XB-IMG-84487
Xenbase Image ID: 84487
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Figure 2. Successful targeting of the tyrosinase gene caused albinism in Xenopus embryos. (a) Different dose combinations of Cas9 mRNA and sgRNA were tested. The severity of the phenotype was directly dependent on the amounts of RNAs injected. bacCas9, the original bacterial-codon Cas9 mRNA (which shows examples with a weak phenotype; a patchy loss of pigmentation in the RPE is indicated by white arrowheads). humCas9, the modified Cas9 using humanized-codons. sgRNA, targeting tyrosinase gene (first target, see Fig. 3b for location relative to ATG). The toxicity of sgRNA seems to vary depending on its sequence and also on the batch of embryos. For example, in one specific experiment, tyrosinase sgRNA (target1) was overall relatively nontoxic, with more than 75% of injected embryos developing normally one day after injection at all tested doses shown here (76.9�97% survivors [n=19�33] as opposed to 84.8% of uninjected embryos [n=46]), whereas in other experiments, survival was between 50�60% (see Supporting Information Table 1). (b) Representative results of conventional sequencing assays (top three panels). Each single embryo injected with indicated RNAs was lysed and the targeted genomic region was PCR-amplified; amplicons were then directly sequenced (DSP assay, see the text). Perturbation of peaks on the 3' side of the PAM region (red arrows) suggests in-del events happen in-between the target sequence (shaded in purple) and the PAM region (shaded in red). PCR amplicons were recloned and sequenced to show the profile of individual mutations found in mosaic individuals (bottom sequence alignments). Dashes (-) indicate gaps. Lowercase green characters indicate insertions or substitutions. The numbers in parentheses indicate the frequency of each mutation pattern seen in total numbers of sequenced clones. Image published in: Nakayama T et al. (2013) Copyright © 2013. Image reproduced with permission of the Publisher, John Wiley & Sons. Larger Image Printer Friendly View |