Abstract
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.
Highlights
Recent development of the CRISPR/Cas[9] gene editing system should allow gene modification and knockout in virtually any species, including X. laevis[25]
We report the successful application of CRISPR/Cas[9] technology to editing of the X. laevis genome
Based on blast searches using previously published X. laevis rhodopsin gene sequence[39], we identified three gene sequences encoding rod opsins, i.e. rhodopsin, rho.S (XB-GENE-17342665) on chromosome 4S, and two paralogous gene sequences, rho.L (XB-GENE-966893) and rho.[2].L (XB-GENE-18034123), corresponding to the sequence identified by Batni et al.[39] located within a 17 kb region on chromosome 4 L
Summary
Recent development of the CRISPR/Cas[9] gene editing system should allow gene modification and knockout in virtually any species, including X. laevis[25]. Indels in early exons can create frame-shift mutations that trigger nonsense mediated decay, acting as loss-of function mutations or “knockouts”, or in-frame deletions and insertions that result in protein instability This technology has previously been successfully applied to generate knockouts of tyrosinase in X. laevis and the related diploid species Xenopus tropicalis[29, 30] to generate F0 albino animals. We anticipated that loss-of-function mutations would cause reduced rhodopsin expression, while mutations that alter the amino acid sequence may cause aggressive RD, and that these phenotypes would be detectable by rhodopsin immunoassays and confocal microscopy[7] By using this technology to create genomic DNA breakpoints and indel mutations, we were able to generate rhodopsin knockout and gain of function phenotypes in X. laevis tadpoles that model the human conditions of recessive and dominant retinitis pigmentosa. These techniques add new versatility to X. laevis as a research subject, and are likely to be highly useful for cell and developmental biologists, including those conducting research on rhodopsin function and rhodopsin gene regulation, and retinal disease mechanisms
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