The function of small RNA-based viral defense system in E. coli

描述（由申请人提供）：原核生物与其病毒（噬菌体）和质粒的相互作用解释了水平基因转移（HGT），这是抗生素耐药性传播和人类病原体出现的基础。细菌进化出许多系统来限制HGT。一种新的原核生物防御外源DNA的系统是基于CRISPR（聚集规则间隔短回文重复）磁带和cas基因。CRISPR卡带由直接重复序列组成，中间穿插着高度可变的序列间隔。与大型Cas蛋白复合物结合的小CRISPR rna （crRNA）识别外源DNA，匹配crRNA中存在的间隔序列，并破坏它。这个过程被称为“CRISPR干扰”。CRISPR磁带中的间隔器不受干扰。在一个被称为“CRISPR适应”的过程中，病毒或质粒衍生的DNA被CRISPR卡带获得，成为间隔物。必须避免获取宿主衍生的间隔，因为它将导致自干扰。CRISPR反应的两个阶段都没有被完全理解。我们建议在研究最多的原核生物大肠杆菌中研究CRISPR的功能。实验室大肠杆菌的CRISPR/cas基因座处于休眠状态。我们开发了遗传系统来研究大肠杆菌CRISPR反应的两个阶段。我们将使用这些系统和遗传、生化、交联、实验室进化和建模方法：目标1。分析CRISPR干扰，确定CRISPR干扰机制对自我和非自我DNA识别的控制规则；鉴定体外Cas蛋白- crrna复合物与靶向降解的外源DNA形成的特征，并定位crrna介导的目标切割位点。实验将用现有的系统靶向M13噬菌体和新的系统干扰大肠杆菌的裂解T-odd噬菌体和RNA噬菌体进行。目标2。分析CRISPR适应并确定i)通过CRISPR适应机制管理自我与非自我DNA区分的规则；ii) CRISPR盒外影响间隔序列获取的序列；以及iii)导致CRISPR卡带中出现额外间隔重复单元的过程的分子细节。与外源DNA形成的Cas蛋白复合物将在体外和体内通过将它们捕获在蛋白质路障上进行表征。为了更好地理解CRISPR介导的病毒-宿主动力学和共同进化，我们将监测宿主CRISPR磁带中的间隔层获取和病毒突变，这些突变使CRISPR干扰在持续感染的培养物中无效，并与一组生物信息学家合作建立这一过程的数学模型。这项工作将揭示在CRISPR应答过程中运行的新的分子机制，并为菌株工程和原核生物基因沉默开辟新的途径。拟议工作的意义并不局限于大肠杆菌，因为在40%以上的真细菌和95%的古细菌中发现了CRISPR位点。

DESCRIPTION (provided by applicant): Interaction of prokaryotes with their viruses (phages) and plasmids accounts for horizontal gene transfer (HGT) that underlies the spread of antibiotic resistance and emergence of human pathogens. Bacteria evolved numerous systems to limit HGT. A novel prokaryotic defense system against foreign DNA is based on CRISPR (clustered regularly interspaced short palindromic repeats) cassettes and cas genes. A CRISPR cassette consists of direct repeats interspersed with spacers of highly variable sequence. Small CRISPR RNAs (crRNAs) bound to a large Cas proteins complex recognize foreign DNA, matching the spacer sequence present in crRNA, and destroy it. This process is referred to as "CRISPR interference". Spacers in CRISPR cassettes are excluded from interference. Viral or plasmid-derived DNA is acquired by CRISPR cassette, becoming a spacer, in a process called "CRISPR adaptation". Acquisition of host-derived spacers must be avoided, for it will lead to self-interference. Neither stage of CRISPR response is fully understood. We propose to study CRISPR function in Escherichia coli, the best-studied prokaryote. CRISPR/cas loci of laboratory E. coli are dormant. We developed genetic systems to study both stages of E. coli CRISPR response. We will use these systems and genetic, biochemical, crosslinking, laboratory evolution, and modeling approaches to: Aim 1. Analyze CRISPR interference and identify rules that govern self versus non-self DNA recognition by CRISPR interference machinery; characterize in vitro Cas protein-crRNA complexes formed with foreign DNA targeted for degradation, and localize the sites of crRNA-mediated target cleavage. Experiments will be performed with existing systems targeting the M13 phage and with new systems interfering with lytic T-odd phages and RNA phages of E. coli. Aim 2. Analyze CRISPR adaptation and determine i) rules that govern self versus non-self DNA discrimination by CRISPR adaptation machinery; ii) sequences outside CRISPR cassette that affect spacer acquisition; and iii) molecular details of the process that leads to appearance of extra spacer-repeat units in CRISPR cassette. Cas protein complexes formed with foreign DNA targeted for adaptation will be characterized in vitro and in vivo by trapping them at protein roadblocks. To better understand CRISPR-mediated viral-host dynamics and co-evolution we will monitor spacer acquisition in CRISPR cassettes of the host and viral mutations that render CRISPR interference ineffective in continuously infected cultures and develop a mathematical model of this process in collaboration with a group of bioinformaticians. As a result of proposed work novel molecular mechanisms operational during CRISPR response will be revealed and new ways for strain engineering and gene silencing in prokaryotes will be developed. The significance of proposed work will not be limited to E. coli, since CRISPR loci are found in more than 40% eubacteria and in 95% of archaea.