利用RNA引导的CRISPR/Cas9系统研究多房棘球绦虫丝氨酸蛋白酶抑制剂的功能

Echinococcosis (commonly known as hydatid disease), caused by the metacestodes (proliferative larvae) of the genus Echinococcus tapeworms, is among the most severe parasitic diseases in humans and animals and is one of the 17 neglected tropical diseases (NTDs) prioritized by the World Health Organization. The disease is difficult to treat due to no effective control measures available up to date. In this project, we will focus on the functions of serine protease inhibitors (Em_serpins) in the stem cells and protoscolices of E. multilocularis, combining with comprehensive analysis of genomic data of E. multilocularis. Firstly, Em_serpins knockout/down experiments will be performed at the genome level in stem cells of E. multilocularis via CRISPR/Cas9 genome editing system for the first time, which will demonstrate the mutation efficiency and off-target effects of Em_serpins target genes. Secondly, the mutation efficiency, expression level, tissue distribution and viability of the new generation of the protoscolices will be determined using qRT-PCR, western-blot, immunohistochemistry and Real-Time Cellular Analysis (RTCA) methods, respectively. The new generation of the protoscolices will be harvested from cysts which are expected to form after the mutated stem cells are injected to mice in abdomen. Lastly, the infectivity and reproductive capacity of the F1 protoscolices will be observed through animal experimental trails. Expected results will be obtained to elucidate the real functions of Em_serpins during the process of development, pathogenicity and so on of the E. multilocularis F1 protoscolices, and to provide ideal drug targets or vaccine candidates for the development of novel interventions for the control of echinococcosis. Meanwhile, the results will confirm that the targeted, heritable genetic alterations can be achieved in E. multilocularis via CRISPR/Cas9 system, suggesting that the system should be a convenient and effective approach for generating loss-of-function mutants in the studies of gene functions of tapeworms.

棘球蚴病(俗称包虫病)是由棘球属绦虫的幼虫—棘球蚴引起的一类危害严重的人兽共患寄生虫病，是国际卫生组织优先防治的17种被忽略的热带疾病之一，目前尚无有效防治措施。本项目以多房棘球蚴干细胞和原头蚴为研究对象，以丝氨酸蛋白酶抑制剂（Em_serpins）为研究靶标，在充分利用多房棘球绦虫基因组研究成果的基础上，首次采用CRISPR/Cas9系统介导的基因组编辑技术对干细胞中的Em_serpins在基因组水平进行突变，并鉴定靶基因的突变效率及脱靶效应。分别采用qRT-PCR、免疫印迹、免疫组化及RTCA技术，探析F1代原头蚴中Em_serpins的表达水平、组织分布及虫体活动性等特点。通过动物感染实验确定F1代原头蚴感染性与繁殖力的变化。阐明Em_serpins在多房棘球绦虫发育和致病等过程中的功能，为新型疫苗和药物研制提供重要的靶分子，同时将CRISPR/Cas9系统拓展到绦虫功能基因研究中。

多房棘球蚴病是由多房棘球绦虫的幼虫-多房棘球蚴寄生人体或动物所致的一种寄生虫病，又称泡球蚴病或泡型包虫病(alveolar echinococcosis，AE)，俗称“虫癌”。本研究基于多房棘球蚴动物模型，开展了多棘球蚴感染宿主后宿主与寄生虫相互作用基因的筛选研究。本研究完成了多房棘球蚴原头蚴腹腔接种小鼠后6个月内不同时间点小鼠肝脏组织样品的采集、RNA提取, 总RNA反转录成双链cDNA，再进一步合成用Cyanine-3-CTP(Cy3)标记的cRNA。标记好的cRNA和芯片杂交，洗脱后利用Agilent Scanner G2505C(Agilent Technologies)扫描得到原始图像。完成了所有样品的表达谱芯片检测及其结果的生物信息学分析，筛选鉴定了不同时间点差异表达基因；功能分析（GO analysis）表明，感染后2天与感染前相比，参与生物学过程（Biological Process）的差异表达基因有1225个表达下调，909个基因表达上调；参与细胞成分（Cellular Component）的差异基因有282个表达下调，204个表达上调；参与分子功能（Molecular Function）的差异表达基因有560个表达下调，439个表达上调；感染后180天与感染前比较，参与生物学过程的差异表达基因有2224个表达下调，2072个表达上调；参与细胞成分（Cellular Component）的差异基因有502个表达下调，385个表达上调；参与分子功能（Molecular Function）的差异表达基因有1215个表达下调，823个表达上调；这些差异基因主要富集于翻译、核糖体结构和生物发生，信号转导机制，蛋白的翻译后修饰、催化及分子伴侣，细胞壁生物发生，能量生产与转换等；归纳总结了变化规律，初步筛选了多房棘球蚴感染定植过程中宿主与寄生虫可能的相互作用基因；分析了差异基因的信号通路及其相互之间的关系。后续将开展显著差异表达基因的qPCR和Western-blot验证，作用分子网络及其相互作用基因在泡球蚴发育过程中的功能研究。本研究为多房棘球蚴发育与致病机理研究提供了丰富的数据资源。