CRISPR/Cas9-Mediated Knockout Reveals the Involvement of CYP304F1 in β-Cypermethrin and Chlorpyrifos Resistance in Spodoptera litura

Functions of insect CYP2 clan P450s in insecticide resistance are relatively less reported. In Spodoptera litura, a gene from the CYP2 clan (CYP304F1) was validated to be up-regulated significantly in a pyrethroid- and organophosphate-resistant population (QJ) than a susceptible population by RNA-Seq and qRT-PCR. Spatial-temporal expression indicated the high expression of CYP304F1 in the fourth, fifth, and sixth instar larvae and the metabolism-related tissue fat body and malpighian tubules. CYP304F1 was knocked out by CRISPR/Cas9, and a homozygous population (QJ-CYP304F1) with a G-base deletion at exon 2 was obtained after selection. Bioassay results showed that the LD₅₀ values to β-cypermethrin and chlorpyrifos in the QJ-CYP304F1 population decreased significantly, and the resistance ratio was both 1.81-fold in the QJ population compared with that in the QJ-CYP304F1 population. The toxicity of fenvalerate, cyhalothrin, or phoxim showed no significant change. These results suggested that CYP304F1 is involved in β-cypermethrin and chlorpyrifos resistance in S. litura.