Small RNA-based immunity by CRISPR-Cas complexes of Pyrococcus furiosus

描述(申请人提供)：所有生物体必须保护自己免受病毒和其他分子入侵者的侵袭，这些病毒和分子入侵者是他们在环境中不可避免地遇到的。最近发现原核生物(古生菌和细菌)具有基于RNA的小适应性免疫系统，称为CRISPR-CAS系统，可以有效地控制病毒和质粒的入侵。CRISPR-Cas系统的功能是捕获并整合寄主基因组CRISPR基因座内的短病毒和质粒序列，处理CRISPR基因座转录本中的单个CRISPR(Cr)RNA，并使用crRNAs引导效应器复合体破坏入侵者的核酸。基于CRISPR-Cas的免疫是由众多不同的Cas(CRISPR-Associated)蛋白介导的，一个特定的生物体可能拥有九套不同的CRISPR-Cas免疫模块中的一套或多套。我们目前对CRISPR-Cas防御途径中的关键步骤是如何在大多数这些系统中发生的知之甚少。这项建议的长期目标是了解控制CRISPR-Cas免疫的分子机制，这是一种为理解CRISPR-Cas生物学而建立的模式生物，它利用三条不同的CRISPR-Cas途径来沉默入侵者。在这个项目中，我们通过以下具体目标来研究入侵者序列获取和沉默的分子基础：确定CMR效应复合体的功能和体内RNA靶向潜力的分子基础。？确定科技委和协调委员会crRNPs的组织和职能。确定如何在CRISPR基因座内捕获外源DNA序列。利用分子、遗传和生化方法的强大组合，我们将描述入侵者沉默的机制，为每一个CRISPR-CAS途径的效应器复合体。对于每个效应复合体，我们将努力了解crRNA负载、靶核酸相互作用和入侵者沉默的分子基础。我们还将承诺获得 首先，通过研究入侵者DNA片段的产生和整合到宿主基因组中，深入了解CRISPR基因座在任何系统中获取入侵者序列的有趣过程。这项工作将定义大量原核生物用来在病毒捕食中生存的CRISPR-Cas通路。在这项研究中获得的知识将直接有助于 目前正在努力利用CRISPR-CAS系统加强驯化细菌(用于生产安全食品、药品和生物燃料)，并与人类病原体作斗争，限制抗生素耐药性的传播。

DESCRIPTION (provided by applicant): All organisms must protect themselves from the viruses and other molecular invaders that they inevitably encounter in their environments. It was very recently discovered that prokaryotes (archaea and bacteria) have small RNA-based adaptive immune systems, called CRISPR-Cas systems that effectively control invasions of viruses and plasmids. CRISPR-Cas systems function by capturing and incorporating short viral and plasmid sequences within the CRISPR locus of the host genome, processing individual CRISPR (cr)RNAs from the CRISPR locus transcripts, and using the crRNAs to guide effector complexes to destroy the nucleic acids of the invader. CRISPR-Cas based immunity is mediated by numerous and diverse Cas (CRISPR-associated) proteins and a given organism may possess one or more of the nine distinct sets of known CRISPR-Cas immune modules. We currently know very little about how the key steps in the remarkable CRISPR-Cas defense pathway occur for most of these systems. The long-term objective of this proposal is to understand the molecular mechanisms that govern CRISPR-Cas immunity in Pyrococcus furiosus, an established model organism for understanding CRISPR-Cas biology that utilizes three distinct CRISPR-Cas pathways for invader silencing. In this project, we address the molecular basis for invader sequence acquisition and silencing through the following specific aims: ¿ Determine the molecular basis for function and the in vivo RNA targeting potential of the Cmr effector complex. ¿ Define the organization and function of Cst and Csa crRNPs. ¿ Determine how foreign DNA sequences are captured within CRISPR loci. Using a powerful combination of molecular, genetic, and biochemical approaches, we will delineate the mechanism of invader silencing for the effector complexes of each of the three CRISPR-Cas pathways. For each effector complex, we will work to understand the molecular basis of crRNA loading, target nucleic acid interaction, and invader silencing. We will also undertake to gain the first insight into the intriguing process of acquisition of invader sequences by the CRISPR locus in any system by investigating the generation of invader DNA fragments and incorporation into the host genome. The work will define the CRISPR-Cas pathways used by multitudes of prokaryotes to survive viral predation. The knowledge gained in this study will contribute directly to ongoing efforts aimed at exploiting CRISPR-Cas systems to both strengthen domesticated bacteria (used to produce safe food, pharmaceuticals and biofuels) and combat human pathogens and limit the spread of antibiotic resistance.