Generation and Characterization of a Zebrafish Model for Associated Retinal Dysfunction Using CRISPR/Cas9 Genome Editing Technology.

Worldwide, around 40,000 people progressively lose their eyesight as a consequence of retinitis pigmentosa (RP) caused by pathogenic variants in the gene, for which currently no treatment options exist. A model organism that mimics the human phenotype is essential to unravel the exact pathophysiological mechanism underlying associated RP, and to evaluate future therapeutic strategies. The introduction of CRISPR/Cas-based genome editing technologies significantly improved the possibilities of generating mutant models in a time- and cost-effective manner. Zebrafish have been recognized as a suitable model to study Usher syndrome-associated retinal dysfunction. Using CRISPR/Cas9 technology we introduced a 4bp deletion in exon 9 (). Immunohistochemical analysis showed that Adgrv1 was absent from the region of the photoreceptor connecting cilium in the zebrafish retina. Here, the absence of Adgrv1 also resulted in reduced levels of the USH2 complex members usherin and Whrnb, suggesting that Adgrv1 interacts with usherin and Whrnb in zebrafish photoreceptors. When comparing zebrafish with wild-type controls, we furthermore observed increased levels of aberrantly localized rhodopsin in the photoreceptor cell body, and decreased electroretinogram (ERG) B-wave amplitudes which indicate that the absence of Adgrv1 results in impaired retinal function. Based on these findings we present the zebrafish as the first mutant model that displays an early retinal dysfunction. Moreover, the observed phenotypic changes can be used as quantifiable outcome measures when evaluating the efficacy of future novel therapeutic strategies for associated RP.