Fig. 8

CRISPR/Cas9 mutagenesis of the third exon of zebrafish Hemogen reduces primitive and definitive erythropoiesis. Embryos were injected with Cas9 mRNA and a guide RNA to establish lines with mutations in exon three of zebrafish Hemogen. (A) 20?hpf. Representative wild-type and mutant siblings with notochord defects (arrow). (B) 48?hpf. Mutant ?12 embryos with an in-frame deletion showing kinked notochords (arrow). (C) 24?hpf. Wild-type and ?5/+ mutant embryos stained with diaminofluorene. Production of erythrocytes was reduced in heterozygotes. (D) Schematic of CRISPR/Cas9 target in the third exon (red arrowhead) of zebrafish Hemogen. (E) Sequences of founder mutations aligned at the CRISPR target site: ?5 (Hemgn^nuz2); ?12 (Hemgn^nuz4). The sequence traces show the ?5 and ?12 mutant alleles. PAM, blue and underlined; ?, deletions (highlighted in red). (F) Relative expression of wild-type and ?5 transcripts in blood from single adult, heterozygous Hemgn^nuz2/+ mutants determined by qRT-PCR with allele specific primers. Three biological replicates were normalized to ?-actin. Error bars represent the standard deviation. (G) Western blot of Hemogen in pooled 33?hpf wild-type embryos or pooled embryos from a ?5 Hemgn^nuz2/+ heterozygous in-cross. We calculated that the protein would run 6.5?kDa above its molecular weight at 28.5?kDa because of its high acidic composition (Guan et al., 2015). Arrows show the calculated sizes of wild-type and truncated alleles. (H) Proportion of genotyped mutants and wild-type sibling embryos at 2?dpf that were anemic (black) or phenotypically normal (white) (*P?0.05, **P?0.005, Chi square). (I) Wild-type and mutant zebrafish heterozygous for the ?5 and ?12 alleles. (J) Red blood cells from adult Hemgn^nuz2/+ mutant zebrafish and wild-type siblings. (K) Erythrocyte counts in adult heterozygous Hemgn^nuz2 (?5, n=12), heterozygous Hemgn^nuz4 (?12, n=4) mutants, homozygous Hemgn^nuz4 (?12, n=2) mutants and wild-type (n=9) siblings (*P?0.05, ANOVA, Tukey post hoc test). Scale bars: 500?µm (A-C); 50?mm (I); 20?µm (J).