Mutation of the cytochrome P450 CYP360A8 gene increased sensitivity to herbicide paraquat in Daphnia magna. Mutation of the cytochrome P450 CYP360A8 gene increased sensitivity to herbicide paraquat in Daphnia magna

Purpose: To create a CYP gene mutant from D. magna as a model for xenobiotic metabolism study Methods: We performed RNA-seq of wildtype 12 days old D. magna to find out the basal expression level of CYP genes. We picked up 5 highest expressed CYP genes and examined the temporal expression level from day 0 to day 12 by qPCR. We chose the highest expressed gene and performed mutagenesis to this gene by CRISPR/Cas9 to creat a mutant line. Results: From RNA-seq result, we found that CYP360A8 is the highest expressed CYP in D. magna. qPCR result showed that CYP360A8 has developmental pattern where its expression is low in juvenile, increase 6 fold by maturation time and stay stable till day 12. Mutagenesis experiment of this gene resulted in the obtaning of a monoallelic mutant. Paraquat exposure to this mutant line showed more sensitivity of the mutant compared to wildtype (showed by 1.3 fold lower of EC50). Conclusions: Our study showed the first mutant model of CYP in D. magna. This may be the start of creating a panel that list D. magna CYP gene with its inducing xenobiotics. Overall design: mRNA profiles of 12 days old wild type D. magna