A pipeline for identifying guide RNA sequences that promote RNA editing of nonsense mutations that cause inherited retinal diseases.

Adenosine deaminases acting on RNA (ADARs) are endogenous enzymes catalyzing the deamination of adenosines to inosines, which are then read as guanosines during translation. This ability to recode makes ADAR an attractive therapeutic tool to edit genetic mutations and reprogram genetic information at the mRNA level. Using the endogenous ADARs and guiding them to a selected target has promising therapeutic potential. Indeed, different studies have reported several site-directed RNA-editing approaches for making targeted base changes in RNA molecules. The basic strategy has been to use guide RNAs (gRNAs) that hybridize and form a double-stranded RNA (dsRNA) structure with the desired RNA target because of ADAR activity in regions of dsRNA formation. Here we report on a novel pipeline for identifying disease-causing variants as candidates for RNA editing, using a yeast-based screening system to select efficient gRNAs for editing of nonsense mutations, and test them in a human cell line reporter system. We have used this pipeline to modify the sequence of transcripts carrying nonsense mutations that cause inherited retinal diseases in the FAM161A, KIZ, TRPM1, and USH2A genes. Our approach can serve as a basis for gene therapy intervention in knockin mouse models and ultimately in human patients.

Dissecting the basis for differential substrate specificity of ADAR1 and ADAR2.

Millions of adenosines are deaminated throughout the transcriptome by ADAR1 and/or ADAR2 at varying levels, raising the question of what are the determinants guiding substrate specificity and how these differ between the two enzymes. We monitor how secondary structure modulates ADAR2 vs ADAR1 substrate selectivity, on the basis of systematic probing of thousands of synthetic sequences transfected into cell lines expressing exclusively ADAR1 or ADAR2. Both enzymes induce symmetric, strand-specific editing, yet with distinct offsets with respect to structural disruptions: -26 nt for ADAR2 and -35 nt for ADAR1. We unravel the basis for these differences in offsets through mutants, domain-swaps, and ADAR homologs, and find it to be encoded by the differential RNA binding domain (RBD) architecture. Finally, we demonstrate that this offset-enhanced editing can allow an improved design of ADAR2-recruiting therapeutics, with proof-of-concept experiments demonstrating increased on-target and potentially decreased off-target editing.