Orson Moritz

Mutations in the rhodopsin gene (RHO) are the most common cause of autosomal dominant retinitis pigmentosa (adRP). Previously, our research group has identified two distinct mechanisms of light-exacerbated retinal degeneration (RD) associated with P23H and T4K RHO mutations. Here, we developed a transgenic X. laevis carrying human/mouse hybrid T4K RHO and compared the light-exacerbated RD phenotype to human/mouse wildtype, human-T4K and mouse-T4K RHO transgenic X. laevis models. For animals reared in cyclic light, expression of T4K rhodopsin in rods caused significant RD regardless of whether the transgene was human, mouse, or a human/mouse hybrid RHO. When raised in the dark, no significant RD was detected in animals expressing T4K RHO. Therefore, the light-exacerbated RD phenotype associated with the RHO T4K mutation is relatively independent of the underlying RHO cDNA. This hybrid animal model allows us to explore treatment strategies directly on the human gene, streamlining the transfer of therapeutics from lab benches into clinical trials.To date, RP remains an incurable disease. Utilizing a previously developed X. laevis model for adRP, we tested multiple CRISPR/Cas9-based gene-editing strategies to prevent RD in our adRP model. We designed highly specific guide RNAs to 1. Edit the mutant allele and allow for the error-prone non-homologous end joining (NHEJ) repair mechanism to result in a premature stop codon, nullifying the mutant allele 2. Induce simultaneous double-strand breaks on both sides of the start codon, generating large inactivating deletions and 3. Edit the mutant allele and utilize the homology-directed recombination repair mechanism to restore the mutant allele to wildtype. Remarkably, the CRISPR-induced NHEJ repair mechanism appeared to be the most efficient treatment in preventing RD. We postulate that in developing gene editing therapeutics for human RP, similar results are likely to occur, suggesting that the simplest approach may be the most effective. Moreover, our X. laevis models can be used to characterize and understand the pathomechanism of human RP mutations, as well as to develop novel gene-editing treatment strategies. Lastly, our findings demonstrate that CRISPR/Cas9 technology is an effective therapeutic tool for adRP with potential clinical implications for other dominant diseases of the human retina.

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CRISPR/Cas9-mediated mutation of the Xenopus laevis genome has enabled the modeling of autosomal recessive (AR) retinal disease for the first time in this species. Here, knockout (KO) methodology specific to X. laevis rhodopsin1 has been optimized for the targeted mutation of three X. laevis genes orthologous to human genes responsible for AR forms of blindness: cdhr1, rpe65, and gnat1. At 14 days post-fertilization (dpf), these KO mutations did not cause retinal degeneration (RD). However, PCDH21 KO led to disorganization of rod outer segment (ROS) discs. RPE65 KO caused a significant reduction in ROS length by 44 dpf (p=8.6E-8) despite normal rod opsin localization, yet no cone death despite cone opsin mislocalization. Electroretinography (ERG) revealed a significant reduction in photoreceptor responses to light in RPE65 KOs (p=1.889E-13). GNAT1 KO also caused a significant reduction in ROS length by 33 dpf (p=0.014). Further characterization of KO phenotypes is needed to determine whether they can serve as models of AR retinal dystrophy. KO technology was applied to transgenic X. laevis lines expressing human T4K (hT4K) or bovine P23H (bP23H) rhodopsin to characterize components of the respective cell death mechanisms. In animals expressing hT4K rhodopsin, RPE65 KO prevented RD (p=1.02E-10) whereas GNAT1 KO exacerbated RD (p=2.21E-4), suggesting that cell death induced by hT4K rhodopsin requires light and bound chromophore, yet is mitigated by heterotrimeric transducin. In contrast, RPE65 KO in animals expressing bP23H rhodopsin exacerbated RD (p=1.0E-3), suggesting that cell death induced by bP23H rhodopsin requires light but is mitigated by bound chromophore. RPE65 KO was applied to transgenic X. laevis expressing XOP-eGFP-mRFP-LC3, which allows for the quantification of autophagic structures in rods, to characterize the mechanism by which light regulates changes in autophagy. RPE65 KO prevented up-regulation of light-induced autophagy in rods (p=2.5E-7), suggesting that this increase cannot be attributed to an increased misfolding of rhodopsin due to a lack of chromophore in the light. Here we show that CRISPR/Cas9-mediated KO in established X. laevis transgenic lines can be used to test hypotheses about disease mechanisms. These results offer an opportunity for new investigations into the cell biology of inherited retinal disorders.

