Allele-Specific CRISPR-Cas9 Genome Editing of the Single-Base P23H Mutation for Rhodopsin-Associated Dominant Retinitis Pigmentosa

Treatment strategies for dominantly inherited disorders typically involve silencing or ablating the pathogenic allele. CRISPR-Cas nucleases have shown promise in allele-specific knockout approaches when the dominant allele creates unique protospacer adjacent motifs that can lead to allele-restricted targeting. Here, we present a spacer-mediated allele-specific knockout approach that utilizes both SpCas9 variants and truncated single-guide RNAs to achieve efficient discrimination of a single-nucleotide mutation in rhodopsin (<i>Rho</i>)-P23H mice, a model of dominant retinitis pigmentosa. We found that approximately 45% of the mutant P23H allele was edited at the DNA level and that the relative RNA expression of wild-type <i>Rho</i> was about 2.8 times more than that of mutant <i>Rho</i> in treated retinas. Furthermore, the progression of photoreceptor cell degeneration in outer nuclear layer was significantly delayed in treated regions of the <i>Rho</i>-P23H retinas at 5 weeks of age. Our proof-of-concept study therefore outlines a general strategy that could potentially be expanded to examine the therapeutic benefit of allele-specific gene editing approach to treat human P23H patients. Our study also extends allele-specific editing strategies beyond discrimination within the protospacer adjacent motif sites, with potentially broad applicability to other dominant diseases.