Gene knockout of P450s CYP392A16 and CYP392D2 reveals unexpected in vivo effects and transcriptional rewiring in the extreme generalist Tetranychus urticae.

The two-spotted spider mite Tetranychus urticae rapidly evolves resistance to acaricides, frequently through the upregulation of detoxification enzymes such as cytochrome P450 monooxygenases. Among these, CYP392A16 and CYP392D2 have repeatedly been implicated as major resistance determinants, yet their in vivo contributions remain incompletely resolved. Here, we used efficient CRISPR/Cas9-mediated genome editing to generate stable knockouts of CYP392A16 and CYP392D2 in a multi-resistant genetic background. Contrary to expectations, loss of either gene resulted in surprisingly limited effects on acaricide susceptibility, with increased sensitivity observed only for a subset of compounds. Experimental evolution assays revealed a moderate, host-dependent fitness cost associated with disruption of CYP392D2, but not CYP392A16. Transcriptomic analyses further uncovered stark contrasts between the knockouts, with CYP392D2 loss triggering extensive regulatory reprogramming suggestive of compensatory detoxification pathways. Together, our results demonstrate that even resistance-associated detoxification genes that are considered functionally validated in vitro, can have strong context-dependent phenotypic effects in vivo, underscoring the complexity of resistance gene networks and highlighting the need to interpret CRISPR/Cas9 knockout phenotypes within the broader genetic and regulatory context.