Structures of RNA Processing and Silencing Enzymes in Prokaryotes

原核生物中 RNA 加工和沉默酶的结构

• 批准号: 9247630
• 负责人: Hong Li
• 金额: $ 35.38万
• 依托单位: FLORIDA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9247630
• 关键词: Antibiotic Resistance; Antibiotics; Archaea; Bacteria; Binding; Biochemical; Biological; Biological Assay; Biology; Biophysics; Biotechnology; Cells; Cellular biology; Cleaved cell; Clostridium tetani; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Community Developments; Cryoelectron Microscopy; Cytosine; Cytosine Nucleotides; DNA; Data; Defense Mechanisms; Disease; Electron Microscopy; Elements; Embryo; Environment; Enzymatic Biochemistry; Enzyme Kinetics; Enzyme Stability; Enzymes; Epidemic; Equilibrium; Escherichia coli; Evolution; Exhibits; Funding; Future; Genetic Materials; Genetic Transcription; Goals; Guanine; Guide RNA; Haemophilus influenzae; Health; Helicobacter pylori; Human; Human Microbiome; Image Analysis; Immunity; In Vitro; Kinetics; Laboratories; Libraries; Maintenance; Mammalian Cell; Mediating; Medical; Methods; Microbe; Microbiology; Molecular; Mycobacterium tuberculosis; Neisseria; Nucleic Acid Biochemistry; Organism; Pathway interactions; Plasmids; Prokaryotic Cells; Property; Proteins; RNA; RNA Binding; RNA Degradation; RNA Interference; RNA Processing; Race; Research; Risk; Salmonella typhi; Scientist; Single-Stranded DNA; Small RNA; Specific qualifier value; Specificity; Staphylococcus aureus; Structure; System; Technology; Tern; Tertiary Protein Structure; Transcript; Vibrio vulnificus; Virulence; Work; X-Ray Crystallography; Yersinia pestis; antimicrobial; arm; base; cryogenics; experience; gene therapy; genetic element; genome editing; in vivo; interest; kidney cell; member; microbial; novel; nuclease; pathogen; structural biology; success; thermophilic organism; three dimensional structure; tool

Description: The recent discovery that bacteria and archaea employ an RNA-guided DNA cleavage mechanism to defend themselves from invasive genetic elements offers an unprecedented opportunity for understanding fundamental microbial biology and for developing biotechnology tools. Clustered, regularly interspaced, short palindromic repeats (CRISPR) loci encode three types of mechanistically different RNA-guided DNA cleavage enzymes that degrade invasive DNA while avoiding self-DNA. Understanding the molecular mechanisms of how these distinct DNA cleavage enzymes control their activities has important implications in basic enzymology, antibiotics resistance epidemics, human microbiome research, and genome editing. The Li laboratory has identified and purified representative members of two major types (Types II and III) of CRISPR-Cas DNA cleavage enzymes and is poised to unveil novel molecular mechanisms as well as to develop useful tools. Though both types are RNA-guided and invader-specific, these nucleases have drastically different in enzyme composition and activation mechanisms. An integrated approach ranging from cell-based assays, to structural biology and to fundamental enzymology will be employed to compare and contrast the mode of DNA interference by these nucleases, leading to an understanding of how microbe impact human health and biosphere and to an ultimate goal of developing CRISPR-based technology. The Li laboratory has assembled a team of scientists with complementary expertise in microbiology, nucleic acid biochemistry, mammalian cell biology, X-ray crystallography, and high-throughput cryogenic electron microscopy, in order to maximize the impact while mitigating risks of the research. Relevance: The CRISPR elements are found in more than 40% bacteria and are critical to maintenance of the overall microbial environment. The frequent occurrence of CRISPR in medically important bacteria that include but not limited to Yersinia pestis, Mycobacterium tuberculosis, Haemophilus influenzae, Helicobacter pylori, Neisseria meningitides, Vibrio vulnificus, Staphylococcus aureus, Salmonella Typhi, Clostridium tetani, and human microbiome relates CRISPR directly to human health. A thorough understanding of the CRISPR immunity has important implications in eradicating virulence and creating new antimicrobial strategies. While one of the CRISPR enzymes, namely Cas9, has been repurposed to serve as a user-specified genome-editing tool with ever-increasing popularity, we are yet to unleash the full potential of the CRISPR-derived tools in biomedical applications. The proposed research is aimed at overcoming current limitations while expanding the capability.

描述：最近发现细菌和古生菌使用RNA引导的DNA切割 防御入侵遗传因素的机制提供了一个前所未有的机会 了解基础微生物生物学和开发生物技术工具。聚集在一起， 规则间隔的短回文重复序列(CRISPR)基因座编码三种类型的机械性 不同的RNA引导的DNA裂解酶可以降解入侵的DNA，同时避免自体DNA。 了解这些不同的DNA裂解酶如何控制的分子机制 它们的活性对基础酶学、抗生素耐药性流行、 人类微生物组研究和基因组编辑。锂实验室已经鉴定并提纯了 CRISPR-Cas DNA裂解酶两种主要类型(类型II和III型)的代表成员 并准备揭开新的分子机制以及开发有用的工具。虽然两者都是 核酸酶的类型是rna引导的，是入侵者特有的，这些核酸酶在酶的作用上有很大的不同。 组成和活化机制。集成的方法，从基于细胞的分析，到 结构生物学和基础酶学将被用来比较和对比模式 这些核酸酶的DNA干扰，导致对微生物如何影响人类的理解 健康和生物圈，以及开发基于CRISPR的技术的最终目标。李氏 实验室组建了一支在微生物学、核酸等领域拥有互补专业知识的科学家团队 酸性生物化学、哺乳动物细胞生物学、X射线结晶学和高通量低温 电子显微镜，以最大限度地发挥影响，同时减轻研究的风险。 相关性：CRISPR成分存在于40%以上的细菌中，对 维护整个微生物环境。CRISPR在医学上的频繁发生 重要的细菌，包括但不限于鼠疫耶尔森氏菌，结核分枝杆菌， 流感嗜血杆菌、幽门螺杆菌、脑膜炎奈瑟菌、创伤弧菌、葡萄球菌 金黄色葡萄球菌、伤寒沙门氏菌、破伤风杆菌和人类微生物群与CRISPR直接相关 健康。彻底了解CRISPR免疫对根除 毒力和创造新的抗微生物策略。而CRISPR的一种酶，即Cas9， 已经被重新用作用户指定的基因组编辑工具，越来越受欢迎， 我们还没有充分释放CRISPR衍生工具在生物医学应用中的潜力。这个 拟议的研究旨在克服目前的限制，同时扩大能力。