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

描述（由申请人提供）：所有生物必须保护自己免受病毒和其他不可避免地在其环境中遇到的分子入侵者的侵害。最近发现，原核生物（古生菌和细菌）具有小的基于rna的适应性免疫系统，称为CRISPR-Cas系统，可有效控制病毒和质粒的入侵。CRISPR- cas系统的功能是在宿主基因组的CRISPR位点内捕获并整合短病毒和质粒序列，处理来自CRISPR位点转录本的单个CRISPR (cr) rna，并使用crrna引导效应复合物破坏入侵者的核酸。基于CRISPR-Cas的免疫是由许多不同的Cas （crispr相关）蛋白介导的，一个给定的生物体可能拥有九组不同的CRISPR-Cas免疫模块中的一组或多组。我们目前对CRISPR-Cas防御途径的关键步骤如何在大多数这些系统中发生知之甚少。本提案的长期目标是了解在炽热焦球菌（Pyrococcus furiosus）中控制CRISPR-Cas免疫的分子机制，这是一种用于理解CRISPR-Cas生物学的已建立的模式生物，它利用三种不同的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解决入侵序列获取和沉默的分子基础：确定Cmr效应复合物的功能和体内RNA靶向潜力的分子基础。定义Cst和Csa crRNPs的组织和功能。确定外源DNA序列如何在CRISPR基因座中被捕获。利用分子、遗传和生化方法的强大组合，我们将描述三种CRISPR-Cas途径中每一种效应复合物的入侵沉默机制。对于每个效应复合物，我们将努力了解crRNA装载、靶核酸相互作用和入侵者沉默的分子基础。我们也将保证获得

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.