Abstract
Purpose :
Retinitis Pigmentosa (RP) affects 1 in 3000-4000 people. Mutations in the RP1 gene accounts for 5-10% of dominant RP cases. Currently there is no treatment for RP1-associated RP. Notably, all the dominant RP1 mutations are clustered on the last exon, which lead to the production of truncated RP1 proteins. Thus, removal or suppression of the truncated RP1 protein is critical in developing an effective therapy for RP1-associated dominant RP. Here we developed a CRISPR/Cas9-based allele-ablation therapy for RP1 diseases. Specifically, we used a dual-sgRNA strategy to remove two or more RP1 exons and its flanking introns by targeting a pair of RP1 haplotype-specific SNPs.
Methods :
We performed linkage analysis and identified 13 distinct SNPs (3 on intron 1, 4 on intron 3, and 6 on exon 4) in the RP1 gene, which designates to three common RP1 haplotypes. We designed multiple sgRNAs that could distinguish the specific bases at each of the SNP sites. Editing efficiency and allele-specificity of individual sgRNA were determined by PCR and amplicon sequencing in HEK or WERi-RB1 cells. The most efficient sgRNAs targeting SNPs in intron 1 or 3 were paired with sgRNAs targeting SNPs in intro 3 or exon 4 and their efficiencies of deleting a large fragment of multiple exons and introns (up to 13kb) were assessed by long read nanopore sequencing. The reduction of RP1 expression in human retinal cells or tissues were examined by RT-qPCR and western-blot. Off-target effect of selected sgRNAs were assessed by deep sequencing.
Results :
Eleven out of 13 SNPs can be targeted in an allele specific manner. Screening and optimization of sgRNAs showed editing efficiencies at ~40-70% on the targeted allele with ~0-5% on the non-targeted allele. Dual-sgRNAs editing resulted in predicted large deletions of targeted RP1 allele with ~20-45% efficiency, which led to ~30-45% reduction of RP1 expression. Off-target analysis showed no off-target editing for all sgRNA except one single sgRNA.
Conclusions :
We have successfully developed a haplotype-based dual-sgRNA gene editing approach. This strategy greatly enhances the feasibility of using the CRISPR/Cas9 system to treat dominant diseases because a small number of sgRNAs are sufficient to specifically target relatively large sets of mutant alleles. Furthermore, it paves the avenue of its application in treating human RP1 patients with heterozygous RP1 haplotypes.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.