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Sci Rep 2017 Jul 31;71:6920. doi: 10.1038/s41598-017-07153-4.
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Modeling Dominant and Recessive Forms of Retinitis Pigmentosa by Editing Three Rhodopsin-Encoding Genes in Xenopus Laevis Using Crispr/Cas9.

Feehan JM , Chiu CN , Stanar P , Tam BM , Ahmed SN , Moritz OL .

The utility of Xenopus laevis, a common research subject for developmental biology, retinal physiology, cell biology, and other investigations, has been limited by lack of a robust gene knockout or knock-down technology. Here we describe manipulation of the X. laevis genome using CRISPR/Cas9 to model the human disorder retinitis pigmentosa, and to introduce point mutations or exogenous DNA sequences. We introduced and characterized in-frame and out-of-frame insertions and deletions in three genes encoding rhodopsin by co-injection of Cas9 mRNA, eGFP mRNA, and single guide RNAs into fertilized eggs. Deletions were characterized by direct sequencing and cloning; phenotypes were assessed by assays of rod opsin in retinal extracts, and confocal microscopy of cryosectioned and immunolabeled contralateral eyes. We obtained germline transmission of editing to F1 offspring. In-frame deletions frequently caused dominant retinal degeneration associated with rhodopsin biosynthesis defects, while frameshift phenotypes were consistent with knockout. We inserted eGFP or point mutations into rhodopsin genes by co-injection of repair fragments with homology to the Cas9 target sites. Our techniques can produce high frequency gene editing in X. laevis, permitting analysis in the F0 generation, and advancing the utility of X. laevis as a subject for biological research and disease modeling.

PubMed ID: 28761125
PMC ID: PMC5537283
Article link: Sci Rep

Species referenced: Xenopus laevis
Genes referenced: gnat1 rho rho.2 rpe
GO keywords: retinal pigment epithelium development [+]
Antibodies: Rho Ab12 Rho Ab3
gRNAs referenced: rho gRNA1 rho gRNA2 rho.2 gRNA1

Disease Ontology terms: retinitis pigmentosa
Phenotypes: Xla Wt + rho CRISPR (Fig.1.A) [+]

Article Images: [+] show captions
References [+] :
Allen, Transgenic Xenopus laevis embryos can be generated using phiC31 integrase. 2005, Pubmed, Xenbase