Knockout of zebrafish gnav1. (a) Schematic representation of knockout strategy using CRISPR/Cas9 method. The target sequence is shown. (b) Trace for homozygous knockout, with 13 b deletion indicated by red overlay in the nucleotide sequence. (c) RT-PCR for 5 dpf larvae shows presence of Gv RNA in the deletion mutant; 1% agarose TAE gel, stained with Midori green (NIPPON Genetics). (d) qPCR for Gv RNA in seven developmental stages. Wildtype (wt), black bars; deletion mutant, red bars. Significance estimated by two tailed unpaired t-test. *, p < 0.05; **, p < 0.01. y axis, fold change is normalized to reference genes rpl8 and rpl37. (e) Structure for Gv was taken from alphafold model AF-B0V3V7-F1-model_v4. The 13 b deletion is indicated by pink rectangle, it leads to a premature stop, pink asterisk. STIR, a potential secondary initiation site of translation. Acylation refers to two closely neighboring sites, for N-linked myristoylation and thio-palmitoylation (5). (f) Five peptides identified as suitable for parallel reaction mass spectrometry (PRM) are shown, together with their position in the exons of the gnav1 gene; the position of the deletion in exon 1 is indicated by arrowhead. Relative amounts present in 48 hpf larvae are shown for wildtype (black line) and deletion mutant (red line). Larvae are progeny of mutants and their wildtype siblings. The significance was determined with two-way ANOVA, **, p = 0.0055. Points represent mean+/-SEM of four independent determinations (each with a pool of 50 embryos)
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