Abstract
Purpose :
We have previously demonstrated that the rhodopsin mutation T4K, which disrupts glycosylation, causes a light-induced retinal degeneration (RD) in X. laevis that is not associated with a biosynthesis defect. The RD requires photoactivation of the mutant rhodopsin. Therefore, it is possible that downstream components of the phototransduction cascade are involved. Notably, stable arrestin/rhodopsin complexes have previously been implicated in retinal degeneration caused by other rhodopsin mutations. Here we examine the involvement of arrestin via CRISPR/Cas9 mediated knockout of arrestin in T4K rhodopsin-expressing and wild type X. laevis.
Methods :
Visual arrestin expression was knocked out in WT and T4K rhodopsin transgenic animals using CRISPR/Cas9 editing methodology. Synthetic guide RNAs (sgRNA) were made to 4 different Cas9 target sites within the X. laevis arrestin coding sequence by in vitro transcription. Cas9 mRNA, GFP mRNA and sgRNAs were co-injected into fertilized X. laevis embryos at the 1 cell stage. On day 2 post-fertilization (dpf), GFP expressing embryos were selected for futher rearing and analysis. PCR amplification of the Cas9 target region was performed and the resulting products sequenced to assess whether editing had occurred. On dpf14, tadpole eyes were enucleated and solubilized for quantitative dot blot and Western blot analysis or fixed for IHC.
Results :
Two sgRNAs were identified which successfully edited the X. laevis arrestin gene, creating small insertions and deletions. Co-injection of the two guides resulted in dramatic reduction of arrestin expression in X. laevis retinas in the F0 generation as assessed by western blotting and immunofluorescence. Retinal degeneration was not evident in retinal sections although there was a small decrease in total rhodopsin levels in arrestin KO eyes. Unexpectedly, arrestin knockout significantly exacerbated RD in X. laevis expressingT4K rhodopsin.
Conclusions :
CRISPR/Cas9 can be used to knock out phototransduction components in F0 animals with very high efficiency, allowing us to examine their importance in X. laevis models of RP. In this case, we determined that the presence of arrestin moderates retinal degeneration caused by T4K rhodopsin, providing new information on the underlying mechanism of retinal degeneration. Future studies will involve knockout of other phototransduction components.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.