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

项目摘要：人体是依赖细菌的复杂生态系统的汇编。噬菌体 扰乱这些微生物群落的感染最近被牵连到广泛的人类 精神错乱。簇状规则间隔短回文重复序列(CRISPR)是 一种RNA引导的基因调控系统，保护细菌免受噬菌体感染并控制其表达 毒力因子。我们研究的长期目标是了解CRISPR中介的影响 免疫系统对人类相关微生物群落的进化和生态学的影响。假单胞菌 铜绿假单胞菌、CRISPR基因座被转录和加工成小RNA，并被整合到CSY中 复合体，是一种大的多亚单位核糖核蛋白，对噬菌体具有保护作用。然而， 我们对csy复合体是如何发现入侵的核酸，以及它们是如何 分子信标招募效应核酸酶(即Cas3)以破坏核酸为目标。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 种类繁多的真核细胞和模式生物，这些细胞和模式生物以前不愿进行基因操作。 在本提案中，我们结合使用生化策略(目标1)和结构策略(目标2)来确定 CRISPR-Cas RNA引导的获得性免疫系统检测外源DNA的机制 铜绿假单胞菌(I-F型)，并确定病毒编码的抑制子如何颠覆这种免疫系统。结果 这些实验将提供机械的见解，有助于我们的长期目标，并具有 这些精密基因组工程新兴工具近期应用的意义。

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.