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Figure 1- Eye development in Xenopus laevis. Representative sections of embryos staged according to Nieuwkoop and Faber,26 hematoxylin–eosin stained. A, Stage 27, 30 hours postfertilization at 23 C. lr, lens rudiment; oc, optic cup. B, Stage 32, 40 hours postfertilization. lv, lens vesicle. C, Stage 38, 2 days 5 hours postfertilization. le, lens epithelium; plfc, primary lens fiber cells; rpe, retinal pigmented epithelium. D, Stage 41, 3 days 4 hours postfertilization. D0, Higher magnification of the lens shown in D, with interpretative diagram. E, Stage 44, 3 days 20 hours postfertilization. lfc, lens fiber cells. F, Stage 47, 5.5 days postfertilization. gcl, ganglion cell layer; inl, inner nuclear layer; oc, outer cornea; onl, outer nuclear layer; pr, photoreceptors; rpe, retinal pigmented epithelium. F0, Higher magnification of the lens shown in F. G, Stage 48, 1 week postfertilization. All scale bars 200 μm except F0 90 μm
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Figure 2-Eye phenotype of pax6 crispants. We injected embryos with Cas9 enzyme and a sgRNA targeted against pax6.L and pax6.S or buffer, and we allowed the embryos to develop until stage 41–42. A, We separately amplified the pax6.L and pax6.S loci from total embryos for Sanger sequencing. We show representative chromatograms of one sgRNA and one buffer-injected embryo. The sgRNA and PAM sequences are underlined and the cleavage sites are shown. B, The percentages of InDels in individual embryos were calculated from Sanger chromatograms with ICE.38 C-E, from top to bottom whole embryo, higher magnification of the eye and histological section of the eye, scale bar 200 μm. C, Buffer-injected embryo. D and E, Two Cas9- and sgRNA-injected embryos. Key for lens and retina layers, see Figure 1. F, Quantification of eye defects, in tadpoles previously injected with the same sgRNA against pax6 as above (sgRNA #1) or an alternative sgRNA against pax6 (sgRNA #2) G, Functional vision assay of stage 45 buffer-injected embryos and pax6 crispants (sgRNA #1). We put 10 tadpoles in a water tank with a black and a white side, we switched the sides and we counted the number of embryos on the white side after 1 minute. We repeated the switching-counting procedure 10 times for each batch of 10 tadpoles. The cumulative number of embryos on white (between 0 and 100) is shown here, for 8 independent pools of 10 tadpoles. The tadpoles spend more time on white if they are able to distinguish white from black, and the bias in favor of the white side reflects their visual acuity.
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Figure 3-Eye phenotype of gja8 crispants. We injected embryos with Cas9 enzyme and a sgRNA targeted against gja8.L and gja8.S, and we allowed the embryos to develop until stage 47. A and B, Representative Sanger chromatograms of one sgRNA and one buffer-injected embryo and percentages of InDels in individual embryos, as in Figure 2A,B. C, We subcloned the gja8.L amplimere from one gja8 crispant and we sequenced individual clones. We show the sequences and their number of occurrences. ICE is the result of the ICE analysis carried out on the same embryo. D and E, From top to bottom whole embryo, higher magnification of the eye, and histological section of the eye, scale bar 200 μm. D, Buffer-injected embryo. E, Cas9- and sgRNA-injected embryo. Note the grayish lens. F, Quantification of cataract, in tadpoles previously injected with the same sgRNA against gja8as above (sgRNA #1) or an alternative sgRNA against gja8 (sgRNA #2). G
and H, From top to bottom, DAPI staining, Phalloidin staining, and Merge of lens sections. A and P highlight the anterior and posterior pole of the lens. G, Buffer-injected embryo. H, Cas9- and sgRNA #1-injected embryo. I, Functional vision assay of stage 47 buffer-injected embryos and gja8 crispants (sgRNA #1), as in Figure 2G.
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Figure 4-Eye phenotype of dnase2b crispants. We injected embryos with Cas9 enzyme and a mixture of sgRNA targeted against dnase2b.S and a putative dnase2b.L locus, and we allowed the embryos to develop. A
and B, Representative Sanger chromatograms of one sgRNA and one buffer-injected embryo and percentages of InDels in individual embryos, as in Figure 2A,B. Since we failed to amplify the putative dnase2b.L locus, only the results for dnase2b.S are shown. C
and D, Stage 41 larvae previously injected with buffer (C) or Cas9-sgRNA (D). Insets are high magnifications of the eyes of the same larvae. E, Functional vision assay of buffer- injected embryos and dnase2b crispants, as in Figure 2F. F, Histological section of the eye of a buffer-injected stage 41 tadpole. G, Section of the eye of a Cas9- and sgRNA-injected stage 41 tadpole with microphthalia. H and I, Same as F and G with stage 45 tadpoles. Scale
bars 200 μm.
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Figure 5-Effect of 4-phenylbutyrate on the vision of gja8 crispants. We injected embryos with buffer or Cas9 enzyme and a sgRNA targeted against gja8.L and gja8.S (sgRNA #1). We allowed them to develop until stage 44, and we incubated half of them for 48 hours in 4PBA. We led them recover overnight in water before testing their vision as in Figure 2G.
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