Fig. S1

CRISPR targeting of urod, but not p53, elicits a fluorescent phenotype in zebrafish embryos (related to Figure 1)

(A) CRISPR target sequences used to knockout the urod and p53 zebrafish genes.

(B) T7E1 mutagenesis assay at the p53 CRISPR target site (left) and at the second urod site (right). The assay was performed on genomic DNA from 2 dpf embryos injected at the one-cell stage with Cas9 mRNA and either the gRNA against p53 or the gRNA against urod. Arrows point to cleavage bands.

(C) Quantification of the fluorescent phenotype obtained after co-injection of Cas9 mRNA and a gRNA against urod ± wt urod mRNA (urod^wt) or yquem urod mRNA (urod mRNA bearing the inactivating mutation found in the yquem mutant, urod^yq). WT: no fluorescent blood cell. Mild: <10 fluorescent blood cells. Intermediate: <50 fluorescent blood cells. Strong: >50 fluorescent blood cells. Data represented as mean percentage ± SD of 3 independent experiments. *: p<0.05, paired t-test.

(D) T7E1 mutagenesis assay at the CRISPR target site in the urod gene. AB embryos were injected at the one-cell stage with Cas9 mRNA and a gRNA against urod, and sorted into 4 groups (WT, mild, intermediate, high) according to their fluorescent phenotypes (see Figure S1C). The assay was performed on genomic DNA from 8 embryos from each group at 2 dpf. Arrows point to cleavage bands.

(E) Confocal images at 50 hpf of embryos injected at the one-cell stage with Cas9 mRNA and either the gRNA against p53 (negative control) or a gRNA against urod.