Additional file 11: of Zebrafish prdm12b acts independently of nkx6.1 repression to promote eng1b expression in the neural tube p1 domain

Generation of bhlhe22 germline mutant. a. Schematic showing genomic sequence of bhlhe22 with the bHLH domain indicated in blue. Note that bhlhe22 is contained on a single exon. The CRISPR target sequence is shown in red with the BstYI restriction site bracketed and the black arrow indicating the BstYI cut site. b. Identification of functional guide RNAs. sgRNA and cas9 mRNA was injected into 1-cell stage embryos. Injected embryos were raised to 24hpf and BstYI digestion of PCR amplicons from pools of embryos was used to identify CRISPR-induced mutations (black arrow). c. Identification of individual F0 founders. sgRNA/cas9 injected embryos were raised to adulthood and crossed to wildtype fish. BstYI digests of PCR amplicons from pools of embryos was used to identify F0 mosaic founders (black arrow). d. Identification of F1 animals. Adult F0 mosaic founders were out-crossed to wildtype fish and the F1 offspring raised to adulthood. BstYI digests of PCR amplicons from fin clip genomic DNA was used to identify heterozygous F1 animals. e. Sequencing of F1 genomic DNA revealed the transmission of one mutant allele (um320) carrying a 5 base pair deletion (black dashes). The CRISPR target sequence is shown in red. f. Predicted amino acid sequence of mutant allele. The um320 peptide shares its first 67 amino acids with the wildtype protein before going out of frame and terminating at a premature stop codon N-terminal to the bHLH domain. (PDF 485 kb)