Fig. 5

Generation of grm8a and grm8b knock-out lines using CRISPR/Cas9. (A) Grm8a exon-intron structure (top) with coding (gray) and non-coding exons (white). The sgRNA (pink) targeting grm8a exon 4 and the primer binding sites for genotyping PCR are displayed. The CRISPR/Cas9-induced strand break (black arrowheads) caused a 17 bp deletion represented by a smaller PCR product (pink asterisk) in grm8a^± and grm8a^–/– animals (bottom). The third band of ∼220 bp corresponds to a heterodimer of wildtype and mutated PCR product (black asterisks). (B) The predicted amino acid sequence of the wildtype (top) and mutated allele (bottom) show that the frameshifted sequence (black box) is interrupted by a premature stop codon (pink asterisk) in the ligand binding domain. (C) Grm8b exon-intron structure (top) with coding (gray) and non-coding exons (white). The sgRNA (pink) targeting grm8b exon 2 and the primer binding sites for genotyping are indicated. The CRISPR/Cas9-induced strand break (black arrowheads) caused a 13 bp deletion represented by a smaller PCR product (pink asterisk) in grm8b^± and grm8b^–/– animals (bottom). The third band of ∼250 bp corresponds to a heterodimer of wildtype and mutated PCR product (black asterisks). (D) The predicted amino acid sequence of the wildtype (top) and mutated allele (bottom) shows that the frameshifted sequence (black box) is interrupted by a premature stop codon (pink asterisk) in the N-terminal domain. (E) Dorsal (top) and ventral (bottom) views of 5 dpf old grm8a^+/+, grm8a^± and grm8a^–/– zebrafish. Anterior is to the left. (F) Dorsal (top) and ventral (bottom) views of 5 dpf old grm8b^+/+, grm8b^± and grm8b^–/– zebrafish. Scale bar, 1 mm.