Abstract
Purpose :
RPE65 is an isomerohydrolase that converts all-trans-retinyl ester to 11-cis-retinol, which is then oxidized into 11-cis-retinal, the ligand for rod and cone opsins, by downstream enzymes. Studies in Rpe65-/- mice have shown an almost complete depletion of 11-cis-retinal in the photoreceptors causing a slow retinal degeneration. In order to test the involvement of bound chromophore in the cell death mechanism in a number of rhodopsin mutations which cause light-exacerbated retinal degeneration, we have generated RPE65 knockout X. laevis.
Methods :
RPE65 knockouts were generated using CRISPR/Cas9 technology by injecting a single sgRNA targeting exon 7 of both the rpe65.L and rpe65.S genes into single cell X. laevis embryos. F0 or F1 inbred animals were raised with or without 0.005% phenylthiourea to prevent melanization to enchance the anti-RPE65 antibody signals in immunohistochemistry. Animals were analyzed at (i) 14 days post fertilization by immunohistochemistry, DNA sequencing, and western blotting; (ii) 6 weeks post fertilization by scotopic, photopic, and flicker electroretinagram, DNA sequencing, and immunohistochemistry; (iii) 4 months post fertilization by difference spectra obtained from retinas isolated from dark adapted animals.
Results :
CRISPR/Cas9 targeted animals showed extensive DNA editing near the predicted cut site in both rpe65 genes. Sequencing analysis indicated that ~25% of F1 inbred animals retained one WT rpe65.S allele, while all analyzed animals were edited in rpe65.L gene.
No RPE65 protein could be detected in edited animals by western blotting at 14 dpf regardless of genotype. By immunohistochemistry, there was markedly reduced RPE65 protein expression at 14 dpf with no changes to retinal morphology observed otherwise. Scotopic ERG response in dark adapted animals was abolished. Photopic ERG response resulted in reduced a-wave and b-wave amplitude. Flicker ERG response peaks were comparable to WT at the highest light intensity, but were much reduced or abolished at lower intensities. Typical rhodopsin absorption maximum at 500nm was absent, suggesting a complete elimination of the rhodopsin chromophore.
Conclusions :
Using CRISPR/Cas9 technology, we have generated RPE65 knockout X. laevis, and transmitted the resulting genotype and phenotype to the F1 generation. These animals are similar to mammalian RPE65 knockouts in that they have minimal ERGs and residual chromophore, with minimal or no retinal degeneration.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.