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Joanna M Feehan, Beatrice M Tam, Orson L Moritz; Rapid high-yield production of rhodopsin knockout Xenopus laevis using CRISPR/Cas9. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3590.
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© ARVO (1962-2015); The Authors (2016-present)
Xenopus laevis is a widely used animal model in vision research, and, although transgenic X. laevis are generated relatively easily; researchers have previously been unable to engineer knockouts (KO) or knockdowns in X. laevis. New techniques for genome editing using CRISPR/Cas9 have been applied to related organisms, including Xenopus tropicalis. Our goal is to develop a CRISPR/Cas9-based technique for genome editing in X. laevis, using the Rhodopsin (RHO) gene as an initial target. RHO KO X. laevis may be useful for investigating trafficking properties of rhodopsin mutants in the absence of wild-type endogenous rhodopsin. Rhodopsin KO animals are anticipated to have a phenotype that is easily detected by histology (no rod outer segments in complete KO), immunoassay (reduced rhodopsin) and physiology (reduced or no rod electroretinogram (ERG)).
We designed sequence-specific single-guide RNAs (sgRNAs) to target the X. laevis RHO gene, and injected them into fertilized X. laevis eggs in combination with mRNA encoding Cas9. At 14 days post-fertilization, rhodopsin levels in the eyes of the resulting animals were determined by immunoassay and compared to rhodopsin levels from uninjected animals and animals injected with null sgRNA. We optimized the procedure by varying the temperature, sgRNA target-site specificity, sgRNA concentration, and use of recombinant Cas9.
Our current procedure results in approximately 50% reduction in the average rhodopsin protein levels in injected embryos, indicating that the majority of animals have at least a partial KO phenotype. The retinal phenotype of these mutants included missing or shortened rod outer segments relative to wild-type embryos. The results suggest chimeric KO in the majority of animals.
CRISPR/Cas9-based techniques can result in very high KO rates in X. laevis. The efficiency is sufficiently high that phenotypes can be successfully examined in primary KO animals.
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