UBC Theses and Dissertations
Development of methodology for genome editing in Xenopus laevis using CRISPR/Cas9, targeting the rhodopsin gene Feehan, Joanna Marie
Xenopus laevis is a commonly used research subject for retinal physiology and cell biology studies, but its utility is limited by the lack of a robust technology for generation of knock-out (KO) or knock-down (KD) phenotypes. However, new genome manipulation techniques involving CRISPR/Cas9 offer an opportunity for generating gene KOs in X. laevis. RNA-guided Cas9 endonuclease introduces double-stranded DNA breaks into the genome, which are either repaired by error-prone non-homologous-end joining (NHEJ), facilitating indel generation, or by less error-prone homology-directed repair (HDR), facilitating insertion of specific sequences. Rhodopsin was targeted for editing as the expected phenotypes, missing/malformed rod photoreceptor outer segments and lower rhodopsin content, are easily assayed. RNA and transgene methods for CRISPR/Cas9-mediated rhodopsin KOs and knock-ins (KI) in rod photoreceptors of X. laevis were tested, and an RNA injection protocol was developed and optimized. KOs were generated by in vitro transcription and microinjection of Cas9 mRNA, eGFP mRNA, and sgRNAs into in vitro fertilized eggs. Cas9 transgene cassettes were built and tested but editing attempts were unsuccessful. Indel mutations were identified by direct sequencing of PCR products and further characterized by sequencing individual clones. The extent of rhodopsin KO was quantified in 14 post-fertilization day-old tadpoles by anti-rod opsin dot blot assay of retinal extracts, and retinal phenotypes were assessed by cryosectioning and immunolabeling contralateral eyes for confocal microscopy. HDR-mediated KIs were generated by co-injection of a DNA repair fragment, with sufficient homology to the genomic region surrounding predicted dsDNA break-site. Heterologous expression of KIs was confirmed by immunohistochemistry. Delivery of Cas9 by RNA injection can produce high frequency homozygous and heterozygous KOs in X. laevis, permitting analysis in the first generation. I was able to obtain extensive KD generating very severe retinal degeneration phenotypes, and germline transmission of Cas9-mediated indels was confirmed. However, KO was never complete. Sequencing results indicate that first generation animals are chimeric containing many independently derived indels. HDR-mediated KI techniques proved possible, but low in efficiency. These techniques significantly advance the utility of X. laevis as an experimental subject for cell biology and physiology studies.
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