Fig. 1

Fig. 1. Generating her3 mutations in zebrafish. (A) Schematic of strategy to generate a her3 CRISPR/Cas9 null mutant zebrafish. Zebrafish embryos were injected in the cytoplasm at the 1-cell stage with both Cas9 protein and her3 gRNA. A subset of the injected clutch was set aside to determine gRNA efficiency. The remaining embryos were grown up to identify adult founders, which were outcrossed to wildtype WIK zebrafish to generate the F₁ generation. (B) HRMA difference RFU graphs of F₁ heterozygous zebrafish generated from three identified founders. Fin clips of adult F₁ zebrafish were taken and HRMA was performed to identify potential mutations, with different melt curves indicating distinct DNA mutations. Wildtype reference fish are labeled black, her3^nch1 fish are labeled green, her3^nch2 fish are labeled orange, and her3^nch3 fish are labeled blue. Each line represents an individual fish. (C) Schematic of full length Her3 protein and identified mutation sequences. To determine the mutation sequence, the her3 gRNA target site from the three prospective mutant F₁ fish was A-tail cloned into the pGEM T-Easy vector. Multiple colonies from each line were Sanger sequenced with an SP6 primer. The mutated sequence was then aligned with the wildtype reference sequence for her3. Colored lines in the schematic indicate where the predicted frameshift mutation occurs for each respective mutation, all of which are in the DNA binding domain. Below, the DNA sequence and amino acid sequence are shown for the three mutations, with mutant DNA sequence on top of the aligned wildtype sequence. For both the DNA sequence and the amino acid sequence, colored and bolded letters highlight the mutated sequence.