Generation and Characterization of a Zebrafish Model for ADGRV1-Associated Retinal Dysfunction Using CRISPR/Cas9 Genome Editing Technology

Abstract

Worldwide, around 40,000 people progressively lose their eyesight as a consequence of retinitis pigmentosa (RP) caused by pathogenic variants in the ADGRV1 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 ADGRV1-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 adgrv1 exon 9 (adgrv1rmc22). Immunohistochemical analysis showed that Adgrv1 was absent from the region of the photoreceptor connecting cilium in the adgrv1rmc22 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 adgrv1rmc22 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 adgrv1rmc22 zebrafish as the first ADGRV1 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 ADGRV1-associated RP.

In Search of a Target Gene for a Desirable Phenotype in Aquaculture: Genome Editing of Cyprinidae and Salmonidae Species

“…Another approach to modeling is to simulate the symptoms of the target disease through mutations in genes with known function. Individuals and lines have been created that exhibit analogs of human somatic diseases such as cataract [ 49 , 50 ], myopia [ 51 ], exfoliative syndrome [ 52 ], retinal dysfunction [ 53 , 54 ], congenital heart defect [ 55 ], cardiac hypertrophy [ 56 ], dilated cardiomyopathy [ 57 ], arrhythmia [ 58 ], autoinflammatory syndrome [ 59 ], metabolic syndrome [ 60 ], diabetes and obesity [ 61 ], tuberculosis [ 62 ], thrombocytopenia [ 63 ], pediatric intestinal pseudoobstruction [ 64 ], pediatric cirrhosis [ 65 ], congenital hypothyroidism [ 66 ], fatty or alcoholic hepatosis [ 67 ], and scoliosis [ 68 , 69 ]. Genome editing has been used to model human tumors such as liver cancer [ 70 ], paraganglioma [ 71 ], skin melanoma [ 72 ], and epithelioid sarcoma [ 73 ].…”