Fig. 6

CRISPR/Cas9 targeted mutation of smyd5 phenocopies morpholino knockdown.

Identification of embryos with CRISPR/Cas9-mediated insertion and/or deletion (indel) mutations in smyd5 genomic region by heteroduplex mobility assay (HMA). Heteroduplex (whitelines) and homoduplex (asterisks) DNA band indicate the presence of indel mutant allele, and wild type allele, respectively. Five embryos (#1-#5) were injected with smyd5 guide RNA (gRNA) and Cas9 mRNA. We detected heteroduplex DNA band in #4 and #5 of embryos. Homoduplex DNA band was detected in #1, #2 and #3 of embryos, which has similar size with that observed in no-injected control. (B) Sequences of smyd5 mutations in #4 embryos. All sequences had indels near the smyd5 target site of gRNA, which is underlined. Deletions and insertions are indicated by dashe and lowercase red letters, respectively. The number of nucleotides deleted (-) and inserted (+) is indicated to the right with the detection number. (C,D) Whole mount in situ hybridization of smyd5-KO or control embryos. The genes involving primitive myelopoiesis in smyd5-KO F0 embryos were examined (C). Expression of pu.1 in smyd5-KO F0 embryos, and those no-injection at 24 hpf. Expression of mpx and l-plastinin in smyd5-KO F0 embryos and those no-injection at 28 hpf. Expression of the genes for definitive myelopoiesis in smyd5-KO F0 embryos was examined (D). Expression of cmyb in smyd5-KO F0 embryos, and those no-injection at 30 hpf. Expression of mpx and l-plastinin in smyd5-KO F0 embryos, and those no-injection at 72 hpf. Numbers on each panel indicate the number of embryos showing the representative phenotype per the total number of embryos. Embryos are depicted in the lateral view. Scale bar, 200 µm.