Flavin-containing Monooxygenases (FMOs) are a family of enzymes that are co-localized with cytochrome P450s (CYPs) in the endoplasmic reticulum of multiple cell-types, organs, and organisms.  FMOs are also similar to CYPs in that they require NADPH and oxygen.  However, the substrate specificities and catalytic reaction mechanism is significantly different in that soft-nucleophilic substrates attack peroxyl-flavins located in the active site of the enzyme. Numerous studies have shown an association with expression and functionality of FMOs in animals residing in saltwater or undergoing saltwater acclimation.  Isolation and sequencing of salmonid FMO1 possessed Osmoregulatory response Elements (OsREs) in the promoter region of the gene, and expression of FMO mRNA was increased following NaCl treatment in isolated hepatocytes of rainbow trout.  

To determine the relevance of the enzyme in osmoregulation and xenobiotic metabolism, we targeted the D.rerio fmo5 for functional disruption using direct microinjection of CRISPR-Cas9 Ribonucleoprotein (RNP) complexes  into two-cell stage embryos. The Schlenk Lab evaluated several small guide RNAs (sgRNAs) to the fmo5 gene for editing efficiency and identified an sgRNA targeting the region encoding the functionally essential FAD binding site which resulted in high embryo editing efficiency and no embryo-lethality.  They evaluated RNP and mock microinjected embryos at 7 days postfertilization, for sensitivity to NaCl, the organophosphate insecticide, phorate, nicotine, and thiourea. Genomic DNA was prepared from each embryo and editing efficiency was evaluated by direct PCR of the targeted region, sanger sequencing, and Inference of CRISPR edits (ICE) analyses. Importantly, all FAD targeted larvae were predicted to have 95% composite loss of function in all injected animals. No significant differences were observed in thiourea, nicotine, or NaCl treatments between mock injected and larvae confirmed to have predicted functional disruption. In contrast, while 100% mortality was observed in mock injected animals treated with phorate at all concentrations, only 42% mortality at the highest concentrations was noted in the larvae with fmo5 disruption.