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

大肠杆菌中基于小RNA的病毒防御系统的功能

基本信息

批准号：
8420796
负责人：
KONSTANTIN V SEVERINOV
金额：
$ 29.45万
依托单位：
RUTGERS, THE STATE UNIV OF N.J.
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-02-01 至 2017-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8420796
关键词：
Accounting Address Affect Antibiotic Resistance Appearance Archaea Bacteria Bacteriophage M13 Bacteriophages Biochemical Cessation of life Cluster Analysis Collaborations Complex DNA DNA Restriction Enzymes DNA Restriction-Modification Enzymes Development Direct Repeats Discrimination Engineered Gene Enzymes Epigenetic Process Escherichia coli Eubacterium Evolution Gene Silencing Gene Silencing Pathway Genes Genetic Genetic Engineering Horizontal Gene Transfer Host Defense Human Immunity In Vitro Infection Laboratories Lead Light Lytic Mediating Methylation Mobile Genetic Elements Modeling Modification Molecular Molecular Cloning Molecular Genetics Monitor Mutation Organism Pathogenicity Island Plasmids Process Prokaryotic Cells Proteins RNA Binding RNA Phages Race Site Small RNA Staging System Toxin Viral Viral Physiology Virus Work arm base crosslink fascinate human BCAR1 protein in vivo interest mathematical model novel pathogen protein complex public health relevance research study response tool

项目摘要

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.

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。一种针对外来 DNA 的新型原核防御系统基于 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间隔区的同向重复序列组成。与大型 Cas 蛋白复合物结合的小型 CRISPR RNA (crRNA) 可识别外来 DNA，与 crRNA 中存在的间隔序列相匹配，并将其破坏。这个过程被称为“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，成为间隔区，这一过程称为“CRISPR 适应”。必须避免获得宿主衍生的间隔区，因为这会导致自我干扰。 CRISPR 反应的两个阶段尚不完全清楚。我们建议研究大肠杆菌（研究最深入的原核生物）中的 CRISPR 功能。实验室大肠杆菌的 CRISPR/cas 位点处于休眠状态。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以及遗传、生化、交联、实验室进化和建模方法来：目标 1. 分析 CRISPR 干扰并确定 CRISPR 干扰机制控制自身与非自身 DNA 识别的规则；表征与目标降解的外源 DNA 形成的体外 Cas 蛋白-crRNA 复合物，并定位 crRNA 介导的靶标切割位点。实验将使用针对 M13 噬菌体的现有系统以及干扰大肠杆菌的裂解性 T-odd 噬菌体和 RNA 噬菌体的新系统进行。目标 2. 分析 CRISPR 适应并确定 i) 通过 CRISPR 适应机制控制自身与非自身 DNA 区分的规则； ii) CRISPR盒外部影响间隔区获取的序列； iii) 导致 CRISPR 盒中出现额外间隔重复单元的过程的分子细节。与针对适应的外来DNA形成的Cas蛋白复合物将通过将它们捕获在蛋白质路障处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，我们将监测宿主 CRISPR 盒中间隔区的获取以及使 CRISPR 干扰在持续感染的培养物中无效的病毒突变，并与一个小组合作开发该过程的数学模型生物信息学家。作为拟议工作的结果，将揭示 CRISPR 响应期间运作的新分子机制，并将开发原核生物菌株工程和基因沉默的新方法。拟议工作的意义不仅限于大肠杆菌，因为在超过 40% 的真细菌和 95% 的古细菌中都发现了 CRISPR 位点。