Feehan JM et al. (2017), Modeling Dominant and Recessive Forms of Retini...

XB-ART-53879

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 .

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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.

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

???displayArticle.disOnts??? retinitis pigmentosa
???displayArticle.omims??? RETINITIS PIGMENTOSA; RP
Phenotypes: Xla Wt + rho CRISPR (Fig.1.A) [+]

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	Figure 1. Injection of Cas9 and guide RNAs causes reduced rod opsin levels and retinal degeneration. (A) Rod opsin levels assayed in individual eyes by dot blot assay at 14 dpf. Each data point represents a different animal. P values are for Tukey multiple comparisons test for comparisons with WT group. P (ANOVA) = 4.1 × 10−19 (B) Phenotypes assessed by confocal microscopy. Retinas from animals injected with Cas9 mRNA and rhosg3 had missing and malformed rod photoreceptors. Green: anti-rhodopsin (B630N). Red: wheat germ agglutinin. Blue: Hoechst 33342. RPE: retinal pigment epithelium. OS: outer segments. IS: inner segments. ONL: outer nuclear layer. INL: inner nuclear layer. Bar = 50 µm.
	Figure 2. Relative to guide RNA rhosg3, rhosg1 causes greater reductions in rod opsin and greater rod photoreceptor loss. 6 ng of Cas9 mRNA and 2 ng of sgRNA were co-injected. (A) Rod opsin levels assayed in individual eyes by dot blot assay at 14 dpf. Each data point represents a different animal. P values are for Tukey multiple comparisons test for comparisons with WT group. P (ANOVA) = 2.9 × 10−9 (B) Phenotypes assessed by confocal microscopy. Green: anti-rhodopsin (B630N). Red: wheat germ agglutinin. Blue: Hoechst 33342. RPE: retinal pigment epithelium. OS: outer segments. IS: inner segments. ONL: outer nuclear layer. INL: inner nuclear layer. Bar = 50 µm.
	Figure 3. Indels are present in the genomic DNA of F0 animals. (A,B) Sample trace reads from dye-termination Sanger sequencing. (A) WT sample. (B) Edited with rhosg1. Cleavage is predicted to occur after the nucleotide highlighted in blue (numbered “zero” on plots below). (C–F) Comparisons of gene editing efficiency between guide RNAs, genes, and stage of development. Calculation of the sequence fidelity score is described under Methods. Each plot shows data derived from three animals, numbered 1–3, 4–6, 7–9 and 10–12.
	Figure 4. Characterization of indels by sequencing genomic DNA clones: (A–H) Sequences obtained from individual pBluescript-SKII+ clones of PCR products derived from genomic DNA isolated from F0 embryos at either 1 dpf or 14 dpf (as indicated) edited with Cas9 mRNA+ rhosg1 or rhosg3 (as indicated). The reverse complement sequence of the guide RNA is shown above the individual genomic DNA sequences in blue. The targeted sequences are also shown in blue. Mismatches with the guide RNA are shown in magenta. Insertions are noted in green. The protospacer adjacent motif (PAM) sequence is shown in red. Bases that are diagnostic of rho.L vs. rho.2.L are shown in orange. The number at the beginning of each sequence corresponds to sample numbers for direct sequencing results shown in Fig. 3.
	Figure 5. Germline transmission of genomic DNA editing and phenotypes of F1 offspring: Plots: Rod opsin levels assayed in individual eyes by dot blot assay at 14 dpf. Each data point represents a different F1 animal. Each X-axis point represents offspring of a single mating, for which the F0 parents are indicated as being WT and/or F0 animals (named Male 1, Male 2, etc.). Each plot represents samples analyzed on a separate dot blot, and all animals on each plot were modified using the same sgRNA, indicated at the bottom of the plot. (A–I) Phenotypes of F1 animals assessed by confocal microscopy. (A) Wildtype. (B–I) genetically modified (genotypes indicated on panels, for frame-conserving mutations the altered amino acid sequence is shown, with inserted amino acid residues shown in red). (B,C) frame-conserving mutations with minimal phenotype. (D–F) frame-conserving indels with significant RD phenotypes. (G–I) frame shifting indels. (A–C) High magnification panels: frame-conserving indels generating minimal or no RD phenotype do not alter rhodopsin localization, which is largely confined to outer segments (OS) and wheat germ agglutinin-positive inner segment (IS) membranes (arrowheads). (D–F) High magnification panels: frame-conserving indels generating significant RD phenotypes alter rhodopsin localization in inner segments causing a punctate distribution (D, arrowhead) or diffuse labeling consistent with ER retention (E,F, arrowheads). Green: anti-rhodopsin (B630N – A-I or 514–18 A-D high mag). Red: anti-rod transducin. Blue: Hoechst 33342. Bars = 20 µm (low mag) or 5 µm (high mag). RPE: retinal pigment epithelium. OS: outer segments. IS: inner segments. ONL: outer nuclear layer. INL: inner nuclear layer.
	Figure 6. Electron microscopy of rod photoreceptors with loss of function mutations in one rho.L allele: (A) images of rods with a 1 bp insertion in one rho.L allele induced by rhosg3. (B) Comparable images from WT rods. Outer segment (OS) disks are densely packed at both the rim and lamellar (LAM) regions. Disk rims are closely apposed to the plasma membrane (PM). No significant abnormalities were identifiable. IS: inner segment. Bar = 400 nm (left) 200 nm (center) or 100 nm (right).
	Figure 7. HDR mediated gene alterations assessed by confocal microscopy: (A) RD in an animal edited using sgRNA rhosg4 (B,C,C’) images derived from an animal in which rhosg4 was used to direct targeted insertion of eGFP into rho.L. A number of eGFP-positive rods are present, while other cells of the retina and lens do not show eGFP expression. Significant RD is present (D,E) images derived from an animal with targeted mutation of residue M13→F in rho.L, and stained with anti-mammalian rhodopsin (2B2). There is no identifiable RD. (A–C) Green: eGFP. (D,E) green: anti-mammalian rhodopsin (2B2). (A,B) Red: anti-rod transducin. C–E: Red: wheat germ agglutinin. (A–E) Blue: Hoechst 33342. OS: outer segments. IS: inner segments. ONL: outer nuclear layer. INL: inner nuclear layer. GCL: ganglion cell layer. (A,B,D) Bar = 200 µm. (C) Bar = 50 µm. (C’) Bar = 10 µm. (E) Bar = 20 µm. Panels C and C’ are confocal projections derived from 10 confocal sections.

References [+] :

Allen, Transgenic Xenopus laevis embryos can be generated using phiC31 integrase. 2005, Pubmed, Xenbase