1. Zebrafish as a Model to Evaluate a CRISPR/Cas9-Based Exon Excision Approach as a Future Treatment Option for EYS-Associated Retinitis Pigmentosa
- Author
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Theo A. Peters, Erik de Vrieze, Pam Graave, Sanne Broekman, Renske Schellens, Erwin van Wijk, Kerstin Nagel-Wolfrum, Rob W.J. Collin, and Hannie Kremer
- Subjects
QH301-705.5 ,Catalysis ,Sensory disorders Donders Center for Medical Neuroscience [Radboudumc 12] ,Article ,Inorganic Chemistry ,Exon ,All institutes and research themes of the Radboud University Medical Center ,EYS ,Protein Domains ,retinitis pigmentosa ,Retinitis pigmentosa ,medicine ,CRISPR ,Coding region ,Animals ,Biology (General) ,Physical and Theoretical Chemistry ,Outer nuclear layer ,Eye Proteins ,QD1-999 ,Molecular Biology ,Zebrafish ,CRISPR/Cas9 ,Spectroscopy ,Genetics ,exon skipping therapy ,biology ,Organic Chemistry ,photoreceptors ,General Medicine ,Exons ,Genetic Therapy ,Zebrafish Proteins ,medicine.disease ,biology.organism_classification ,zebrafish ,Exon skipping ,Computer Science Applications ,Chemistry ,Open reading frame ,Disease Models, Animal ,medicine.anatomical_structure ,Phenotype ,CRISPR-Cas Systems ,antisense oligonucleotides - Abstract
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 eysΔexon40-44 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, EysΔexon40-44 protein expression could not be detected. Visual motor response experiments revealed that eysΔexon40-44 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.
- Published
- 2021