Modeling Dominant and Recessive Forms of Retinitis Pigmentosa by Editing Three Rhodopsin-Encoding Genes in Xenopus Laevis Using Crispr/Cas9

• Published in: Scientific reports Vol. 7; no. 1; pp. 6920 - 14
• Main Authors: Feehan, Joanna M., Chiu, Colette N., Stanar, Paloma, Tam, Beatrice M., Ahmed, Sheikh N., Moritz, Orson L.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 31.07.2017; Nature Publishing Group; Nature Portfolio
• Subjects: 14/19; 14/28; 38/1; 42/41; 42/70; 45/23; 45/41; 631/1647/1513/1967/3196; 631/1647/334/1874/761; 631/1647/767/1424; 631/45/612/1237; 64/114; 692/699/3161/3165; Animals; Biological research; Biosynthesis; Cloning; Confocal microscopy; CRISPR; CRISPR-Associated Protein 9 - genetics; CRISPR-Cas Systems; Developmental biology; Disease Models, Animal; Eggs; Female; Gene Editing - methods; Genes; Genes, Dominant; Genes, Recessive; Genome editing; Genomes; Green Fluorescent Proteins - genetics; Homology; Humanities and Social Sciences; Humans; Injection; Male; mRNA; multidisciplinary; Mutation; Nucleotide sequence; Offspring; Phenotype; Point Mutation; Retina; Retinal degeneration; Retinitis; Retinitis pigmentosa; Retinitis Pigmentosa - genetics; Retinitis Pigmentosa - pathology; Rhodopsin; Rhodopsin - genetics; RNA, Guide, CRISPR-Cas Systems - genetics; Science; Science (multidisciplinary); Sequence Deletion; Xenopus laevis; Xenopus laevis - embryology; Xenopus Proteins - genetics
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-017-07153-4

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.