Mechanisms and Applications of CRISPR-Cas Enzymes

描述：细菌和古菌利用RNA引导的免疫机制来 保护自己免受入侵的遗传因素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。群集式，定期 间隔短回文重复序列(CRISPR)基因座编码两类主要的机械重复序列 不同的RNA引导的酶可以降解入侵的核酸，同时避免自身核酸 或通过合成第二信使诱导二次免疫。理解分子 这些不同类别的酶的作用机制在基础研究中具有重要意义 酶学、抗生素耐药性流行、人类微生物组研究、病毒与癌症 检测和基因组编辑。锂实验室已经确定并建立了 研究两类CRISPR-Cas酶的表型成员，并有望推出新的 分子机制以及开发有用的工具。这两个阶级都有作为RNA的交易- 引导和特定于入侵者的。然而，它们在酶组成和生化方面有很大的不同。 因此，这需要广泛的调查工具和专门知识。一个完整的 从基于细胞的分析，到结构生物学，再到基础酶学的方法将是 用来比较和对比1类和2类酶的干扰模式，导致 对微生物如何影响人类健康和生物圈的理解，以及对 开发基于CRISPR的技术。李实验室组建了一支科学家团队， 在微生物学、核酸生物化学、哺乳动物细胞生物学、病毒方面的互补专业知识 检测、X射线结晶学和高通量低温电子显微镜，以便 最大限度地发挥研究的影响，同时降低研究的风险。 相关性：CRISPR成分存在于超过40%的细菌中，对维持生命至关重要 对整个微生物环境的影响。CRISPR在医学重要细菌中的频繁出现 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、金黄色葡萄球菌、伤寒沙门氏菌、 破伤风杆菌和人体微生物群直接关系到人类的健康。一次彻底的 了解CRISPR免疫对于消除毒力和创造 新的抗菌策略。而CRISPR酶之一，即Cas9，已被重新用于 作为一个用户指定的基因组编辑工具，我们还没有充分释放出 CRISPR衍生工具在生物医学应用中的潜力。拟议的研究旨在 在扩展能力的同时克服当前的限制。

Description: The discovery that bacteria and archaea employ an RNA-guided immunity 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 two major classes of mechanistically different RNA-guided enzymes that can degrade invasive nucleic acids while avoiding self-nucleic acids or elicit secondary immunity through synthesis of second messengers. Understanding the molecular mechanisms of these distinct classes of enzymes has important implications in basic enzymology, antibiotics resistance epidemics, human microbiome research, virus and cancer detection and genome editing. The Li laboratory has identified and established the conditions for studying phenotypical members of the two classes of CRISPR-Cas enzymes and is poised to unveil novel molecular mechanisms as well as to develop useful tools. Both classes share the trade of being RNA- guided and invader-specific. However, they differ drastically in enzyme composition and biochemical mechanisms and, therefore, require a broad range of investigative tools and expertise. 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 interference by the Class 1 and 2 enzymes, 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, virus detection, 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 utility, 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.