Mechanisms and Applications of CRISPR-Cas Enzymes

CRISPR-Cas酶的机制和应用

• 批准号: 10609066
• 负责人: Hong Li
• 金额: $ 37.14万
• 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 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10609066
• 关键词: Address; Adopted; Antibiotic Resistance; Archaea; Bacteria; Binding; Biochemical; Biogenesis; Biological Assay; Biology; Biophysical Process; Biophysics; Biotechnology; CRISPR interference; Cancer Detection; Cells; Cellular biology; Clinical; Clostridium tetani; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complex; Coupled; Cryoelectron Microscopy; Cytosine Nucleotides; DNA; DNA Binding; DNA Methylation; Defense Mechanisms; Development; Disease; Early Diagnosis; Elements; Engineering; Environment; Enzymatic Biochemistry; Enzymes; Epidemic; Epigenetic Process; Eukaryotic Cell; Evolution; Funding; Genes; Genetic Transcription; Goals; Guide RNA; Haemophilus influenzae; Health; Helicobacter pylori; Human; Human Microbiome; Immune response; Immunity; Infection; Knowledge; Laboratories; Lactococcus lactis; Maintenance; Mammalian Cell; Mediating; Medical; Methylation; Microbe; Microbiology; Modeling; Molecular; Molecular Conformation; Mycobacterium tuberculosis; Neisseria; Nucleic Acid Biochemistry; Nucleic Acids; Periodicity; Property; Protein Dynamics; Proteins; RNA; Reaction; Regulation; Research; Salmonella typhi; Scientist; Second Messenger Systems; Specific qualifier value; Specificity; Staphylococcus aureus; Structure; System; Technology; Therapeutic; Vibrio vulnificus; Viral; Virulence; Virus; Work; X-Ray Crystallography; Yersinia pestis; antimicrobial; biophysical properties; biophysical techniques; detection platform; diagnostic tool; enzyme activity; experience; genetic element; genome editing; improved; in vivo; member; microbial; microbiome; microbiome research; novel; nuclease; nucleic acid detection; oligoadenylate; reconstitution; risk mitigation; structural biology; tool; viral RNA; viral detection

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.

描述：发现细菌和古细菌利用 RNA 引导的免疫机制来 保护自己免受入侵性遗传因素的侵害，为人类提供了前所未有的机会 了解基础微生物生物学并开发生物技术工具。成簇、有规律 间隔短回文重复序列 (CRISPR) 位点机械地编码两大类 不同的RNA引导酶可以降解侵入性核酸，同时避免自身核酸 或通过第二信使的合成引发二次免疫。了解分子 这些不同类别的酶的机制在基础研究中具有重要意义 酶学、抗生素耐药性流行病、人类微生物组研究、病毒和癌症 检测和基因组编辑。李实验室已经确定并建立了条件 研究两类 CRISPR-Cas 酶的表型成员，并准备揭开新的面纱 分子机制以及开发有用的工具。这两类人都有一个共同点：RNA- 引导和入侵者特定。然而，它们在酶组成和生化方面存在巨大差异。 机制，因此需要广泛的调查工具和专门知识。一个集成的 从基于细胞的测定到结构生物学和基础酶学的方法将是 用于比较和对比 1 类和 2 类酶的干扰模式，导致 了解微生物如何影响人类健康和生物圈以及最终目标 开发基于 CRISPR 的技术。李实验室组建了一支科学家团队 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 射线晶体学和高通量低温电子显微镜，以便 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，对于维持细菌至关重要 整体微生物环境。 CRISPR 在医学上重要的细菌中频繁出现 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、 破伤风梭菌和人类微生物组将 CRISPR 直接与人类健康联系起来。彻底的 了解 CRISPR 免疫对于消除毒力和创造新的病毒具有重要意义。 新的抗菌策略。虽然其中一种 CRISPR 酶，即 Cas9，已被重新用于 作为用户指定的基因组编辑工具，其实用性不断增加，但我们尚未释放全部功能 CRISPR 衍生工具在生物医学应用中的潜力。拟议的研究旨在 克服当前的限制，同时扩展能力。