Abstract
Purpose :
Loss-of-function mutations in Retinitis Pigmentosa 1-Like 1 (RP1L1) cause autosomal recessive retinitis pigmentosa (RP). RP is a genetically heterogeneous photoreceptor degenerative disease characterized by the progressive loss of rod cells. Patients with RP develop night blindness, loss of peripheral vision, and eventual blindness due to death of cones post rod cell loss. The reason for cone death after rod degeneration is poorly understood. We aimed to model RP1L1-associated RP in zebrafish to characterize the mechanism of rod pathology and cone response. Zebrafish are an ideal model of photoreceptors, as they have a density of cone photoreceptors similar to what can be observed in the human macula. This allows for the impact of rod degeneration on cone photoreceptors to be observed in the zebrafish, which has proven challenging in nocturnal rodent models. Additionally, zebrafish have early elicited visually-mediated behaviours that can be utilized and molecular tools available for genome editing and transgenesis.
Methods :
A CRISPR/Cas9 genome editing strategy was developed to target zebrafish rp1l1in its first exon. Guide RNA was synthesized and injected into Tg(sws1:GFP;sws2:mCherry) zebrafish embryos at the single cell stage. Mosaic animals were grown to adulthood and bred with wild-type individuals. Progeny were finclipped at the larval stage and sequenced. Larvae with chromatograms suggesting heterozygosity were grown, finclipped, a fragment of rp1l1 PCR amplified, TOPO cloned, and sequenced. Fish with the same deletion were crossed to generate homozygous individuals, genotyped via RFLP. Eyes from homozygous fish were cryosectioned and immunofluorescence carried out to label photoreceptors.
Results :
Three stable mutant lines were generated: a 5bp deletion, a 7bp deletion, and a 16bp deletion, all of which are predicted to create a severely truncated protein. Homozygous fish for the 16bp deletion were obtained and grown, and they are fertile, viable, and do not have any overt ocular anomalies. Cone photoreceptors appear grossly normal within mutant retina. Preliminary observations suggest that the mutant retinas may have a reduction in rod photoreceptors, but this needs to be confirmed.
Conclusions :
We generated zebrafish with predicted loss-of-function mutations in rp1l1 that have cone photoreceptors that appear normal and a potential reduction in rod photoreceptors.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.