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Yael Kinarty, Samer Khateb, Dror Sharon, Adi Inbal; Analysis of CEP78 knockout in zebrafish generated by CRISPR-Cas9. Invest. Ophthalmol. Vis. Sci. 2018;59(9):375.
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Cone-rod degeneration (CRD) is a retinal disorder characterized initially by cone photoreceptor degeneration followed by rod dysfunction which causes loss of visual acuity, color vision impairment, and photophobia. Centrosomal protein 78 (CEP78) encodes a protein that is expressed in the cilia of photoreceptors as well as in some other tissues. We previously reported that CEP78 mutations cause CRD with a sensorineural hearing loss in humans. Aiming to study the function of CEP78, we generated cep78 mutations in zebrafish using the CRISPR-Cas9 system.
Primers targeting two different regions of cep78 in zebrafish were designed using the online tool CHOPCHOP. A Guide-RNA was injected together with Cas9 protein into single-cell stage WT embryos. Screening for mutations was performed by restriction enzyme analysis followed by Sanger sequencing. Histological analysis and a color preference test were performed on adult knockout fish.
We injected 165 single-cell embryos and raised 150 fish that are potential chimeras for different cep78 sequence alterations. Mutation analysis revealed 7 out of 9 fish with suspected mutations. Sequencing analysis of their offspring revealed 5 different variants at the expected CRISPR site, including a +9-2 bp indel and a deletion of 4 bp, both causing a frameshift. The histological analysis of 6 WT and 7 KO fish did not reveal clear evidence of photoreceptor degeneration; however, KO fish retinae at ages 7, 8.5, and 9.5 months were less organized and tight, double cones were disfigured and seemed bend, and the photoreceptor layer seemed elongated. Color preference test revealed that adult KO fish distinguish between different colors and have similar preferences as WT.
Zebrafish can serve as a quick and relatively easy animal model for studying the function of genes of interest. In combination with the novel CRISPR-Cas9 system, it provides a powerful tool for analysis of retinal disease genes, such as CEP78. The results suggest that cep78 loss of function does not cause early-onset retinal degeneration but seems to have a role in maintaining the correct structure of photoreceptors in zebrafish over time. Follow up is required to find if retinal function is affected at older age. We anticipate that the zebrafish model we developed will further aid to understand the function of CEP78 and hopefully to develop gene therapy for CEP78 disease.
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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