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（CMV）和质粒的相互作用导致了水平基因转移（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。针对外源DNA的一种新的原核防御系统是基于CRISPR（成簇规则间隔短回文重复序列）盒和cas基因。CRISPR盒由散布有高度可变序列的间隔区的同向重复序列组成。小CRISPR RNA（crRNA）与大Cas蛋白复合物结合识别外来DNA，匹配crRNA中存在的间隔序列，并将其破坏。该过程被称为“CRISPR干扰”。CRISPR盒中的间隔区不受干扰。病毒或质粒衍生的DNA通过CRISPR盒获得，在称为“CRISPR适应”的过程中成为间隔区。必须避免获得宿主衍生的间隔区，因为这将导致自干扰。CRISPR反应的两个阶段都没有被完全理解。我们建议在大肠杆菌中研究CRISPR功能，大肠杆菌是研究得最好的原核生物。实验室E.大肠杆菌处于休眠状态。我们开发了遗传系统来研究E. coli CRISPR反应。我们将使用这些系统和遗传，生物化学，交联，实验室进化和建模方法：目标1。分析CRISPR干扰并通过CRISPR干扰机制确定控制自身与非自身DNA识别的规则;表征与靶向降解的外源DNA形成的体外Cas蛋白-crRNA复合物，并定位crRNA介导的靶切割位点。实验将用现有的靶向M13噬菌体的系统和新的干扰E.杆菌目标2.分析CRISPR适应并确定i）通过CRISPR适应机制控制自我与非自我DNA区分的规则; ii）影响间隔子获取的CRISPR盒外部序列;以及iii）导致CRISPR盒中出现额外间隔子重复单元的过程的分子细节。与靶向适应的外源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路障处来表征。为了更好地了解CRISPR介导的病毒-宿主动力学和共同进化，我们将监测宿主CRISPR盒中的间隔区获取以及使CRISPR干扰在连续感染的培养物中无效的病毒突变，并与一组生物信息学家合作开发该过程的数学模型。 作为拟议工作的结果，将揭示CRISPR反应期间的新分子机制，并将开发原核生物中菌株工程和基因沉默的新方法。建议的工作的意义将不仅限于E。因为CRISPR基因座存在于超过40%的真细菌和95%的古细菌中。

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.