ZFIN Image: Rosello et al., 2022, Fig. 4

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Figure Caption

Fig. 4

Endogenous activation of nras oncogene and knock-out of tp53 tumor-suppressor gene led to an increase in melanocyte numbers.

a Lateral view of 3 dpf larvae. The injected embryos edited only for nras do not present any defects, whereas 50% of the injected embryos targeted for nras and tp53 were hyperpigmented. Scale bar = 100 µm. b DNA sequencing chromatogram of the targeted nras gene from a pool of 80 injected embryos with the CBE4max-SpRY mRNA and the nras NAN sgRNA. C-to-T conversion shows 83% of efficiency for the C in position 16, 81% for the C in position 15, 78% for the C in position 13, and 16% for the C in position 12 bp away from the PAM. Numbers in the boxes represent the percentage of each base at that sequence position. In red are highlighted the base substitutions introduced by base editing while the original bases are in blue. The color code of the chromatogram is indicated in the upper left corner (Adenine green, Cytosine blue, Thymine red, Guanine black). The distance from the PAM sequence of the targeted C base is indicated below the chromatogram. The efficiencies have been calculated using EditR software and chromatograms from Sanger sequencing of PCR products. c Box plot showing the base editing efficiency for nras and tp53 genes for the 4 “wt-like” larvae and the 8 larvae with increased pigmentation from Supplementary Fig.. Statistical significance was determined using a two-tailed Mann–Whitney test. p value = 0.0162 (nras C1), 0.0081 (nras C2), 0.0081 (nras C3), 0.0040 (nras C4), 0.0020 (tp53). The boxes range from the 25th to 75th percentiles. The box plot whiskers down to the minimum and up to the maximum value, the center shows the median, and each individual value is represented by a point. d Western blot made on 3 dpf control larvae, nras targeted larvae, tp53 targeted larvae or nras and tp53 targeted larvae using CBE4max-SpRY mRNA and antibodies targeting phosphorylated ERK1/2 (pERK) and Histone 3 as loading control. The molecular weight is in kDa. Source data are provided as a Source data file. e Dot plot showing the means and standard errors of the mean (SEM) of the relative band intensity of 3 independent western blots as seen in d. Statistical significance was determined using a two-tailed unpaired t test. p value= 0.0027 (**, control vs nras), 0.3160 (n.s., control vs tp53), 0.0313 (*, control vs tp53 + nras). f, g Dot plots showing the means and SEM of quantitative real-time PCR of p21, puma, baxa (f) and of bcl2, mcl1a, mdm2 (g) expression levels in 3 dpf control larvae, nras targeted larvae, tp53 targeted larvae or nras and tp53 targeted larvae using CBE4max-SpRY mRNA. Statistical significance was determined using a two-tailed unpaired t test. For p21, p value = 0.4881 (n.s., control vs nras), 0.0082 (**, control vs tp53), 0.0011 (**, control vs nras + tp53). For puma, p value = 0.8653 (n.s., control vs nras), 0.0196 (*, control vs tp53), 0.0465 (*, control vs nras + tp53). For baxa, p value = 0.4695 (n.s., control vs nras), 0.0015 (**, control vs tp53), 0.0121 (*, control vs nras + tp53). For bcl2, p value = 0.5810 (n.s., control vs nras), 0.2163 (n.s., control vs tp53), 0.0033 (**, control vs nras + tp53). For mcl1a, p value = 0.0004 (***, control vs nras), 0.0445 (*, control vs tp53), 0.0423 (*, control vs nras + tp53). For mdm2, p value= 0.8956 (n.s., control vs nras), 0.1014 (n.s., control vs tp53), 0.0037 (**, control vs nras + tp53). n = 3 for (f) and n = 6 for (g) independent experiments.

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