Delineation of CRISPR-Cas invader defense pathways in Streptococcus thermophilus

描述(申请人提供)：CRISPR-CAS系统是最近发现的，基于RNA的免疫系统，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基因座内的短入侵序列，从CRISPR基因座产生的小RNA(CRISPR(Cr)RNAs)被认为引导Cas蛋白识别和沉默入侵的核酸；然而，我们对CRISPR-Cas免疫反应通路中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研究CRISPR-Cas防御所需三个步骤的分子基础：确定CRISPR基因座如何获得外源DNA序列确定产生功能性CRISPR RNA的机制|具体描述入侵沉默的机制，我们的目的是从分子上了解CRISPR-Cas防御途径在嗜热链球菌中的作用。我们将确定CRISPR-Cas防御的三个关键阶段中的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检验这一假设和一些特定的相关假设。这些实验将确定大量原核生物用来在病毒攻击下生存的CRISPR-Cas通路。这项研究中获得的信息将直接有助于正在进行的旨在利用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)、削弱人类病原体和限制抗生素耐药性传播的努力。

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.