Advances in guide RNA engineering for endogenous ADAR-mediated therapeutic RNA editing

Adenosine deaminases acting on RNA (ADARs) convert adenosine positioned within double-stranded RNA (dsRNA) to inosine (A-to-I). This reaction can be redirected to a specific target transcript by exogenous guide RNAs (gRNAs) that recruit abundant endogenous ADARs without the need for exogenous protein delivery. Approximately 16,000 pathogenic G > A mutations are correctable by A-to-I conversion, thus representing a broad scope for therapeutic A-to-I editing. This review covers the key advances in gRNA engineering for endogenous ADAR-mediated RNA editing. We first discuss how ADAR biology informs rational gRNA design. We then survey gRNA design strategies that increase on-target editing efficiency and diminish bystander editing. Delivery modalities are then discussed for both unmodified and modified gRNAs as a strategy to minimize transcriptome-wide off-target editing. We conclude by outlining the outstanding challenges and future opportunities, including computational gRNA design, to guide the next generation of gRNAs towards the path of approved RNA editing medicines.