Figure 6

qRT-PCR results for the expression of transcripts with high (> 75%) sequence similarity to the main zebrafish hcn4 transcript, with and without CRISPR/Cas9 targeting of hcn4, hcn4l, or hcn4 & hcn4l. Each sample consists of five pooled, 5-day-old embryos, which at the single cell stage had been injected with: (1) hcn4 and hcn4l gRNAs, or Cas9 mRNA (controls, in blue, n = 26); or with Cas9 mRNA together with (2) hcn4 gRNA (red, n = 10); (3) hcn4l gRNA (green, n = 12); or (4) hcn4 and hcn4l gRNA (orange, n = 21). In all samples, technological triplicates of quantification cycles (Cq) were averaged, and normalized using expression in un-injected controls as calibrator, and expression of mob4 as a reference gene, using the Pfaffl method. For hcn4 and hcn4l, expression was quantified at the CRISPR/Cas9 targeted site and at the last exon. Differences between the three experimental conditions and controls were examined in a multiple linear regression analysis, adjusting for batch (n = 2). CABZ01086574.1 is likely an orthologue of the human HCN2. Significant differences with controls are highlighted by * (p < 0.05) or # (P < 1 × 10^–4). Boxes show mean ± 1 SD. Genes are ordered by sequence similarity to the main hcn4 transcript.