Structure, Function and Application of CRISPR RNA-guided Immunity in Bacteria

描述（由申请人提供）：噬菌体是地球上最丰富的生物实体，这些无处不在的捕食者施加的选择压力对每个生态环境中微生物群落的组成和行为产生深远影响。人体是一个依赖细菌的复杂生态系统的汇编。事实上，生活在健康人体内的细菌数量估计是人体细胞数量的十倍，而扰乱这些微生物群落的噬菌体感染最近被认为与多种人类疾病有关。此外，大肠杆菌是赋予毒力和抗生素抗性的基因的主要提供者，因此大肠杆菌在细菌发病机制的进化中起主要作用。规则间隔短回文重复序列（CRISPR）是最近发现的基于核酸的适应性免疫系统的重要组成部分，该系统广泛存在于细菌和古细菌中，并且这些免疫系统在控制毒力相关基因的水平传播中发挥着核心作用。我们研究的长期目标是了解CRISPR介导的免疫系统对人类相关微生物群落的进化和生态的影响。具体而言，该提案中概述的工作旨在阐明大肠杆菌中适应性免疫系统的CRISPR RNA引导的监视和靶向消除外源DNA的机制。我们预计这项工作将导致大肠杆菌CRISPR RNA指导的监视复合物的高分辨率结构，这些结构对于我们理解这些监视机器如何工作将是非常宝贵的。然而，结构只会捕捉特定姿势的复合体的快照。为了了解这些机器的动力学，我们补充了我们的结构研究与实时动力学分析，旨在探测目标监视的机制和速率。最后，我们实施我们的结构和生物化学的见解，设计可编程的基因沉默系统。CRISPR介导的基因沉默为系统地控制原核和真核系统中的基因表达提供了一种新方法。总的来说，这项提案为公众和科学界提供了关于细菌适应性免疫机制的有希望的新见解，这些机制将对生物技术和医学产生重大影响。

DESCRIPTION (provided by applicant): Bacteriophages are the most abundant biological entities on earth and the selective pressures imposed by these pervasive predators have a profound impact on the composition and the behavior of microbial communities in every ecological setting. The human body is a compilation of complex ecosystems that rely on bacteria. In fact, the number of bacteria living in a healthy human is estimated to be ten times greater than the number of human cells and phage infections that perturb these microbial communities have recently been implicated in a wide range of human disorders. Furthermore, phages are major purveyors of genes that confer virulence and antibiotic resistance and thus phages play a major role in the evolution of bacterial pathogenesis. Clustered regularly interspaced short palindromic repeats (CRISPR) are essential components of a recently discovered, nucleic-acid-based adaptive immune system that is widespread in bacteria and archaea, and these immune systems play a central role in controlling the horizontal dissemination of virulence associated genes. The long-term goal of our research is to understand the impact of CRISPR-mediated immune systems on the evolution and ecology of human associated microbial communities. Specifically, the work outlined in this proposal is aimed at explicating the mechanisms of CRISPR RNA-guided surveillance and targeted elimination of foreign DNA by the adaptive immune system in Escherichia coli. We anticipate that this work will result in high-resolution structures of the CRISPR RNA-guided surveillance complex from Escherichia coli and that these structures will be invaluable to our understanding of how these surveillance machines work. However, structures will only capture snap-shots of the complex in specific poses. To understand the dynamics of these machines we complement our structural studies with real-time kinetic analysis aimed at probing the mechanism and rates of target surveillance. Finally, we implement our structural and biochemical insights to design programmable gene silencing systems. CRISPR-mediated gene silencing offers a novel approach for systematically controlling gene expression in both prokaryotic and eukaryotic systems. Overall, this proposal provides promising new insight to the public and scientific community regarding the mechanisms of adaptive immunity in bacteria that will have significant impacts in biotechnology and medicine.