Comparing and optimising the editing efficiency of three RNA editor families targeting a pathogenic CRB1 nonsense variant

Abstract

Purpose : Mutations in the Crumbs homologue 1 (CRB1) gene are associated with early onset inherited retinal degeneration (IRD), for which no therapy currently exists. Since the CRB1 gene is expressed in multiple isoforms, a therapeutic approach requires mutation correction. RNA editing harnesses the Adenine Deaminase acting on DNA (ADAR) enzyme for correction of G>A mutations in the human transcriptome without the risk of creating permanent genomic off-target edits. Here, we compare and optimise the efficiency of three families of RNA editors to correct a pathogenic nonsense variant in CRB1 in-vitro.

Methods : RNA editor families differed in the intermediate protein used to link the catalytically active ADAR deaminase domain (ADAR_DD) to the guide RNA (gRNA). RNA editor components, including the gRNA length, A-C mismatch position and ADAR_DD were optimised. On target editing efficiencies were assessed with a dual luciferase reporter assay. Editing rates were confirmed and bystander edits analysed by Sanger sequencing and EditR analysis.

Results : In the PspCas13b RNA editor, a 50 nucleotide (nt) gRNA with an AC mismatch at position 24 and carrying a hypermutated ADAR_DD was able to achieve 39% (±8) editing in the luciferase assay and 48% (±12) on EditR analysis. No bystander editing was observed. In GluR2 RNA editors, lengthening the gRNA to 100bp and adding a second copy of the gRNA significantly improved editing efficiency. The highest editing rate of 73% (±12) in the luciferase assay and 51% (±3) on EditR analysis was achieved using an editor with two copies the 100bp gRNA and wild-type, full length ADAR2 protein. Notably, no bystander edits were observed. The best editing rates of 23% (±4) in the luciferase assay and 41% (±10) on EditR analysis were achieved when paring two copies of a short, 20bp gRNA with a hypermutated ADAR_DD and a NES. Due to the short length of the gRNA, bystander edits were not observed, even when using a hypermutated ADAR_DD.

Conclusions : In this in-vitro screen, all three RNA editor families showed highly significant editing rates, highlighting the translational potential of RNA editors for the treatment of CRB1-associated IRDs. GluR2 and the MS2-MCP RNA editors are packageable into a single Adeno-associated virus and are therefore of particular translational interest.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.