Mechanisms and Applications of CRISPR-Cas Enzymes

CRISPR-Cas酶的机制和应用

• 批准号: 10446170
• 负责人: Hong Li
• 金额: $ 37.94万
• 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 2026-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10446170
• 关键词: Address; Adopted; Antibiotic Resistance; Archaea; Bacteria; 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; Electron Microscopy; 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; Phenotype; Property; Proteins; RNA; Reaction; Regulation; Research; Risk; 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; base; biophysical properties; biophysical techniques; cancer genome; cryogenics; detection platform; diagnostic tool; enzyme activity; enzyme mechanism; experience; genetic element; genome editing; improved; in vivo; member; microbial; microbiome; microbiome research; novel; nuclease; nucleic acid detection; oligoadenylate; reconstitution; 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：细菌和古细菌采用RNA引导的免疫机制的发现 捍卫自己免受侵入性遗传元件的侵害，为前所未有的机会 了解基本的微生物生物学和开发生物技术工具。定期聚集 间隔短的，短的回文重复序列（CRISPR）基因座在机械上编码两个主要类别 不同的RNA引导的酶可以降解浸润性核酸，同时避免自核酸 或通过合成第二使者而引起次要免疫。了解分子 这些不同类别的酶的机制在基本中具有重要意义 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。 LI实验室已经确定并确定了 研究两类CRISPR-CAS酶的表型成员，并有望揭开新颖性 分子机制以及开发有用的工具。这两个班级都共享rna的贸易 - 指导和入侵者特定的。但是，它们在酶组成和生化方面截然不同 因此，机制需要广泛的调查工具和专业知识。一个集成的 从基于细胞的测定到结构生物学和基本酶学的方法将是 用于比较和对比1级和2类酶的干扰方式，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。 LI实验室已经与 微生物学，核酸生物化学，哺乳动物细胞生物学，病毒的互补专业知识 检测，X射线晶体学和高通量低温电子显微镜，以便 在减轻研究风险的同时最大程度地发挥影响。 相关性：CRISPR元素在40％以上的细菌中发现，对维护至关重要 整体微生物环境。在医学上重要的细菌中，CRISPR经常出现 其中包括但不限于耶尔森氏念珠菌，结核分枝杆菌，流感嗜血杆菌， 幽门螺杆菌，奈瑟氏菌，弧菌，金黄色葡萄球菌，沙门氏菌鼠伤寒，沙门氏菌，幽门螺杆菌 Tetani梭状芽胞杆菌和人类微生物组直接将CRISPR与人类健康联系起来。彻底 对CRISPR免疫的理解在消除毒力和创造方面具有重要意义 新的抗菌策略。虽然一种CRISPR酶，即Cas9，已重新使用为 用作用户指定的基因组编辑工具，具有越来越多的实用程序，我们尚未释放完整的 CRISPR衍生工具在生物医学应用中的潜力。拟议的研究针对 克服当前局限性，同时扩展功能。