Structure and function of CRISPR RNA-guided foreign DNA surveillance systems in P. aeruginosa

铜绿假单胞菌中 CRISPR RNA 引导的外源 DNA 监测系统的结构和功能

基本信息

批准号：
9173121
负责人：
Blake A Wiedenheft
金额：
$ 31.16万
依托单位：
MONTANA STATE UNIVERSITY - BOZEMAN
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：
2016-09-23 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9173121
关键词：
Address Affinity Animal Model Bacteria Bacterial Genome Bacteriophages Base Pairing Behavior Binding Biochemical Biotechnology Cell model Chemicals Clustered Regularly Interspaced Short Palindromic Repeats Complementary DNA Complex Conserved Sequence Cryoelectron Microscopy DNA Data Detection Disease Ecology Ecosystem Escape Mutant Eukaryotic Cell Evolution Feedback Gene Expression Regulation Genome Genome engineering Goals Guide RNA Human Human body Immune system Immunity Infection Infection Control Intercept Invaded Kinetics Knowledge Lead Libraries Major Groove Measures Mediating Medicine Methods Minor Groove Modeling Modification Molecular Mutate Mutation Nucleic Acids Nucleotides Plasmids Process Proteins Pseudomonas aeruginosa RNA Recognition Motif Recruitment Activity Research Resolution Ribonucleoproteins Small RNA Structure System Testing Time Update Virulence Factors base design genetic manipulation insight microbial community nuclease particle prevent public health relevance reconstruction research study tool

项目摘要

Project Summary: The human body is a compilation of complex ecosystems that rely on bacteria. Phage infections that perturb these microbial communities have recently been implicated in a wide range of human disorders. Clustered regularly interspaced short palindromic repeats (CRISPRs) are essential components of an RNA-guided gene regulation system that protects bacteria from phage infection and controls the expression of virulence factors. 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. In Pseudomonas aeruginosa, CRISPR loci are transcribed and processed into small RNAs that are incorporated into the Csy complex, a large multi-subunit ribonucleoprotein required for protection against bacteriophages. However, there is a critical gap in our understanding of how the Csy complex finds invading nucleic acids and how these molecular beacons recruit effector nucleases (i.e., Cas3) to target nucleic acids for destruction. Recently, these CRISPR RNA-guided surveillance systems have been repurposed for programmable genome engineering in a wide variety of eukaryotic cells and model organisms that were previously recalcitrant to genetic manipulation. In this proposal we use a combination of biochemical (Aim 1) and structural (Aim 2) strategies to determine the mechanisms of CRISPR-Cas RNA-guided detection of foreign DNA by the adaptive immune system from P. aeruginosa (Type I-F) and determine how virally encode suppressors subvert this immune system. Results from these experiments will provide mechanistic insights that contribute to our long-term goal and have significance implications in the near-term application of these emerging tools for precise genome engineering.

项目摘要：人体是依赖细菌的复杂生态系统的集合。噬菌体最近，扰乱这些微生物群落的感染与广泛的人类感染有关。失调。成簇的规则间隔的短回文重复序列 (CRISPR) 是 RNA引导的基因调控系统，保护细菌免受噬菌体感染并控制表达毒力因子。我们研究的长期目标是了解 CRISPR 介导的影响免疫系统对人类相关微生物群落进化和生态的影响。在假单胞菌中铜绿假单胞菌中，CRISPR 位点被转录并加工成小 RNA，并整合到 Csy 中复合物，一种大的多亚基核糖核蛋白，用于防御噬菌体。然而，我们对 Csy 复合物如何发现入侵核酸以及这些核酸如何分子信标招募效应核酸酶（即 Cas3）来靶向核酸以进行破坏。最近，这些 CRISPR RNA 引导的监测系统已被重新用于可编程基因组工程以前难以进行基因操作的各种真核细胞和模型生物。在本提案中，我们结合使用生化（目标 1）和结构（目标 2）策略来确定 CRISPR-Cas RNA引导的适应性免疫系统检测外源DNA的机制铜绿假单胞菌（I-F 型）并确定病毒编码抑制因子如何颠覆该免疫系统。结果这些实验将提供有助于我们长期目标的机制见解，并具有这些新兴工具对精确基因组工程的近期应用具有重要意义。