Structures of RNA Processing and Silencing Enzymes in Prokaryotes

描述：最近发现细菌和古细菌采用RNA引导的DNA切割 保护自己免受入侵遗传因素的机制提供了前所未有的机会 了解基础微生物生物学和开发生物技术工具。被激怒了， 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切割酶，降解侵入性DNA，同时避免自身DNA。 了解这些不同的DNA切割酶如何控制 它们的活性在基础酶学，抗生素耐药性流行病， 人类微生物组研究和基因组编辑。李实验室已经鉴定并提纯了 CRISPR-Cas DNA切割酶的两种主要类型（II型和III型）的代表性成员 并准备揭示新的分子机制以及开发有用的工具。尽管两 类型是RNA引导的和入侵特异性的，这些核酸酶在酶 组成和活化机制。一种综合方法，从基于细胞的测定， 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，导致了解微生物如何影响人类 我们的目标是实现人类健康和生物圈的可持续发展，以及开发基于CRISPR的技术的最终目标。李 实验室已经组建了一个科学家团队，他们在微生物学、核酸、 酸生物化学、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，以最大限度地提高影响，同时降低研究的风险。 相关性：CRISPR元件存在于超过40%的细菌中，对 维持整个微生物环境。CRISPR在医学上的频繁出现 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 健康对CRISPR免疫的深入了解对根除癌症具有重要意义。 毒力和创造新的抗菌策略。而其中一种CRISPR酶，即Cas9， 已经被重新利用，作为一个用户指定的基因组编辑工具，越来越受欢迎， 我们还没有释放CRISPR衍生工具在生物医学应用中的全部潜力。的 拟议的研究旨在克服目前的局限性，同时扩大能力。

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.