A new CRISPR-Cas9-induced mutation of the zebrafish GR. (A) Schematic of the nr3c1 gene and encoded GR showing location of the targeted sequence and resulting premature stop codon with respect to the protein domains: N-terminal domain (NTD), DNA binding domain (DBD), and ligand binding domain (LBD). (B) Nucleotide sequence of nr3c1 exon 3, showing sequence targeted by the gRNA (underlined), the 20 base deletion resulting from injection of that gRNA and Cas9 mRNA (gray), and the resulting premature stop codon (bold italic). (C) Predicted amino acid sequence of the full-length zebrafish GR, plus the extra amino acids introduced by the frameshift shown in (B) (red font). The sequence eliminated by the premature stop is shown in gray, with the DBD underlined. Methionines corresponding to potential alternative initiation sites are shown in green. The positions of all reported frameshift mutations (including that reported here, E369) are boxed. The arginine that is changed to a cysteine in the grs357 mutation is shown in orange. (D) Schematic of the GR showing locations of the two previously reported exon 2 frameshift mutations resulting in truncations at amino acids 178 and 310, and the one in exon 3 reported here truncating at amino acid 369, and residues included in N310 and N369 mRNA constructs produced for microinjection. (E) Relative expression of fkbp5 in 6 hpf homozyogous GR369- embryos, either uninjected or injected with mRNA encoding full-length GR (GR-FL), N-terminally truncated GR isoforms (GR-N310 and GR-N369), or a nonspecific control mRNA encoding Xenopus elongation factor 1α (Xeno EF).
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