A Novel CRISPR-based Series of Retinitis Pigmentosa Models in Zebrafish

Abstract

Purpose : Retinitis pigmentosa (RP) is a hereditary retinal degeneration characterized by progressive rod photoreceptor death followed by cone cell loss. Although over 70 genes have been reported to be responsible for RP, the underlying disease mechanisms are largely unclear. Moreover, no therapy can substantially treat most patients. To better understand RP-causative gene function and seek effective treatment, we utilized CRISPR to disrupt zebrafish homologs of major human RP-linked genes, and generated a series of novel genetic mutant models of RP that are highly amenable to small molecule screening for new neuroprotectants.

Methods : In non-syndromic RP, the most common disease-causing genes are RHO, PDE6A and PDE6B. In this study, zebrafish homologs (rho, pde6a, and pde6b) were disrupted by co-injecting Cas9 mRNA with one or two guide RNAs targeting the 5’-end of each gene into zebrafish embryos at one cell stage. Mutant founders identified by fluorescence PCR and sequencing were crossed to a transgenic line in which rod photoreceptors are labeled with yellow fluorescence protein (YFP). YFP-expressing heterozygous progeny carrying frameshift mutations predicted to interrupt gene function were propagated and pair bred. Collected embryos underwent qualitative visual screen and confocal imaging, as well as an automated YFP quantification assay using a microplate reader from 4 to 8 days post-fertilization (dpf). At 8 dpf, larval tails were collected for genotyping and heads for immunofluorescence assays.

Results : Frameshift mutations were identified in rho, pde6a, and pde6b genes. The selected mutant alleles (rho^JH600E, rho^JH600F, pde6a^JH602I, and pde6b^JH603E) were further characterized. Phenotyping/genotyping showed that reductions of rod-YFP expression were highly associated with either heterozygous or homozygous mutations. Confocal intravital imaging confirmed rod cell loss in mutants. Time-series YFP quantification displayed a range of rod cell loss with varying severity and temporal progression. Rod marker 1d1 staining verified rod photoreceptor degeneration, but cone marker zpr1 staining showed no obvious cone cell loss out to 8 dpf.

Conclusions : We successfully generated a series of novel zebrafish RP models, exhibiting varying severity of rod photoreceptor degeneration. These genetic mutant models will be valuable for mechanistic studies of mutation effects and in vivo reporter-based phenotypic drug discovery.

This is a 2020 ARVO Annual Meeting abstract.