Expression pattern of dstyk and knock out dstyk by CRISPR/Cas9.aDstyk ISH at 1-cell/2-cell, 4-somite and 8-somite stage. b From 18-somite, 24 hpf, 38 hpf to 54 hpf, notochordal expression of dstyk was dynamically changed as detected by ISH. c The Cas9 target site for knockout dstyk gene worked in knock out F0 generation. d Top panel showed the DNA sequences for WT and two Cas9 mutant lines. TGG (red) is the PAM sequence. Deletion is represented by a red dashed line and insertion is highlighted in green. Bottom panel diagramed DSTYK protein structures of WT and predicted mutants. Arrows indicate amino acid at the mutation site. e Bright-field images showing the WT and two Cas9 mutant lines at 4 dpf. f Cas9 mutant lines showed similar scoliosis phenotypes with smt mutant. Scale bar represent 200 μm in (a and b), 400 μm in (e).
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