Zebrafish as a Model to Evaluate a CRISPR/Cas9-Based Exon Excision Approach as a Future Treatment Option for...

Retinitis pigmentosa (RP) is an inherited retinal disease (IRD) with an overall prevalence of 1 in 4000 individuals. Mutations in EYS (Eyes shut homolog) are among the most frequent causes of non-syndromic autosomal recessively inherited RP and act via a loss-of-function mechanism. In light of the recent successes for other IRDs, we investigated the therapeutic potential of exon skipping for EYS-associated RP. CRISPR/Cas9 was employed to generate zebrafish from which the region encompassing the orthologous exons 37-41 of human EYS (eys exons 40-44) was excised from the genome. The excision of these exons was predicted to maintain the open reading frame and to result in the removal of exactly one Laminin G and two EGF domains. Although the eys transcript was found at levels comparable to wild-type eys, and no unwanted off-target modifications were identified within the eys coding sequence after single-molecule sequencing, Eys protein expression could not be detected. Visual motor response experiments revealed that eys larvae were visually impaired and histological analysis revealed a progressive degeneration of the retinal outer nuclear layer in these zebrafish. Altogether, the data obtained in our zebrafish model currently provide no indications for the skipping of EYS exons 37-41 as an effective future treatment strategy for EYS-associated RP.