Zhang Zhongjie, Aslam Abu F M, Liu Xiaojing, Li Muwang, Huang Yongping, Tan Anjiang
Sericultural Research Institute, Jiangsu University of Science and Technology, Zhenjiang 212018, Jiangsu, China; Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
J Insect Physiol. 2015 Aug;79:73-9. doi: 10.1016/j.jinsphys.2015.06.004. Epub 2015 Jun 9.
Recently established, custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system provide attractive genome editing tools. Targeted gene mutagenesis using the CRISPR/Cas9 system has been achieved in several orders of insects. However, outside of studies on Drosophila melanogaster and the lepidopteron model insect Bombyx mori, little success has been reported, which is largely due to a lack of effective genetic manipulation tools that can be used in other insect orders. To create a simple and effective method of gene knockout analysis, especially for dissecting gene functioning during insect embryogenesis, we performed a functional analysis of the Bombyx Wnt1 (BmWnt1) gene using Cas9/sgRNA-mediated gene mutagenesis. The Wnt1 gene is required for embryonic patterning in various organisms, and its crucial roles during embryogenesis have been demonstrated in several insect orders. Direct injection of Cas9 mRNA and BmWnt1-specific sgRNA into Bombyx embryos induced a typical Wnt-deficient phenotype: injected embryos could not hatch and exhibited severe defects in body segmentation and pigmentation in a dose-dependent manner. Quantitative real-time PCR (qRT-PCR) analysis revealed that Hox genes were down-regulated after BmWnt1 depletion. Furthermore, large deletion, up to 18Kb, ware generated. The current study demonstrates that using the CRISPR/Cas9 system is a promising approach to achieve targeted gene mutagenesis during insect embryogenesis.
最近建立的定制设计核酸酶技术，如成簇规律间隔短回文重复序列（CRISPR）相关系统，提供了有吸引力的基因组编辑工具。利用CRISPR/Cas9系统进行靶向基因诱变已在多个昆虫目中实现。然而，除了对黑腹果蝇和鳞翅目模式昆虫家蚕的研究外，鲜有成功报道，这主要是由于缺乏可用于其他昆虫目的有效基因操作工具。为了创建一种简单有效的基因敲除分析方法，特别是用于剖析昆虫胚胎发育过程中的基因功能，我们使用Cas9/sgRNA介导的基因诱变对家蚕Wnt1（BmWnt1）基因进行了功能分析。Wnt1基因在多种生物体的胚胎模式形成中是必需的，并且其在胚胎发育过程中的关键作用已在多个昆虫目中得到证实。将Cas9 mRNA和BmWnt1特异性sgRNA直接注射到家蚕胚胎中会诱导典型的Wnt缺陷表型：注射后的胚胎无法孵化，并以剂量依赖的方式在身体节段化和色素沉着方面表现出严重缺陷。定量实时PCR（qRT-PCR）分析显示，BmWnt1缺失后Hox基因下调。此外，还产生了长达18Kb的大片段缺失。当前研究表明，使用CRISPR/Cas9系统是在昆虫胚胎发育过程中实现靶向基因诱变的一种有前景的方法。
