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

• 批准号: 8995211
• 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
• 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/8995211
• 关键词: Accounting; Address; Affect; Antibiotic Resistance; Appearance; Archaea; BCAR1 gene; Bacteria; Bacteriophage M13; Bacteriophages; Biochemical; Cessation of life; Clustered Regularly Interspaced Short Palindromic Repeats; 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; Health; 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; in vivo; interest; mathematical model; novel; pathogen; protein complex; research study; response; tool

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）：原核生物与病毒（噬菌体）和质粒的相互作用是水平基因转移（HGT），这是抗生素耐药性和人类病原体的出现的基础。细菌发展了许多系统以限制HGT。针对外国DNA的一种新型的原核防御系统是基于CRISPR（群集的定期间隔短的回文重复序列）和CAS基因的。 CRISPR盒子由直接重复插入，并散布着高度可变序列的间隔者。与大型CAS蛋白复合物结合的小CRISPR RNA（CRRNA）识别出异物DNA，与CRRNA中存在的间隔序列相匹配并破坏它。该过程称为“ CRISPR干扰”。 CRISPR录像带中的垫片不受干扰。病毒或质粒衍生的DNA是在称为“ CRISPR适应”的过程中被CRISPR盒变成垫片的Crispr盒。必须避免获得宿主衍生的间隔者，因为这将导致自我干扰。 CRISPR响应的阶段都没有完全理解。我们建议研究最久经考验的原核生物大肠杆菌中的CRISPR功能。实验室大肠杆菌的CRISPR/CAS基因座休眠。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，实验室进化以及建模方法：AIM 1。分析CRISPR干扰并确定控制自我与非自我DNA识别的规则，由CRIS PRSPR干扰机械识别；表征由靶向降解的外国DNA形成的体外CAS蛋白 - crrna络合物，并定位CRRNA介导的靶裂解位点。实验将使用针对M13噬菌体的现有系统以及新的系统干扰大肠杆菌的裂解T-ODD噬菌体和RNA噬菌体。目标2。分析CRISPR适应并确定i）根据CRISPR适应机制来控制自我与非自我DNA歧视的规则； ii）crispr盒外的序列会影响间隔者的获取； iii）该过程的分子细节导致在CRISPR盒中出现额外的间隔重复单元。 CAS蛋白复合物由靶向适应的异物形成的CAS蛋白复合物在体外和体内都将在蛋白质障碍物上捕获。为了更好地了解CRISPR介导的病毒宿主动力学和共同进化，我们将监测宿主和病毒突变的crispr盒中的间隔者获取，这些突变使CRISPR干扰在连续感染的培养物中无效，并在与一群生物信息学家协作中开发了该过程的数学模型。 由于提出的工作，将揭示在CRISPR响应期间运作的新型分子机制，并将开发用于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Eubacteria和95％的古细菌中发现了CRISPR基因座。