Abstract
Purpose :
Mutations in GNAT1 (which encodes rod a-transducin) are associated with Congenital Stationary Night Blindness. We characterized Xenopus laevis with CRISPR-edited GNAT1 genes to survey the range of possible phenotypes.
Methods :
We previously generated genetically chimeric GNAT1 edited animals using CRISPR. We interbred these F0 animals to produce F1 animals of defined genotypes with the intention of generating full knockouts. The resulting animals have mutations in both X. laevis GNAT1 genes (GNAT1.S and GNAT1.L)
We investigated visual responses of the F1 animals and age-matched unedited controls via electroretinography (ERG). Subsequently, one eye was sectioned and stained for confocal microscopy while the other was solubilized for western blot analysis. Genomic DNA was sequenced to establish phenotype/genotype correlations.
Results :
Reduction in the level of GNAT1 protein lead to a suppression of the ERG as expected. However, we also noted a delay in the timing of the A and B-waves. Greater reduction in GNAT1 protein was associated with greater suppression and delay of the ERG.
X. laevis tadpoles with nonsense mutations in all four GNAT1 alleles presented with greatly reduced and delayed dark-adapted ERGs. Histology and western blot confirmed the absence of GNAT1 protein, indicating full knockout.We also identified tadpoles carrying a point mutation (Q186L) that conferred an ERG phenotype similar to full knockout, but with minimally reduced GNAT1 protein. GNAT1 Q186L appeared non-functional, as it was largely confined to rod inner segments under dark conditions.
In a final group of animals we observed abnormal rod morphology at the connecting cilium associated with a three base pair deletion (T183,Q184-->K183). Structural abnormalities in photoreceptors linked to GNAT1 mutations have not been reported previously.
We did not observe significant retinal degeneration in any edited animals.
Conclusions :
Decreased GNAT1 expression affects not only the amplitude but the time course of the dark-adapted ERG in CRISPR-edited X.laevis, possibly indicating a role for GNAT1 in rhodopsin inactivation. X. laevis with four nonsense alleles do not express GNAT1. The majority of in-frame GNAT1 mutations cause misfolding and reduced expression. However, we identified two specific mutations that respectively produce non-functional GNAT1 without reduced expression, and structurally abnormal photoreceptors.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.