Genetic and molecular characterization of adult nox2 zebrafish. a) Sanger sequencing chromatogram of genomic DNA at gRNA 1 (intron 2/exon 3) and gRNA 2 (exon 4) target sites in adult WT (upper panel) and nox2 (lower panel) homozygous zebrafish. DNA sequence disruptions are around the protospacer adjacent motif (PAM)—NGG. b) Interpretation of nucleotide sequences in panel a, at the gRNA 1 and gRNA 2 target sites. c) Schematic of PCR forward primer (FP) and reverse primer (RP) binding sites for amplifying cDNA transcripts (above). Gel image showing RT-PCR products from nox2 WT, heterozygous, and homozygous zebrafish embryos (below). d) Sanger chromatogram showing cDNA sequence traces of nox2 WT, heterozygous, and homozygous zebrafish embryos from RT-PCR in panel c. Exons separated by colored broken lines. e) Interpretation of nucleotide sequence and predicted amino acid sequence of WT and nox2 homozygous in panel d. f) Schematic of predicted protein domains in WT and nox2 zebrafish. nox2, NADPH oxidase 2; gRNA, guide RNA; PAM, protospacer adjacent motif; 6TM-FR, heme containing 6 transmembrane ferritin reductase domain; FAD, flavine adenine dinucleotide domain; NADPH, nicotinamide adenine dinucleotide phosphate domain; +/+, WT; +/−, heterozygous; −/−, homozygous; green font, exon 3 sequence; blue font, sequence mismatch; asterisk (*), premature stop; purple arrows, direction of sequencing; FP, forward primer; RP, reverse primer; RT, reverse transcriptase.
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