Bacterial delivery of RNAi and CRISPRs for modulation of mosquito transcription

用于调节蚊子转录的 RNAi 和 CRISPR 的细菌传递

基本信息

批准号：
9090789
负责人：
Grant Leslie Hughes
金额：
$ 23.25万
依托单位：
UNIVERSITY OF TEXAS MED BR GALVESTON
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-03-15 至 2018-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9090789
关键词：
Address Adult Anopheles Genus Anopheles gambiae Arthropods Attention Bacteria Biology Biotechnology CRISPR interference Cells Clustered Regularly Interspaced Short Palindromic Repeats Complement Culicidae Cytolysis Development Disease Double-Stranded RNA Endocytosis Engineering Escherichia coli Gene Activation Gene Expression Gene Silencing Genes Genetic Genetic Materials Genetic Transcription Genetic study Genome Green Fluorescent Proteins In Vitro Insect Genes Insect Vectors Insecta Laboratories Larva Life Listeria Listeria monocytogenes hlyA protein Mediating Medical Methods Molecular Mutate Plasmids Process Promoter Regions Proteins Protocols documentation RNA RNA Interference Research Staging System Techniques Technology Tissues Transfer RNA Transgenic Organisms Vaccination Vesicle Water base cancer therapy extracellular flexibility gene function genome editing genome-wide analysis human disease in vitro Model in vivo knock-down news novel pathogen promoter public health relevance scale up small hairpin RNA stable cell line sugar theories tool vector vector control

项目摘要

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.

描述：细菌转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而受到广泛关注。在这种方法中，大肠杆菌被改造了两个基因，使它们能够将 RNA 发夹或质粒传递给宿主。细菌内吞作用，因此细胞外细菌自然进入细胞内，细菌裂解，使其遗传内容物分散，从而使细胞内细菌得以分散。退出宿主囊泡，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中逃逸，然后它们可以在宿主囊泡中调节宿主基因表达或被转录在这里，我们将把细菌转染的原理应用于蚊子。对这些医学上重要的昆虫进行细菌递送 RNAi 和成簇规则间隔短回文重复序列 (CRISPR) 在具体目标一中，我们将优化细菌递送系统。冈比亚按蚊并使用 RNAi 操纵蚊子基因表达为了优化昆虫的递送系统，除了检查 LLO 的各种突变形式外，我们还将检查细菌或蚊子启动子表达短发夹 RNA (shRNA) 时的击倒效率。蛋白质，已被证明可以增加哺乳动物系统中的细菌转染，然后我们将使用这种昆虫优化的细菌转染系统来沉默幼虫和成年蚊子中的基因，这是一个令人兴奋的结果。不断扩展的分子工具最近经过修改，可以通过将死 Cas9 (dCas9) 蛋白定位到启动子区域来实现，该蛋白可以抑制或增强转录。在第二个目标中，我们将开发 CRISPR。干扰（CRISPRi）和CRISPR激活（CRISPRa）用于蚊子，并使用优化的细菌转染系统将这些分子传递给蚊子，这将使按蚊中的两种shRNA基因特异性敲除。和 CRISPR 传递，只需将细菌接种到幼虫水中或添加糖粉，该技术将为操纵蚊子基因表达提供灵活而简单的系统，并且是对现有方法的巨大改进。此外，该系统有可能扩大用于基因沉默和基因激活的全基因组筛选，并且除了作为一种系统外，还可以在体外和体内使用。作为一种宝贵的实验室工具，这种方法有可能用作一种新型的病媒控制策略。