Testing the causality between CYP9M10 and pyrethroid resistance using the TALEN and CRISPR/Cas9 technologies.

Recently-emerging genome editing technologies have enabled targeted gene knockout experiments even in non-model insect species. For studies on insecticide resistance, genome editing technologies offer some advantages over the conventional reverse genetic technique, RNA interference, for testing causal relationships between genes of detoxifying enzymes and resistance phenotypes. There were relatively abundant evidences indicating that the overexpression of a cytochrome P450 gene CYP9M10 confers strong pyrethroid resistance in larvae of the southern house mosquito Culex quinquefasciatus. However, reverse genetic verification has not yet been obtained because of the technical difficulty of microinjection into larvae. Here, we tested two genome editing technologies, transcription activator-like effector nucleases (TALEN)s and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), to disrupt CYP9M10 in a resistant strain of C. quinquefasciatus. Additionally, we developed a novel, effective approach to construct a TALE using the chemical cleavage of phosphorothioate inter-nucleotide linkages in the level 1 assembly. Both TALEN and CRISPR/Cas9 induced frame-shifting mutations in one or all copies of CYP9M10 in a pyrethroid-resistant strain. A line fixed with a completely disrupted CYP9M10 haplotype showed more than 100-fold reduction in pyrethroid resistance in the larval stage.