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 (PAMs) 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 (trusgRNAs) to achieve efficient discrimination of a single-nucleotide mutation in rhodopsin (Rho)-P23H mice, a model of dominant retinitis pigmentosa (RP). We found that approximately 45% of the mutant P23H allele was edited at 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 five 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 patient. Our study also extends allele-specific editing strategies beyond discrimination within the PAM sites, with potentially broad applicability to other dominant diseases.

Special Issue Introduction: Inherited Retinal Disease: Novel Candidate Genes, Genotype–Phenotype Correlations, and Inheritance Models

“…In addition to the new functional assays, the recent entrance of CRISPR/Cas opens great opportunities for IRD research, as it enables a more efficient introduction of specific mutations into animal models or cells [ 72 ]. This technology also offers a new therapeutic potential [ 73 ].…”

Gene editing prospects for treating inherited retinal diseases

“…However, the proposed strategy was designed to be allele specific relating to the murine gene without distinguishing the WT and the mutant allele, thus could not be translated to future clinical applications. More practical and physiological approaches to knock down dominant mutation in rhodopsin gene were described by Broccoli41 and Liu teams 82. Both groups designed a P23H-specific gRNA to knock down the mutant murine allele in P23H knock-in mice, and employed the engineered SpCas9 VQR variant coupled to the mutation-specific gRNA generating indels predominantly at the mutant allele 41.…”

“…Both groups designed a P23H-specific gRNA to knock down the mutant murine allele in P23H knock-in mice, and employed the engineered SpCas9 VQR variant coupled to the mutation-specific gRNA generating indels predominantly at the mutant allele 41. Li et al 82 obtained a higher and more efficient discrimination between the mutant and the WT allele by combining a 5′-truncated 17-nucleotide gRNA together with the SpCas9 VRQR, by subretinal injected plasmids followed by electroporation. However, clinical translation to humans of this approach may be difficult due to the requirement of invasive surgery to place microelectrodes in close proximity of target cells to limit tissue damage.…”

AsCRISPR: a web server for allele-specific sgRNA design in precision medicine

“…So far, allele-specific CRISPR has been increasingly employed in treating various diseases such as retinitis pigmentosa [5][6][7][8], corneal dystrophy [9], dominant progressive hearing loss [10] and multiple cancers [11][12][13], as well as genome imprinting diseases [14]. And it was also used to alleviate haploinsufficiency by allele-specific CRISPR activation of wildtype alleles [15], and even was designed for manipulating human leukocyte antigen (HLA) locus [16].…”