Fig. 6

Generation of klf8 mutants by CRISPR-Cas9 gene editing and the effect on spaw expression. a klf8 genomic structure with klf8 sgRNA (blue lettering) targeted to exon 2. Protospacer adjacent motif (PAM) sequence is shown in red. b Nucleotide and predicted amino acid sequences of klf8 in wild type, klf8 ^I17 and klf8d25 mutants are shown. Deleted nucleotides are shown by a red dashed line, while inserted nucleotides are shown in green lettering. Representative images of embryos with different spaw expression patterns in the LPM at 18 s stage (arrow; c-f). g Percentage of embryos displayed left (L), right (R), decreased (D) or bilateral (B) expression of spaw in the LPM from intercross of respective klf8 ^d25 or klf8 ⁱ¹⁷ F2 heterozygous mutants. Deduced percentage of wild type (+/+), heterozygote (+/−) or homozygote (−/−) genotype of embryos from intercross of respective klf8 ^d25 or klf8 ⁱ¹⁷ F2 heterozygous mutants exhibited bilateral spaw expression pattern. Expression levels of klf3 (h, k), klf12a (i, l) or klf12b (j, m) were compared between wild type and respective klf8 ^d25 and klf8 ⁱ¹⁷ F5 homozygous mutant embryos at 10–12 s stages (h-m). Knockdown of klf12b reduced the percentage of embryos with bilateral spaw expression in the LPM of klf8 ^d25 F6 homozygous mutant embryos, but the reduction did not reach significance (p = 0.45 for the comparison between klf8 ^d25 and klf8 ^d25 + 5 ng klf812b MO, p = 0.05 for the comparison between klf8 ^d25 and klf8 ^d25 + 10 ng klf812b MO) (n). Statistical significance was determined by Student’s t-test. * p < 0.05. Error bars indicate standard deviation