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.

Retinitis pigmentosa (RP) is an inherited retinal degeneration (RD) that leads to blindness for which no treatment is available. RP is frequently caused by mutations in ; in some animal models, RD is exacerbated by light. Valproic acid (VPA) is a proposed treatment for RP and other neurodegenerative disorders, with a phase II trial for RP under way. However, the therapeutic mechanism is unclear, with minimal research supporting its use in RP. We investigated the effects of VPA on models of RP expressing human P23H, T17M, T4K, and Q344ter rhodopsins, which are associated with RP in humans. VPA ameliorated RD associated with P23H rhodopsin and promoted clearing of mutant rhodopsin from photoreceptors. The effect was equal to that of dark rearing, with no additive effect observed. Rescue of visual function was confirmed by electroretinography. In contrast, VPA exacerbated RD caused by T17M rhodopsin in light, but had no effect in darkness. Effects in T4K and Q344ter rhodopsin models were also negative. These effects of VPA were paralleled by treatment with three additional histone deacetylase (HDAC) inhibitors, but not other antipsychotics, chemical chaperones, or VPA structural analogues. In WT retinas, VPA treatment increased histone H3 acetylation. In addition, electron microscopy showed increased autophagosomes in rod inner segments with HDAC inhibitor (HDACi) treatment, potentially linking the therapeutic effects in P23H rhodopsin animals and negative effects in other models with autophagy. Our results suggest that the success or failure of VPA treatment is dependent on genotype and that HDACi treatment is contraindicated for some RP cases. Retinitis pigmentosa (RP) is an inherited, degenerative retinal disease that leads to blindness for which no therapy is available. We determined that valproic acid (VPA), currently undergoing a phase II trial for RP, has both beneficial and detrimental effects in animal models of RP depending on the underlying disease mechanism and that both effects are due to histone deacetylase (HDAC) inhibition possibly linked to autophagy regulation. Off-label use of VPA and other HDAC inhibitors for the treatment of RP should be limited to the research setting until this effect is understood and can be predicted. Our study suggests that, unless genotype is accounted for, clinical trials for RP treatments may give negative results due to multiple disease mechanisms with differential responses to therapeutic interventions.

PURPOSE. The rhodopsin mutation P23H is responsible for a significant portion of autosomaldominant retinitis pigmentosa, a disorder characterized by rod photoreceptor death. The mechanisms of toxicity remain unclear; previous studies implicate destabilization of P23H rhodopsin during light exposure, causing decreased endoplasmic reticulum (ER) exit and ER stress responses. Here, we probed phototransduction in Xenopus laevis rods expressing bovine P23H rhodopsin, in which retinal degeneration is inducible by light exposure, in order to examine early physiological changes that occur during retinal degeneration. METHODS. We recorded single-cell and whole-retina responses to light stimuli using electrophysiology. Moreover, we monitored morphologic changes in rods after different periods of light exposure. RESULTS. Initially, P23H rods had almost normal photoresponses, but following a brief light exposure varying from 4 to 32 photoisomerizations per disc, photoresponses became irreversibly prolonged. In intact retinas, rods began to shed OS fragments after a rodsaturating exposure of 12 minutes, corresponding to approximately 10 to 100 times more photoisomerizations. CONCLUSIONS. Our results indicate that in P23H rods light-induced degeneration occurs in at least two stages, the first involving impairment of phototransduction and the second involving initiation of morphologic changes.

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Retinitis pigmentosa (RP) is an inherited retinal degeneration (RD) that leads to blindness for which no treatment is available. RP is frequently caused by mutations in Rhodopsin; in some animal models, RD is exacerbated by light. Valproic acid (VPA) is a proposed treatment for RP and other neurodegenerative disorders, with a phase II trial for RP under way. However, the therapeutic mechanism is unclear, with minimal research supporting its use in RP. We investigated the effects of VPA on Xenopus laevis models of RP expressing human P23H, T17M, T4K, and Q344ter rhodopsins, which are associated with RP in humans. VPA ameliorated RD associated with P23H rhodopsin and promoted clearing of mutant rhodopsin from photoreceptors. The effect was equal to that of dark rearing, with no additive effect observed. Rescue of visual function was confirmed by electroretinography. In contrast, VPA exacerbated RD caused by T17M rhodopsin in light, but had no effect in darkness. Effects in T4K and Q344ter rhodopsin models were also negative. These effects of VPA were paralleled by treatment with three additional histone deacetylase (HDAC) inhibitors, but not other antipsychotics, chemical chaperones, or VPA structural analogues. In WT retinas, VPA treatment increased histone H3 acetylation. In addition, electron microscopy showed increased autophagosomes in rod inner segments with HDAC inhibitor (HDACi) treatment, potentially linking the therapeutic effects in P23H rhodopsin animals and negative effects in other models with autophagy. Our results suggest that the success or failure of VPA treatment is dependent on genotype and that HDACi treatment is contraindicated for some RP cases.

Aim: To examine the utility of gene editing therapies for retinitis pigmentosa using Xenopus laevis carrying a mutation in Rhodopsin. Methods: Xenopus laevis were genetically modified using CRISPR-Cas9 based methods and characterized by Sanger sequencing, dot blot, electroretinography, and confocal microscopy. Results: We identified genetically modified Xenopus laevis carrying a net 12 base pair deletion in the Rho.L gene. These animals have a retinal degeneration that is apparent by 14 days, with abnormal or missing rod outer segments, and a reduced electroretinogram signal. We prevented the majority of this retinal degeneration via a treatment strategy using a single sgRNA to neutralize the mutant allele via non-homologous end joining, yielding long-term improvements in histology and the electroretinogram. A second strategy using two sgRNAs to generate large deletions in the mutant allele was also successful, but did not significantly improve outcomes relative to the single-guide strategy as it was less efficient. We found limited evidence of success with a third strategy dependent on homology-directed repair; this treatment was also too inefficient to generate an outcome superior to the single-guide strategy. Conclusion: Our results demonstrate the utility of this new Xenopus laevis model for rapidly assessing and comparing multiple gene-editing based treatment strategies. We conclude that it would be technically difficult to improve on the simple single-guide based strategy, as strategies requiring multiple successive events (such as cleavage followed by homology-directed repair) are likely to be less efficient.