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Chris Zelinka, James M Fadool; In vivo gene editing of zebrafish rho to model human photoreceptor disease. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4472.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in RHO are among the most prevalent causes of hereditary retinal degeneration. RHO alterations lead to the death of rods, and secondary loss of cones. The conservation of photoreceptor structure, gene expression and function among vertebrates has led us to hypothesize that zebrafish rho mutations will model human photoreceptor disease. The purpose of this study was to apply in vivo genome editing to disrupt zebrafish rho.
CRISPR/Cas9 was used to induce mutations within the zebrafish rho gene. One- and two-cell staged embryos were injected with a solution containing a single guide (g) RNA targeting rho plus in vitro transcribed, capped RNA encoding Cas9. Embryos were reared for several days. Genomic DNA was isolated from injected larvae and used as the template to PCR amplify the entire coding sequence for rho. Mutations were detected with Sanger sequencing and/or restriction fragment length polymorphism. Retinal cryosections of injected and uninjected siblings were immunostained with the 1D1 monoclonal antibody which recognizes rhodopsin or 4C12, an independent rod-specific marker to verify the loss of rhodopsin expression.
rho in teleosts is characterized by a single exon with no introns. DNA sequencing of a PCR product spanning the entire rho exon showed that the larvae injected with the gRNA/Cas9 mRNA are mosaic for mutations in rho. The most common mutations are predicted to disrupt the rho coding sequence. Immunolabeling of serial sections of larvae at 6 days post fertilization showed absent or altered expression of rhodopsin in injected-larvae, however immunolabeling with an antibody to a second protein expressed by rods showed that the rod photoreceptors were present. To test for germline transmission, a subset of injected larvae were reared to adults and outcrossed. Novel rho alleles were identified in the F1 generation.
Our recovery of novel mutations shows that rho can be successfully targeted in zebrafish using the CRISPR/Cas9 system. These offer a proof-of-concept for the successful targeted mutagenesis of rho in zebrafish and their potential as models of disease.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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