Abstract
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.
Highlights
An important goal of insecticide resistance research is to identify the gene responsible for the resistance phenotype
Genome editing employs programmable artificial DNA endonucleases such as zinc-finger nuclease[9], transcription activator-like effector nuclease (TALEN)[10,11] and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas[9] system[12], which are designed to target a specific sequence in a genome
We disrupted a detoxification enzyme gene in a pest insect using TALENs and CRISPR to validate the causality of an insecticide resistance phenotype
Summary
An important goal of insecticide resistance research is to identify the gene (or allele) responsible for the resistance phenotype. Much evidences support the causal relationship between CYP9M10 and pyrethroid resistance[2,16,17,19], RNAi verification had not been conducted because the gene overexpression and resistance are specific to the larval stage, which is highly vulnerable to microinjection[20]. Both TALENs and gRNA targeting CYP9M10 introduced frame-shift mutations in the JPP strain. Dramatic reduction (110-fold) in permethrin (one of pyrethroid insecticides) resistance was observed in a line in which all CYP9M10 copies were disrupted
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