Structures of RNA Processing and Silencing Enzymes in Prokaryotes

原核生物中 RNA 加工和沉默酶的结构

基本信息

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
项目类别：
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

项目摘要

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：最近发现细菌和古细菌采用 RNA 引导的 DNA 切割防御入侵遗传因素的机制提供了前所未有的机会用于了解基础微生物生物学和开发生物技术工具。簇状，规则间隔的短回文重复序列 (CRISPR) 位点机械地编码三种类型不同的 RNA 引导 DNA 切割酶可降解侵入性 DNA，同时避免自身 DNA。了解这些不同 DNA 切割酶如何控制的分子机制它们的活动对基础酶学、抗生素耐药性流行病、人类微生物组研究和基因组编辑。李实验室已鉴定并纯化 CRISPR-Cas DNA切割酶两大类（II型和III型）的代表成员并准备揭示新的分子机制并开发有用的工具。虽然两者类型是RNA引导的和入侵者特异性的，这些核酸酶在酶方面有很大的不同组成和激活机制。一种综合方法，范围从基于细胞的测定到将采用结构生物学和基础酶学来比较和对比模式这些核酸酶对 DNA 的干扰，有助于了解微生物如何影响人类健康和生物圈，以及开发基于 CRISPR 的技术的最终目标。黎族实验室组建了一支在微生物学、核酸学等领域具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 射线晶体学和高通量低温电子显微镜，以最大限度地发挥影响，同时降低研究风险。相关性：CRISPR 元件存在于超过 40% 的细菌中，对于维持整体微生物环境。 CRISPR在医学领域的频繁出现重要的细菌，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、破伤风梭菌和人类微生物组将 CRISPR 直接与人类联系起来健康。深入了解 CRISPR 免疫对于根除病毒具有重要意义毒力并创造新的抗菌策略。而其中一种 CRISPR 酶，即 Cas9，已被重新定位为用户指定的基因组编辑工具，并且越来越受欢迎，我们尚未充分发挥 CRISPR 衍生工具在生物医学应用中的全部潜力。这拟议的研究旨在克服当前的局限性，同时扩展能力。