment were to evaluate the A-to-G RNA editing index and the global editing precision of the novel guide RNA design.To achieve this, Rett syndrome mice harboring a Mecp2 W104Amber mutation were treated either with a circular CLUSTER guide RNA targeting the mutant Mecp2 transcript or a scrambled and thus non-targeting control guide RNA. The targeting guide RNA would form three GU wobble base pairs adjacent to known bystander off-target sites when binding to the Mecp2 transcript. Both the targeting and the non-targeting guide RNA were encoded as AAV and delivered via retro-orbital injection of 4x10^12 viral genomes per mouse. The used AAV serotype PHP.eB allows cargo delivery to the mouse brain after systemic administration. Four weeks after injection the brain regions olfactory bulb, cerebellum, hippocampus, cortex, midbrain, thalamus, and brainstem were isolated for NGS analysis. Deep amplicon sequencing of the Mecp2 transcript in all brain regions showed that on target editing yields of up to 19 % could be achieved with very high precision. Bystander editing at GU wobble adjacent sites was >100-fold lower than the respective on-target yield. Whole transcriptome sequencing of thalamus showed that the A-to-G RNA editing index was unaffected by treatment with the targeting guide RNA compared to the scrambled non-targeting control. We were unable to identify any global off-target events, excluding mouse to mouse variability, which suggests a very high precision of our approach on the transcriptome-wide level.Harnessing endogenous ADAR with permanent, AAV-driven CLUSTER guide RNAs in the CNS is an important next step towards the development of novel drug modalities that fight neurological diseases.