Systematic empirical evaluation of individual base editing targets: Validating therapeutic targets in USH2A and...

Base editing shows promise for the correction of human mutations at a higher efficiency than other repair methods and is especially attractive for mutations in large genes that are not amenable to gene augmentation therapy. Here, we demonstrate a comprehensive workflow for in vitro screening of potential therapeutic base editing targets for the USH2A gene and empirically validate the efficiency of adenine and cytosine base editor/guide combinations for correcting 35 USH2A mutations. Editing efficiency and bystander edits are compared between different target templates (plasmids vs. transgenes) and assays (next-generation sequencing vs. Sanger), as well as comparisons between unbiased empirical results and computational predictions. Based on these observations, practical assay recommendations are discussed. Finally, a humanized knockin mouse model was created with the best-performing target, the nonsense mutation c.11864G>A p.(Trp3955∗). Split-intein AAV9 delivery of editing reagents resulted in the restoration of USH2A protein and a correction rate of 65% ± 3% at the mutant base pair and of 52% ± 3% excluding bystander amino acid changes. This efficiency is higher than that seen in a retinal gene editing program testing in a clinical trial. These results demonstrate the effectiveness of this overall strategy to identify and test base editing reagents with the potential for human therapeutic applications.