Figure 1

Figure 1

Sgcd^−/− zebrafish lines, mutations introduced, and consequences at the protein level. (A) Genomic organization of the wild type z-sgcd gene: boxes, exons; lines, introns. The red arrow points the site in exon 2 targeted by Cas9. (B) Nucleotide and amino acid sequences of the wild type and 4-bp and 14-bp deletion mutants (δ-SG_ex2KO^Δ4 and δ-SG_ex2KO^Δ14, respectively), as revealed by DNA sequencing analysis of the sgcd CRISPR target site. Each deleted nucleotide is represented by a dash, the CRISPR target site is highlighted in grey with the PAM sequence in bold. The amino acid sequence of the wild type (black amino acid) and of the mutants is reported under the nucleotide sequence. For both mutants, the consequence of the deletions is a frame shift (red amino acids) with the appearance of a premature stop codon (after 12 amino acids in the δ-SG_ex2Δ4bp and 42 amino acids in the δ-SG_ex2Δ14). (C) Scheme of the primary sequence of the wild type δ-SG protein and of the predicted truncated form with the different topological domains. ∆, deletion (D) Western blot (WB) analysis, showing the absence of the δ-SG protein in the lysates from embryos, 72 h post fertilization (hpf), of both sgcd^−/− zebrafish lines. Lysates from HEK293 expressing the zebrafish δ-SG sequence (Hek z-δSG) and from wild type zebrafish embryos were used as positive controls.