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Xenopus laevis is a commonly used research subject for retinal physiology and cell biology studies, but its utility is limited by the lack of a robust technology for generation of knock-out (KO) or knock-down (KD) phenotypes. However, new genome manipulation techniques involving CRISPR/Cas9 offer an opportunity for generating gene KOs in X. laevis. RNA-guided Cas9 endonuclease introduces double-stranded DNA breaks into the genome, which are either repaired by error-prone non-homologous-end joining (NHEJ), facilitating indel generation, or by less error-prone homology-directed repair (HDR), facilitating insertion of specific sequences. Rhodopsin was targeted for editing as the expected phenotypes, missing/malformed rod photoreceptor outer segments and lower rhodopsin content, are easily assayed. RNA and transgene methods for CRISPR/Cas9-mediated rhodopsin KOs and knock-ins (KI) in rod photoreceptors of X. laevis were tested, and an RNA injection protocol was developed and optimized. KOs were generated by in vitro transcription and microinjection of Cas9 mRNA, eGFP mRNA, and sgRNAs into in vitro fertilized eggs. Cas9 transgene cassettes were built and tested but editing attempts were unsuccessful. Indel mutations were identified by direct sequencing of PCR products and further characterized by sequencing individual clones. The extent of rhodopsin KO was quantified in 14 post-fertilization day-old tadpoles by anti-rod opsin dot blot assay of retinal extracts, and retinal phenotypes were assessed by cryosectioning and immunolabeling contralateral eyes for confocal microscopy. HDR-mediated KIs were generated by co-injection of a DNA repair fragment, with sufficient homology to the genomic region surrounding predicted dsDNA break-site. Heterologous expression of KIs was confirmed by immunohistochemistry. Delivery of Cas9 by RNA injection can produce high frequency homozygous and heterozygous KOs in X. laevis, permitting analysis in the first generation. I was able to obtain extensive KD generating very severe retinal degeneration phenotypes, and germline transmission of Cas9-mediated indels was confirmed. However, KO was never complete. Sequencing results indicate that first generation animals are chimeric containing many independently derived indels. HDR-mediated KI techniques proved possible, but low in efficiency. These techniques significantly advance the utility of X. laevis as an experimental subject for cell biology and physiology studies.

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Protocadherin-21 (pcdh-21) is a transmembrane protein concentrated at nascent disks in mouse photoreceptors and thought to regulate disk synthesis. PCDH-21 mutations are associated with retinal degenerative diseases. Pcdh-21 undergoes proteolytic cleavage that may be essential for disk synthesis. In mice, Pcdh-21 interacts with prominin-1 (prom-1) and their interaction may be required for their localization and function in disk synthesis.To compare pcdh-21 localization across species, we performed immunofluorescence microscopy using an antibody raised against the N-terminus of X.laevis pcdh-21 (xpcdh-21). In rods and cones of all species, pcdh-21 was localized to nascent disks at the base of the outer segment, suggesting a conserved role in disk assembly. However, in contrast with the idea that pcdh-21 localizes only to the basal outer segment, pcdh-21 was localized to other outer segment regions, and this localization was different across cell types and species, suggesting that pcdh-21 has cell type- and species- specific structural roles. Pcdh-21 was restricted to open disk rims in X.laevis cones. Prom-1, an interacting partner of pcdh-21 in mice, shows identical labeling at the open disk rims. Pcdh-21 and prom-1 may therefore interact to maintain open disk structure. Immunoblots showed that proteolytic cleavage of pcdh-21 may be unique to mice. In X.laevis rods, pcdh-21 labeling in the nascent disks did not vary with disk synthesis rate.We attempted to inhibit pcdh-21 function using a dominant negative approach. Full length pcdh-21 (FL) and deletion constructs consists of mouse (mpcdh-21) and xpcdh-21 were overexpressed in X.laevis rods. Retinal degeneration and disk defects were only observed in retinas overexpressing mpcdh-21 FL. Mpcdh-21 FL was retained in the ER, caused abnormal ER structure, and was not cleaved in X. laevis retinas. Xpcdh-21 variants were correctly localized and did not cause retinal degeneration.This study illustrated that pcdh-21 localization, processing and properties may not be conserved across species. Differences in pcdh-21 localization may reflect differences in disk synthesis mechanisms or disk ultrastructure. However, the conserved association of pcdh-21 and prom-1 with open disk rims and nascent disks suggests that they may form a complex involved in regulating disk synthesis and/ or in maintaining disk structure.

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