Fig. 2

The generation ofkeap1a-andkeap1b-knockout zebrafish.

(A) Gene knockout of keap1a and keap1b using CRISPR–Cas9 technology. CRISPR target sites were designed in exon 4 of the keap1a and keap1b loci (black arrowheads). In keap1a^it302 and keap1b^it308 lines, 70- and 20-amino acids-extra peptides were added after the original Val215 in Keap1a and after the original Ala347 in Keap1b proteins, respectively (diagonal stripes).

(B) keap1a- and keap1b-homogygous knockout larvae at 5 dpf. No obvious differences were observed between wild-type and knockout larvae. Right panels show PCR genotyping of keap1a^it302-and keap1b^it308-knockout larvae. WT, +/– and −/− indicate wild-type, heterozygous and homozygous fish, respectively. Open and closed arrowheads denote knockout and wild-type alleles, respectively.

(C) keap1a^it302-and keap1b^it308-homozygous knockout adults at 4 mpf. No obvious differences were observed between wild-type and knockout adults (females in this picture). The genotypes of 4-mpf adults derived from heterozygous parents were roughly according to the expected Mendelian ratio.

(D) The relative expression of keap1a and keap1b in wild-type, homozygous keap1a^it302-and keap1b^it308-knockout larvae at 5 dpf analyzed by qPCR. The expression of wild-type specimens was normalized to 1. Each experiment was conducted at least four times with duplicate samples. Asterisks indicate significant differences (*p < 0.05). ns, not significant.