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

描述(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都有深远的影响。人体是依赖细菌的复杂生态系统的集合。事实上，据估计，生活在健康人体中的细菌数量是人类细胞数量的十倍，扰乱这些微生物群落的噬菌体感染最近被认为与一系列人类疾病有关。此外，噬菌体是赋予毒力和抗生素耐药性的基因的主要提供者，因此在细菌发病机制的进化中发挥着重要作用。簇状规则间隔短回文重复序列(CRISPR)是最近发现的一种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生菌中，这些免疫系统在控制毒力相关基因的水平传播方面发挥着核心作用。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。具体地说，这项建议中概述的工作旨在阐明CRISPR RNA引导的监视和通过适应免疫系统在大肠杆菌中靶向消除外来DNA的机制。我们预计，这项工作将导致从大肠杆菌中获得CRISPR RNA引导的监视复合体的高分辨率结构，这些结构将对我们了解这些监视机器的工作原理非常有价值。然而，结构将只捕捉特定姿势下的综合体的快照。为了了解这些机器的动力学，我们用实时动力学分析来补充我们的结构研究，旨在探索目标监视的机制和速度。最后，我们应用我们的结构和生化见解来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的方法。总体而言，这项建议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.