Bacterial delivery of RNAi and CRISPRs for modulation of mosquito transcription

 产品说明：细菌感染是一种很有前途的生物技术，作为许多人类疾病的替代医学治疗方法，正受到相当大的关注。在这种方法中，大肠杆菌被改变了两个基因，使它们能够将RNA发夹或质粒传递给宿主。侵袭素基因促进细菌内吞作用，因此细胞外细菌自然变为细胞内细菌。一旦细胞内，细菌裂解，使其遗传内容物分散。李斯特菌溶血素O（LLO）允许细胞内细菌离开宿主囊泡，在该系统中用于促进细菌来源的RNA发夹或质粒从宿主囊泡逃逸，然后它们可以调节宿主基因表达或被转录。在这里，我们将使细菌感染的原理适用于蚊子，使细菌递送RNAi和成簇的规则间隔短回文重复序列（CRISPR）能够用于这些医学上重要的昆虫。具体目标之一是优化冈比亚按蚊的细菌载体系统，并利用RNAi技术调控蚊基因的表达。为了优化昆虫的递送系统，我们将检查当短发夹RNA（shRNA）从细菌或蚊子启动子表达时的敲低效率，此外还检查LLO蛋白的各种突变形式，这些突变形式已显示出增加哺乳动物系统中的细菌感染。然后，我们将使用这种昆虫优化的细菌感染系统来沉默幼虫和成年蚊子中的基因。CRISPR是一种令人兴奋且不断扩展的分子工具，最近已被修改以操纵基因表达。这是通过将死Cas9（dCas9）蛋白靶向启动子区域来实现的，其可以抑制或增强转录。在第二个目标中，我们将开发用于蚊子的CRISPR干扰（CRISPRi）和CRISPR激活（CRISPRa），并使用优化的细菌感染系统将这些分子递送给蚊子。这将使按蚊中的基因特异性敲高和敲低成为可能。对于shRNA和CRISPR递送，细菌将通过简单地将幼虫水浸泡或将它们的糖粉掺入蚊子体内。该技术将提供一个灵活而简单的系统来操纵蚊子基因表达，是对现有方法的一个巨大改进。该方法适用于调控幼虫、蛹和成虫生命阶段的基因表达。此外，该系统具有扩大用于基因沉默和基因激活的全基因组筛选的潜力，并且可以在体外和体内使用。除了作为一种宝贵的实验室工具外，这种方法还具有用作新型病媒控制策略的潜力。

 DESCRIPTION: Bactofection is a promising biotechnology that is gaining considerable attention as an alternative medical treatment for many human diseases. In this approach, Escherichia coli are altered with two genes that enable them to deliver RNA hairpins or plasmids to a host. The Invasin gene facilitates bacterial endocytosis so naturally extracellular bacteria become intracellular. Once intracellular, the bacteria lyse enabling their genetic contents to disperse. Listeriolysin O (LLO), which allows intracellular bacteria to exit host vesicles, is used in this system to facilitate the escape of bacterially derived RNA hairpins or plasmids from the host vesicle, where they can then modulate host gene expression or be transcribed. Here, we will adapt the principles of bactofection to mosquitoes enabling bacterial delivery RNAi and clustered regularly interspaced short palindromic repeats (CRISPRs) to these medically important insects. In specific aim one, we will optimize the bacterial delivery system for Anopheles gambiae and manipulate mosquito gene expression using RNAi. To optimize the delivery system to insects, we will examine knock down efficiency when a short hairpin RNA (shRNA) is expressed from a bacterial or mosquito promoter in addition to examining various mutated forms of the LLO protein, which have been shown to increase bactofection in mammalian systems. We will then use this insect optimized bactofection system to silence genes in both larvae and adult mosquitoes. CRISPRs are an exciting and ever-expanding molecular tool that has recently been modified to manipulate gene expression. This is accomplished by targeting a dead Cas9 (dCas9) protein to the promoter regions, which can either inhibit or enhance the transcription. In the second aim, we will develop CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) for use in mosquitoes and use the optimized bactofection system to deliver these molecules to mosquitoes. This will enable gene specific knock up and knock down in Anopheles mosquitoes. For both shRNA and CRISPR delivery, bacteria will be administered to mosquitoes by simply inoculating the larval water or spiking their sugar meal. This technology will provide a flexible and simple system for manipulating mosquito gene expression and be a dramatic improvement on current methods. This approach is suitable for modulating gene expression in larval, pupal and adult life stage. Additionally, this system has the potential to be scaled up for genome wide screening for gene silencing and gene activation and can be used both in vitro and in vivo. In additional to being an invaluable laboratory tool, this approach has the potential for use as a novel vector control strategy.