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

Abstract

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 (Rho)-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 Rho was about 2.8 times more than that of mutant Rho in treated retinas. Furthermore, the progression of photoreceptor cell degeneration in outer nuclear layer was significantly delayed in treated regions of the Rho-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.

53BP1 nuclear body-marked replication stress in a human mammary cell model of BRCA2 deficiency

“…While alleles can be effectively distinguished via CRISPR approaches in cases where a unique PAM is present in one allele [19,20], discrimination between alleles with single nucleotide mismatches within the sgRNA site is conceptually challenging given the promiscuity of sgRNA recognition [16,17,18]. In this regard, previous studies have shown promise in utilizing SNPs in the sgRNA target regions to achieve allele-specific mutagenesis [21,22,23,24,25]. Our results now extend allele-specific applications to HR-mediated gene targeting.…”

“…Our results now extend allele-specific applications to HR-mediated gene targeting. Notably, allele selectivity can be further improved with a truncated sgRNA design [25], since it displays an even lower tolerance to mismatches [26]. These efforts have substantially broadened the scope of allele-specific applications to achieve various desired outcomes.…”

Exon 13-skipped USH2A protein retains functional integrity in mice, suggesting an exo-skipping therapeutic approach to treat USH2A-associated disease

“…The reaction was stopped by adding 2.5 μL of 200 mM EDTA. Indel frequencies were estimated as previously described71 by calculating band intensities with ImageJ software (NIH) and applying the following equation: % indels=100×[1−(1−fcut) 1/2 ], where fcut is the fraction cleaved, corresponding to the sum of intensities of the cleaved bands divided by the sum of total band intensities.…”

Single AAV-mediated scarless genome editing in dysfunctional retinal neurons mediates robust visual restoration in mice

“…3 CRISPR-Cas9-mediated allele knock-out based on error-prone non-homologous end joining (NHEJ) can correct gain-of-function mutations with AAV. 4–7 However, many loss-of-function mutations in larger genes still require local replacement with a wildtype sequence (scarless genome editing).…”