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Giulia Ascari, Thomas Naert, Tom Van Nieuwenhuysen, Suzan Demuynck, Chris Guerin, Frauke Coppieters, Kris Vleminckx, Elfride De Baere; CRISPR/Cas9 mediated knockout of rcbtb1 in Xenopus tropicalis to model RCBTB1-associated retinal disease. Invest. Ophthalmol. Vis. Sci. 2018;59(9):377.
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Biallelic mutations in RCBTB1 were recently described in human as a cause of autosomal recessive non-syndromic and syndromic inherited retinal disease (iRD). The retinal phenotype varies from late-onset macular dystrophy with peripheral reticular dystrophy to retinitis pigmentosa. To model RCBTB1-associated iRD and to study its pathogenesis, we aimed to generate a stable rcbtb1 knockout in Xenopus tropicalis, a true diploid amphibian.
We performed CRISPR/Cas9 injection of early Xenopus embryos. High rcbtb1 knockout efficiency was confirmed using targeted next-generation sequencing (MiSeq, Illumina). Mosaic knockout founders as well as stable knockout off-spring animals were evaluated. Optical coherence tomography (OCT) of the eye, hematoxylin and eosin (H&E) staining, modified Richardson staining, proliferating cell nuclear antigen (PCNA) and caspase-3 immunohistochemistry and electron microscopy (EM) were performed to functionally characterize the Xenopus model.
At 10 months of age in the mosaic knockout animals, histology and OCT revealed abnormalities in the retinal outer plexiform and photoreceptor layers. H&E and modified Richardson staining in the stable knockout showed disorganization of the retinal pigment epithelium. PCNA and active caspase-3 immunostaining showed abnormal reactivity in comparison with wild type animals. Interestingly, no retinal phenotypes were detectable in the juvenile tadpole stages, reflecting the late onset of the disease in human. EM on retina sections is currently ongoing and histopathology will be evaluated on different Xenopus organs (ovary, thyroid, inner ear) to investigate additional syndromic features reported in human RCBTB1-associated disease.
We were able to confirm a detrimental effect of rcbtb1 loss-of-function on retinal structures in CRISPR/Cas9 mediated knockout in Xenopus tropicalis, recapitulating the late-onset RCBTB1-associated retinal phenotype in human. Our study highlights the importance of Xenopus tropicalis as model organism for iRD, as Xenopus allows identification and targeted injection of blastomeres that contribute primarily to the retinal cells and as it has a true diploid genome. Further experiments will focus on the molecular mechanistic aspects of the disease in order to identify possible anchor points for future therapy.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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