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

• 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
• 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

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.

人体是一个复杂的生态系统的汇编，依赖于细菌。噬菌体 扰乱这些微生物群落感染最近已经涉及到大范围的人类 紊乱重复的规则间隔短回文重复序列（CRISPR）是CRISPR的重要组成部分。 一种RNA引导的基因调控系统，可保护细菌免受噬菌体感染，并控制 毒力因子。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学的影响。假单胞 在铜绿假单胞菌中，CRISPR基因座被转录并加工成小RNA，这些小RNA被掺入到Csy中。 复合物，一种大的多亚基核糖核蛋白，需要保护免受噬菌体。然而，在这方面， 在我们对Csy复合体如何发现入侵核酸以及这些核酸如何被发现的理解上， 分子信标募集效应物核酸酶（即，Cas 3）以靶向核酸进行破坏。最近这些 CRISPR RNA引导的监视系统已被重新用于可编程基因组工程， 各种各样的真核细胞和模式生物，以前是难以遗传操纵。 在本提案中，我们使用生化（目标1）和结构（目标2）策略的组合来确定 通过来自P. 铜绿假单胞菌（I-F型），并确定病毒编码抑制剂如何破坏这种免疫系统。结果 这些实验将提供有助于我们长期目标的机械见解， 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。