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Fig. 4 - Supplemental 1 Examples of successful disruption of sterlet Bmp5 by CRISPR/Cas9-mediated mutagenesis in G0-injected embryos. (A) Schematic showing the exon structure of the sterlet Bmp5 gene relative to conserved domains and the target sites of Bmp5 sgRNAs (Table 1). (B?D) Sterlet crispants at stage 45 after in situ hybridisation for the hair cell and electroreceptor marker Cacna1d (also expressed in taste buds on the barbels). In comparison to the control Tyr crispant (B), the two Bmp5 crispants (C,D) have fewer ampullary organs (this is particularly clear on the operculum). These images are also shown in Figure 4A, C and E. (E?I) Outputs are shown from Synthego?s 'Inference of CRISPR Edits' (ICE) tool (Conant et al., 2022). This tool was used to analyse Sanger sequence data for the targeted region of genomic DNA extracted from the trunk of each of the Bmp5 crispants shown in panels C and D. ?Indel %? (E) gives the percentage of insertions and/or deletions among the inferred sequences in the CRISPR-edited population. ?Knockout-Score? (E) shows the proportion of indels introducing a frameshift, or that are at least 21 bp in length. Discordance plots (F,G) show the level of discordance between the control sample trace file (orange) and the edited sample Sanger trace file (green). The expected cut sites for the sgRNAs are shown by vertical dotted lines. A successful CRISPR edit is indicated by the increase in discordance near the expected cut site. Panels H and I show nucleotide sequences from the Sanger trace files and their inferred relative contributions to the edited mosaic population. Vertical dotted lines indicate the expected cut sites for the sgRNAs. The wild-type sequence (0) is marked by an orange ?+? symbol in H, but is absent in panel I because 100% of the sequence was edited in this crispant. Abbreviation: e, eye. Scale bar: 250 ?m.