Fig. S4

Fig. S4

Analysis of the urod phenotype in stable F1 embryos (related to Figure 4).

(A) Confocal images of 50 hpf embryos from the F1 generation of fish stably expressing the indicated vectors.

(B) FACS analysis of the blood of 36 hpf F1 embryos stably expressing the pcmlc2:GFP, U6:gRNA urod, gata1:Cas9 vector (bottom panels). Blood from F1 siblings not presenting green hearts was used as a negative control (top panels). Red fluorescence due to urod inactivation was detected in the PE-Cy5 channel (right panels). Cytox blue was used to mark dead or dying cells (left panels).

(C) T7E1 mutagenesis assay at the CRISPR target site in the urod gene. The assay was performed on genomic DNA from the blood of 2 dpf F1 embryos stably expressing the pcmlc2:GFP, U6:gRNA urod, gata1:Cas9 vector. Blood from F1 siblings not presenting green hearts was used as a negative control.

(D) Sequences of the mutant urod alleles found in the blood of embryos stably expressing the pcmlc2:GFP, U6:gRNA urod, gata1:Cas9 vector. The CRISPR target sequence is in green, mutations in red. The type of mutation and the associated number of detected alleles are indicated.

(E) Quantification of the fluorescent phenotype observed in F1 embryos. Mild: <10 fluorescent blood cells. Full denotes a number of fluorescent cells comparable to that observed in age-matched LCR:GFP embryos. Ubi:Cas9 (n=37), gata1:Cas9 (n=80).

(F) qPCR analysis of Cas9 expression in F1 embryos stably expressing pcmlc2:GFP, U6:gRNA urod, ubi:Cas9 vector. Four embryos showing no or few fluorescent cells (mild) and four embryos showing a full phenotype were compared. Data represented as mean ± SD.