Novel Zebrafish Autosomal Recessive Retinitis Pigmentosa Disease Models Created by CRISPR/Cas9 Gene Editing

Abstract

Purpose : Retinitis pigmentosa (RP) is a group of inherited retinal degeneration diseases which cause irreversible vision loss. Of ~90 genes associated with RP to date, underlying disease mechanisms are often unclear. To better understand RP-linked gene function and create novel disease models, we utilized CRISPR/Cas9-based genome editing technology to disrupt homologs of RP-linked genes in zebrafish.

Methods : Eight zebrafish homologs of five human genes linked to autosomal recessive RP (PDE6A, PDE6B, PDE6G, ABCA4 and RHO) were selected for CRISPR/Cas9 targeting. One or two guide RNAs targeting the 5’-end of each gene were co-injected with Cas9 mRNA into zebrafish embryos at the one cell stage. To facilitate phenotyping, genotyped founders (F0) were crossed to a transgenic line in which rod photoreceptors are labeled with YFP. F1 progeny carrying predicted loss-of-function frameshift mutations were pair mated, YFP+ offspring collected, and YFP expression levels monitored from 4 to 8 days post-fertilization using fluorescence microscopy. At day 8, F2 progeny were split into high and low expressing YFP+ groups. Representative larvae from each group were genotyped and confocal imaging performed to assess changes in rod cell numbers and morphology.

Results : All eight genes were successfully targeted. Predicted frameshift mutations were identified in all genes. Here, we report on novel phosphodiesterase 6b (pde6b) autosomal recessive mutants and autosomal dominant and recessive rhodopsin (rho) mutants. Decreased YFP expression was observed to be highly associated with mutant genotypes. Confocal imaging of mutant fish from 4 to 8 dpf confirmed the loss of rod photoreceptors, a hallmark of RP. Interestingly, autosomal dominant rho mutants undergo severe rod cell loss at early developmental stages, while recessive rho and pde6b mutants exhibit gradual rod cell loss.

Conclusions : In summary, we demonstrate a simple transgene-assisted screening method for identifying RP-like phenotypes in larval zebrafish. Novel zebrafish lines carrying mutations in RP-implicated genes can serve as models for future studies focusing on disease gene mechanisms and for large-scale drug screening.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.