Abstract
Purpose :
P23H mutation in the rhodopsin (RHO) gene is the most common cause (15-18%) of autosomal dominant retinitis pigmentosa (adRP) in USA. Strategies for treating RHO-P23H RP have predominantly involved the suppression of the wild-type and mutant alleles, which can therefore require supplementation of a wild-type gene. In this study, we aim to develop an allele-specific CRISPR/Cas9 system to knock out only the P23H mutant allele at its native locus in a well-characterized Rho-P23H knock-in mouse model.
Methods :
Three sgRNAs were designed with PAM sequences for alternative Cas9 variants to target the P23H mutation site. The sgRNAs, together with CAG-Cas9-T2A-EGFP encoding plasmids, were transfected into retinal cells of the wild type or mutant mice at P0-P2 by sub-retinal injection and in vivo electroporation. The targeted cleavage efficiency in the EGFP-positive retinal cells was determined 5-7 days post injection by next generation sequencing (NGS). The selected sgRNA with the highest efficiency and specificity, along with its Cas9-EGFP variants, were co-transfected into Rho-P23H heterozygous mice. The effect of this CRISPR/Cas9-sgRNA treatment on the retinal structure was evaluated by comparing the outer nuclear layer (ONL) of the EGFP-positive area with the adjacent EGFP-negative region on the frozen sections from the injected retinas.
Results :
Of the three sgRNAs tested, sgRNA2 with SpCas9 variants VQR or VRQR specifically cleaved the P23H mutant allele, with a cleavage efficiency of 43.20 ± 1.7% (n=3) in Rho-P23H homozygous retinas, and 0.94 ± 0.35% (n=3) in the wild type littermates. However, sgRNA1 and sgRNA3 showed no allele specificity between P23H mutant and wild type allele, with cutting efficiencies at 13.66% vs. 38.29% and 33.16% vs. 38.38%, respectively. NGS results showed that 70-90% of the Indels are out-of-frame. Preservation of photoreceptor degeneration in the transfected retina of Rho-P23H heterozygous mice was observed with 1-2 more rows of ONL than the adjacent non-transfected area at age of 40 days.
Conclusions :
Our study showed that CRISPR/Cas9 can selectively disrupt the Rhodopsin-P23H allele, which differed by only a single nucleotide from the wild type allele in mouse retina. This demonstrates the feasibility of an allele-specific CRIPSR-Cas9 gene editing approach that could be applied to a wide range of dominant diseases.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.