Abstract
Purpose :
Xenopus laevis frogs are useful for the study of rhodopsin (RHO) variants due to the ease with which transgenic animals can be generated. RHO knockout X. laevis would provide a useful background for the generation of transgenic animals in which the properties of RHO mutants could be studied in the absence of WT RHO. However, generation of knockouts is complicated by the fact that there are three functional X. laevis RHO genes totaling six alleles. Therefore, we have used a multi-generation approach to develop RHO knockouts, using multiple rounds of CRISPR/Cas9-based gene editing.
Methods :
Single-cell X. laevis embryos were injected with Cas9 mRNA and sgRNAs designed to target and edit the X. laevis RHO genes. The resulting animals were raised to maturity and bred to identify animals with null RHO alleles caused by frameshift mutations in the first exon. A second round of editing was used to achieve full knockout of remaining functional RHO alleles by editing the second exon. Rod opsin expression was examined by dot blot, and retinal degeneration and photoreceptor morphology were investigated by confocal microscopy.
Results :
By characterizing partial RHO knockout; we found that the RHO.L gene was the highest expressed of the three X. laevis RHO genes, and is responsible for 80% of the rod opsin in a tadpole retina. Partial RHO knockouts did not cause extensive retinal degeneration, but resulted in smaller outer segments. Complete RHO knockouts were generated from a background genotype in which RHO.L and RHO.2.L were knocked out and the RHO.S gene carried an in-frame 12 bp deletion that caused retinal degeneration. Editing of the remaining mutant RHO.S gene reduced retinal degeneration. Photoreceptors with complete RHO knockout as assessed by antibody labeling survived to 14 days, and elaborated morphologically abnormal outer segment-like membranes that lacked RHO.
Conclusions :
We have generated X. laevis in which a subset of rod photoreceptors lack RHO expression. Further breeding should result in complete RHO knockout lines. Photoreceptors lacking RHO survive to at least 14 days post-fertilization, and elaborate outer segment-like membranes, a result that differs significantly from previous reports in knockout mice. Further characterization of these membranes may demonstrate the role of RHO in outer segment structure; these animals will also be useful for examining the properties of RHO mutants in the absence of wildtype RHO.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.