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

描述(申请人提供)：原核生物与其病毒(噬菌体)和质粒的相互作用是水平基因转移(HGT)的原因，HGT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。基于CRISPR(簇状规则间隔短回文重复序列)盒和cas基因构建了一种新的原核生物对外来DNA的防御系统。CRISPR盒由散布着高度可变序列的间隔区的直接重复序列组成。与大的Cas蛋白复合体结合的小CRISPR RNAs(CrRNAs)识别外来DNA，与crRNA中存在的间隔区序列匹配，并破坏它。这个过程被称为“CRISPR干扰”。CRISPR盒中的间隔物不受干扰。病毒或质粒衍生的DNA是通过CRISPR盒获得的，成为一个间隔区，这个过程被称为“CRISPR适应”。必须避免获得宿主衍生的间隔区，因为这将导致自我干扰。CRISPR反应的两个阶段都不是完全了解的。我们建议研究CRISPR在原核生物--大肠杆菌中的功能。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来：目的1.分析CRISPR干扰，并通过CRISPR干扰机制识别控制自体和异体DNA识别的规则；在体外表征与外源DNA形成的Cas蛋白质-crRNA复合体以降解为目标，并定位crRNA介导的靶向切割位点。将使用针对M13噬菌体的现有系统以及干扰大肠杆菌裂解T-奇噬菌体和RNA噬菌体的新系统进行实验。目的2.分析CRISPR适应并确定：1)CRISPR适应机制对自身和异体DNA的区分规则；2)影响间隔区获得的CRISPR盒外序列；以及3)导致CRISPR盒中出现额外间隔区-重复单位的过程的分子细节。以适应为目标的与外源DNA形成的CAS蛋白质复合体将通过将它们捕获在蛋白质路障上来在体外和体内进行表征。为了更好地了解CRISPR介导的病毒宿主动态和共同进化，我们将监测CRISPR盒中宿主的间隔区获取和使CRISPR干扰在持续感染的培养中无效的病毒突变，并与一组生物信息学家合作开发这一过程的数学模型。作为拟议工作的结果，将揭示在CRISPR反应过程中起作用的新的分子机制，并将开发新的方法在原核生物中进行菌株工程和基因沉默。拟议工作的意义将不仅限于大肠杆菌，因为CRISPR基因座在超过40%的真细菌和95%的古生菌中被发现。

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.