CRISPR/Cas9 Allele-Specific Targeting of Autosomal Dominant Retinitis Pigmentosa Disease Variants

Autosomal dominant Retinitis Pigmentosa (adRP) is an important cause of progressive, irreversible blindness. Clinically and genetically heterogeneous, adRP is known to be caused by a myriad of heterozygous variants in at least 29 genes. Where adRP is caused by point mutations in haplosufficient genes with dominant-negative and/or toxic gain-of-function disease mechanisms, selective ablation of the mutant allele using CRISPR/Cas9 technology represents a tantalising prospect for treatment. The Pro23His mutation in Rhodopsin (RHO) and the Gly56Arg mutation in Nuclear Receptor Subfamily 2 Group E Member 3 (NR2E3) are particularly attractive targets, representing the two most common causes of non-syndromic adRP, respectively. However, guide RNA design is heavily restricted due to minimal availability of – or in the case of RHO Pro23His, the absence of – canonical SpCas9 5’-NGG-3’ PAM sequences in these regions. In this study, we have explored the application of alternative naturally occurring or engineered CRISPR/Cas9 platforms to target both the RHO Pro23His and NR2E3 Gly56Arg mutations. To assess disease-allele targeting activity we generated clonal cell models of both mutations using a novel PRIME editing construct. Analysis of gene editing outcomes identified several candidate gRNAs with selective targeting of the mutant allele, with a prevalence of frame-shifting indels or larger deletions observed. These data provide evidence that our approach has the potential to mediate specific gene editing events that will ablate the mutant allele whilst sparing the wild-type allele, positing these strategies as therapeutic candidates for adRP.