Testing the causality between CYP9M10 and pyrethroid resistance using the TALEN and CRISPR/Cas9 technologies.
Testing the causality between CYP9M10 and pyrethroid resistance using the TALEN and CRISPR/Cas9 technologies.
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DOI：
10.1038/srep24652
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发表时间：
2016-04-20
影响因子：
4.6
通讯作者：
Tomita T
中科院分区：
文献类型：
--
作者：
Itokawa K;Komagata O;Kasai S;Ogawa K;Tomita T
最近出现的基因组编辑技术甚至在非模式昆虫物种中也实现了靶向基因敲除实验。对于杀虫剂抗性研究，基因组编辑技术提供了一些优于传统反向遗传技术，RNA干扰，用于测试解毒酶基因与抗性表型之间的因果关系。有比较丰富的证据表明，细胞色素P450基因CYP 9 M10的过表达使致倦库蚊幼虫对拟除虫菊酯具有较强的抗性。但由于显微注射入幼虫的技术难度较大，目前尚未获得反向遗传验证。在这里，我们测试了两种基因组编辑技术，转录激活因子样效应物核酸酶（TALEN）和成簇规则间隔短回文重复序列（CRISPR/Cas9），以破坏耐药菌株中的CYP 9 M10。致倦肌此外，我们开发了一种新的，有效的方法来构建一个TALE使用的化学裂解的硫代磷酸酯核苷酸间的连接在1级组装。TALEN和CRISPR/Cas9均诱导拟除虫菊酯耐药菌株中CYP 9 M10的一个或所有拷贝发生移码突变。一个完全破坏的CYP 9 M10单倍型固定的线表现出超过100倍的减少拟除虫菊酯抗性在幼虫阶段。
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.