The utility of Xenopus laevis, a common research subject for developmental biology, retinal physiology, cell biology, and other investigations, has been limited by lack of a robust gene knockout or knock-down technology. Here we describe manipulation of the X. laevis genome using CRISPR/Cas9 to model the human disorder retinitis pigmentosa, and to introduce point mutations or exogenous DNA sequences. We introduced and characterized in-frame and out-of-frame insertions and deletions in three genes encoding rhodopsin by co-injection of Cas9 mRNA, eGFP mRNA, and single guide RNAs into fertilized eggs. Deletions were characterized by direct sequencing and cloning; phenotypes were assessed by assays of rod opsin in retinal extracts, and confocal microscopy of cryosectioned and immunolabeled contralateral eyes. We obtained germline transmission of editing to F1 offspring. In-frame deletions frequently caused dominant retinal degeneration associated with rhodopsin biosynthesis defects, while frameshift phenotypes were consistent with knockout. We inserted eGFP or point mutations into rhodopsin genes by co-injection of repair fragments with homology to the Cas9 target sites. Our techniques can produce high frequency gene editing in X. laevis, permitting analysis in the F0 generation, and advancing the utility of X. laevis as a subject for biological research and disease modeling.

We previously reported autophagic structures in rod photoreceptors expressing a misfolding RHO (rhodopsin) mutant (RHO P23H ), suggesting that autophagy may play a role in degrading the mutant RHO and/or be involved in photoreceptor cell death. To further examine autophagy in normal and diseased rods, we generated transgenic Xenopus laevis tadpoles expressing the dually fluorescent autophagy marker mRFP-eGFP-LC3 in rods, which changes from green to yellow and finally red as autophagic structures develop and mature. Using transgenic lines with constitutive and inducible expression, we determined the time-course of autophagy in rod photoreceptors: autophagosomes last for 6 to 8 hours before fusing with lysosomes, and acidified autolysosomes last for about 28 hours before being degraded. Autophagy was diurnally regulated in normal rods, with more autophagic structures generated during periods of light, and this regulation was non-circadian. We also found that more autophagosomes were produced in rods expressing the misfolding RHO P23H mutant. The RHO chromophore absorbs photons to initiate phototransduction, and is consumed in this process; it also promotes RHO folding. To determine whether increased autophagy in light-exposed normal rods is caused by increased RHO misfolding or phototransduction, we used CRISPR/Cas9 to knock out the RPE65 and GNAT1 genes, which are essential for chromophore biosynthesis and phototransduction respectively. Both knockouts suppressed light-induced autophagy, indicating that although light and misfolded rhodopsin can both induce autophagy in rods, light-induced autophagy is not due to misfolding of RHO, but rather due to phototransduction.

Mutations in prominin-1 (prom1) and photoreceptor cadherin (cdhr1) are associated with inherited retinal degenerative disorders but their functions remain unknown. Here, we used CRISPR-Cas9 to generate prom1-null, cdhr1-null, and prom1 plus cdhr1 double-null Xenopuslaevis and then documented the effects of these mutations on photoreceptor structure and function. Prom1-null mutations resulted in severely dysmorphic photoreceptors comprising overgrown and disorganized disc membranes. Cone outer segments were more severely affected than rods and had an impaired electroretinogram response. Cdhr1-null photoreceptors did not appear grossly dysmorphic, but ultrastructural analysis revealed that some disc membranes were overgrown or oriented vertically within the plasma membrane. Double-null mutants did not differ significantly from prom1-null mutants. Our results indicate that neither prom1 nor cdhr1 are necessary for outer segment disc membrane evagination or the fusion event that controls disc sealing. Rather, they are necessary for the higher-order organization of the outer segment. Prom1 may align and reinforce interactions between nascent disc leading edges, a function more critical in cones for structural support. Cdhr1 may secure discs in a horizontal orientation prior to fusion and regulate cone lamellae size.This article has an associated First Person interview with the first author of the paper.

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Mutations in prominin-1 (prom1) and photoreceptor cadherin (cdhr1) are associated with inherited retinal degenerative disorders such as retinitis pigmentosa, cone-rod dystrophy, and juvenile macular dystrophy. The proteins encoded by these genes are hypothesized to regulate photoreceptor outer segment disc morphogenesis, but their functions remain unknown. We used CRISPR/Cas9 to generate prom1-, cdhr1-, and prom1 + cdhr1-null X. laevis and then documented the effects of these mutations on photoreceptor structure and function. Prom1-null mutations resulted in dysmorphic photoreceptors comprised of overgrown and disorganized disc membranes. Cones were more severely affected than rods; outer segments were elongated and fragmented, and ERG response was impaired. Autofluorescent deposits in the outer segment layer of aging prom1-null animals indicate that secondary toxic effects to the retina or RPE drive retinal degeneration for this mutation, instead of direct effects on outer segment disc morphogenesis. Cdhr1-null photoreceptors did not appear grossly dysmorphic, but ultrastructural analysis revealed that some disc membranes were overgrown or aligned vertically within the plasma membrane. Prom1 + cdhr1-null mutants did not differ significantly from prom1-null mutants. Our results indicate that neither prom1 nor cdhr1 are necessary for outer segment disc membrane evagination or the membrane fusion event involved in disc sealing. Rather, they are necessary for higher-order organization of the nascent outer segment discs. Prom1 may align and reinforce interactions between the disc leading edges, a function more critical in cone photoreceptors for structural support. Cdhr1 may help to align nascent discs and maintain horizontal disc orientation prior to fusion.

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