Fig. S7

Generation of zebrafish mymk^insT/insT line using CRISPR-Cas9 technology.

(a) Zebrafish mymk gene schematic with chromosomal position based on danRer10 genome build and exon numbering. Vertical black and red arrows indicate the target location of the sgRNAs used to generate CRISPR-KOs, and horizontal black and blue half-arrows indicate the location of the two sets of primers used to confirm the CRISPR induced NHEJ in exon 3 and 5, respectively. A red asterisk indicates the position of the frameshift, mutation at amino acid 106. (b) Electropherograms using primers indicated in black in (a) of WT, heterozygous and homozygous F2 mymk^insT CRISPR loss-of-function lines reveal the insertion of a second thymidine (T) after the T at position c.434. Amino acid single base letter codes and relative position of mymk WT sequence are indicated above the electropherograms. The position of the insertion is highlighted by a red rectangle. The inserted T is indicated inside the solid black box within the nucleotide sequence beneath the bottom sequence, with the altered protein sequence below. (c) Alignment of WT and mymk^insT zebrafish protein using Blast2Seq algorithm. The insertion of a T at position c.434 is predicted to substitute a cytosine (C) for a valine (V) at position 106 and generation of 33 novel amino acids before encountering a premature stop codon. (d) Maximum projection of Slow-twitch myofibers in mymk WT and homozygous insT at 48 hpf. Myofibers are stained for F59, staining Myosin heavy chain. Mutant appears larger because of the flattening due to less developed fast twitch-fibers. Myofibers size and orientation is similar between mutant and WT. Scale bar=50 μm.