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CRISPR‐Cas9 editing of the zebrafish <italic>rab13</italic> 3′UTR perturbs mRNA polarisationCRISPR‐Cas9 strategy to generate the Tg(kdrl:EGFP) rab13+/∆3′UTR zebrafish strain. The wild‐type (Wt) rab13 exon 8 is represented with its coding sequence in dark and the 3′UTR in clear boxes; the 5′ and 3′ gRNA‐targeted regions are represented with green lines. Arrows: relative positions of the forward (f) and reverse (r) PCR primers used to identify animals with CRISPR‐Cas9-mediated deletions (∆) in the rab13 3′UTR. Representative genotyping PCR demonstrates the band size shift in zebrafish harbouring a ∆482 rab13 3′UTR. Asterisk marks a heteroduplex formed between Wt and ∆482 rab13 3′UTR PCR amplicons. Detailed DNA sequence depicting nucleotide positions within the Wt and ∆482 rab13 3′UTR. RNAseq mapped reads depicting rab13 exon usage in Tg(kdrl:EGFP) rab13+/+ and rab13∆3′UTR/∆3′UTR zebrafish embryos. Coloured lines indicate SNPs. qPCR analysis of rab13 mRNA levels in individual 26–28 hpf clutch‐matched sibling embryos (n ≥ 9 embryos; ns: not significant; Kruskal–Wallis test with Dunn's correction). smFISH detection of rab13 and kdr mRNA in cultured GFP‐expressing endothelial cells extracted from 48 hpf Tg(kdrl:EGFP) rab13+/+ and rab13∆3′UTR/∆3′UTR zebrafish embryos. Polarisation Index (PI) of rab13 and kdr detected by smFISH in individual zebrafish cells (n ≥ 8 cells; **P < 0.01, ns: not significant; one‐way ANOVA with Bonferroni's correction). rab13 PI plotted against respective kdr PI. The slope of the coloured lines represents the average rab13/kdr PI ratio; the dashed grey line represents a 1:1 ratio (n ≥ 8 cells).
Data information: +/+, +/∆ and ∆/∆ represent Tg(kdrl:EGFP) rab13+/+, rab13+/∆3′UTR and rab13∆3′UTR/∆3′UTR embryos, respectively (E, G, H). Arrows indicate orientation of RNA localisation; yellow dashed lines outline cell borders; red circles highlight smFISH spots; scale bars = 10 μm (F). Bar charts are presented as means ± s.d.
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