Xenbase Image ID: 158282

Fig. S1. Assessment of mutagenesis on vegetal PGC-containing explants relative to the rest of the embryo. (A) Dissection scheme. Embryos were injected animally at the 1-cell stage with tyr sgRNA-complexed Cas9 protein. At blastula stage 9 they were dissected into animal cap and vegetal PGC transplant regions, and the embryo remainder composed of vegetal and marginal zone tissues (mesoderm and endoderm; Meso/endo). Individual embryo fragments were lysed and the tyr target region was PCR amplified. The amplicon population was subjected to direct sequencing (DSP; Nakayama et al., 2014). (B) DSP assay results are shown for each region from a single embryo. The position of the predicted Cas9 cleavage site is shown as a vertical line. One can see that, in comparison to the sequence profile of an uninjected embryo at top, all three regions from one injected embryo show similar profiles of “mixed” peaks beginning just upstream of the cleavage site and extending downstream to it. These results suggest that the PGC explant has been successfully mutagenized and that the remainder of the embryo (carcass) can be used as a proxy for the PGCs to determine which embryos carrying leapfrog transplants likely have successfully CRISPR/Cas9 mutated PGCs. Image published in: Blitz IL et al. (2016) Copyright © 2016. Image reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License. Larger Image Printer Friendly View

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