Delineation of CRISPR-Cas invader defense pathways in Streptococcus thermophilus

描述（由申请人提供）：CRISPR-Cas系统是最近发现的基于RNA的免疫系统，可控制原核生物中病毒和质粒的入侵。具有 CRISPR-Cas 系统的原核生物捕获其基因组中 CRISPR 位点内的短入侵序列，并且由 CRISPR 位点产生的小 RNA (CRISPR (cr)RNA) 被认为可以引导 Cas 蛋白识别并沉默入侵的核酸；然而，我们对非凡的 CRISPR-Cas 免疫反应途径的关键步骤如何发生知之甚少。在这个项目中，我们通过以下具体目标来解决 CRISPR-Cas 防御所需三个步骤的分子基础： ¿ 确定 CRISPR 位点如何获取外源 DNA 序列 ¿ 确定功能性 CRISPR RNA 的产生机制 ¿ 描述入侵者沉默的机制 具体来说，我们的目标是获得对嗜热链球菌中发挥作用的 CRISPR-Cas 防御途径的分子理解。我们将确定 CRISPR-Cas 防御的三个关键阶段中每个阶段所涉及的基本细胞机制（蛋白质和 RNA）和分子机制。我们假设 Cas 蛋白在防御途径的每个主要步骤中发挥作用，我们将在拟议的研究中测试这一点以及一些具体的相关假设。这些实验将定义多种原核生物用来抵御病毒攻击的 CRISPR-Cas 途径。这项研究中获得的信息将直接有助于持续努力，旨在利用 CRISPR-Cas 系统来增强驯化细菌（用于生产安全食品、药品和生物燃料）并削弱人类病原体并限制抗生素耐药性的传播。

DESCRIPTION (provided by applicant): CRISPR-Cas systems are recently discovered, RNA-based immune systems that control invasions of viruses and plasmids in prokaryotes. Prokaryotes with CRISPR-Cas systems capture short invader sequences within the CRISPR loci in their genomes, and small RNAs produced from the CRISPR loci (CRISPR (cr)RNAs) are thought to guide Cas proteins to recognize and silence the invading nucleic acids; however, we know very little about how the key steps in the remarkable CRISPR-Cas immune response pathways occur. In this project, we address the molecular basis for the three steps required for CRISPR-Cas defense through the following specific aims: ¿ Determine how CRISPR loci acquire foreign DNA sequences ¿ Determine the mechanisms by which functional CRISPR RNAs are produced ¿ Delineate the mechanisms of invader silencing Specifically, we aim to obtain a molecular understanding of the CRISPR-Cas defense pathways that function in Streptococcus thermophilus. We will identify the essential cellular machinery (proteins and RNAs) and molecular mechanisms involved in each of the three key phases of CRISPR-Cas defense. We hypothesize that Cas proteins function in each of the major steps in the defense pathway, and we will test this and a number of specific related hypotheses in the proposed studies. The experiments will define the CRISPR-Cas pathways used by multitudes of prokaryotes to survive viral attack. The information gained in this study will contribute directly o ongoing efforts aimed at exploiting CRISPR-Cas systems to both strengthen domesticated bacteria (used to produce safe food, pharmaceuticals and biofuels) and weaken human pathogens and limit the spread of antibiotic resistance.