Abstract
Purpose :
Retinitis Pigmentosa (RP) is an inherited retinal disease characterized by progressive visual impairment, which affects 1:4,000 individuals worldwide. Mutations in C2orf71, encoding a retinal ciliary protein, are causative for autosomal recessive RP. Previously, we have shown that, based on its interaction repertoire, C2orf71 might be important for maintaining the photoreceptor outer segment morphology. Here, we report the identification of two copies of the C2orf71 gene in zebrafish, C2orf71a and C2orf71b. To study the role of the largest C2orf71 paralogue, C2orf71a, we have generated a stable C2orf71a-/- zebrafish model using CRISPR/Cas9 technology.
Methods :
The zebrafish C2orf71 paralogues, C2orf71a and C2orf71b, were identified by bioinformatic analysis. To study the expression of these genes, RT-PCR on zebrafish eyes cDNA was performed. For the generation of C2orf71a-/- zebrafish, C2orf71a sequence-specific guide RNA and Cas9 mRNA were co-injected into single-cell zebrafish embryos. Mosaic fish were then outcrossed with wild-type TLF fish to determine germline transmission and the exact introduced genomic lesion. Subsequently, heterozygous mutants were incrossed to generate homozygous C2orf71a mutants. Histological analysis was performed using paraformaldehyde fixed zebrafish eyes stained with TR methyl ester (bodipy). Optokinetic responses were measured at low (5 rpm) and high (8 rpm) speeds.
Results :
Orthologous C2orf71a and C2orf71b genes, of 1122 and 859 amino acids respectively, were identified. A C2orf71a-/- zebrafish line was established that contains a 29-basepair deletion at the start of exon 1 of C2orf71a (c. 21_49del). This leads to the premature termination of translation after amino acid 16, presumably resulting in a functional C2orf71a knockout. Analysis of the morphology of the C2orf71a-/- zebrafish larval retina (5dpf) revealed that the photoreceptor outer segments are shorter and disorganized as compared to those of strain- and age-matched wild-type larvae. Additionally, optokinetic responses of C2orf71a-/- larvae were significantly decreased as compared to control larvae.
Conclusions :
We have successfully generated a C2orf71a knockout zebrafish using CRISPR/Cas9 technology. Defects observed in morphological and functional assays point towards an important role for C2orf71a in zebrafish photoreceptor outer segment morphogenesis and function.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.