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Joanna M Feehan, Beatrice M Tam, Orson L Moritz; CRISPR/Cas9-mediated generation of heterozygous and homozygous rhodopsin knockout photoreceptors in Xenopus laevis. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1156.
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© ARVO (1962-2015); The Authors (2016-present)
Xenopus laevis is a commonly used research subject for retinal physiology and cell biology studies, but its utility is limited by lack of a robust technology for generation of knockout or knockdown phenotypes. However, new genome manipulation techniques involving CRISPR/Cas9 should permit the generation of gene knockouts in Xenopus laevis. We are optimizing CRISPR/Cas9 gene inactivation techniques for Xenopus laevis using RNA injection and transgene expression to generate homozygous and heterozygous knockouts of the rhodopsin gene.
Cas9, eGFP, and sgRNAs were translated in vitro and injected into fertilized Xenopus laevis eggs, or included on transgene constructs used for restriction enzyme mediated integration transgenesis procedures. Insertions and deletions were identified by direct sequencing of PCR products and further characterized by sequencing individual clones. The extent of rhodopsin knockout was quantified in 14 day-old tadpoles by anti-rhodopsin dot blot assay of retinal extracts, and retinal phenotypes were assessed by cryosectioning and immunolabeling contralateral eyes for confocal microscopy.
We have designed sgRNAs to target the Xenopus laevis rhodopsin gene at two sites within the first exon, one of which contains a common polymorphism. Sequencing results suggest that these produce homozygous and heterozygous knockouts respectively. Using RNA injection methods, we were able to obtain extensive knockdown generating very severe retinal degeneration phenotypes. However, knockout was never complete. Sequencing results indicate that each animal is chimeric containing many independently derived indels. We are further comparing the efficiencies of RNA injection delivery methods with transgene-based expression of Cas9 and sgRNA.
Delivery of Cas9 by RNA injection techniques can produce high frequency homozygous and heterozygous knockouts in X. laevis, permitting analysis in the first generation. This technique significantly advances the utility of Xenopus laevis as an experimental subject for cell biology and physiology studies.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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