Generation and molecular characterisation of a myoc KO zebrafish line using CRISPR/Cas9 genome editing. (A) Localisation of the crRNA designed to target myoc exon 1. The PAM sequence is indicated on a green background. Red and blue nucleotides indicate deleted and inserted nucleotides identified in mutant fishes used to establish the KO line, respectively. (B) Stepwise procedure that was followed to establish the KO line. F0 animals were raised to adulthood, crossed with wild-type AB zebrafish and the offspring was genotyped by PAGE to identify germline transmission of myoc deletions (F0 founders). F0 founders were crossed with wild-type AB animals to obtain mutant F1 heterozygotes that were further outbred to segregate off-targets mutations and to obtain the F2 generation. F2 heterozygotes were inbred to produce the F3 generaton. The Biorender tool was used to create this scheme. (C) Genotyping of a myoc indel using 8% PAGE. Three representative samples are shown. White arrowhead: wild-type allele (265 bp). Black arrowhead: mutant allele (275 bp). (D) Sanger sequencing of the selected myoc mutation. The arrow in the electropherogram indicate the nucleotide where the mutation starts. Blue and red letters indicate inserted (14 bp) and deleted (4 bp) nucleotides, respectively. (E) Decreased myoc mRNA levels in myoc mutant zebrafish (+/− and −/−). mRNA levels in pools of 15 F3 zebrafish larvae (144 hpf) were measured by qRT-PCR. The results are expressed as relative expression levels and normalised to control wild-type (+/+) larvae. Values represent the average of three different experiments. Asterisks indicate statistical significance compared to +/+. * p = 00051. ** p =0.00017.
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