Molecular Mechanisms of Invader Silencing in Type III CRISPR-Cas Systems

III 型 CRISPR-Cas 系统中入侵者沉默的分子机制

• 批准号: 9400861
• 负责人: Kawanda A Foster
• 金额: $ 3.76万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9400861
• 关键词: Adaptive Immune System; Address; Archaea; BCAR1 gene; Bacteria; Base Pairing; Basic Science; Biogenesis; Biological; Biological Assay; Biology; Biotechnology; CRISPR/Cas technology; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; DNA Sequence; Deoxyribonucleases; Development; Disease; Electron Transport Complex III; Endoribonucleases; Gene Expression; Genes; Genetic Transcription; Guide RNA; Health; Heritability; Human; Immune system; In Vitro; Industrialization; Industry; Invaded; Knowledge; Laboratories; Medical; Medicine; Mobile Genetic Elements; Molecular; Mutation; Natural regeneration; Nucleic Acid Cleavage; Nucleic Acids; Organism; Planets; Plasmids; Play; Population; Process; Prokaryotic Cells; Proteins; Public Health; Publishing; RNA; RNA Binding; Research; Research Design; Role; Shapes; Societies; Source; System; Technology; Testing; Transcript; Translational Research; Virus; Work; base; combat; experimental study; genome editing; immune system function; in vivo; insight; interest; knock-down; novel; nuclease; precise genome editing; tool

Project Summary CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) systems are RNA-based immune systems that function in prokaryotes to protect against viruses and other invasive mobile genetic elements. Our understanding of these adaptive immune systems function has increased exponentially in the last ten years. Importantly, this information has been exploited and harnessed into exceptionally powerful, novel research tools for genome editing and predictable control of gene expression with widespread industrial and medical applications. There are multiple diverse CRISPR-Cas systems that combat viruses by distinct mechanisms and with distinct machinery, each providing unique biology and potential applications to explore. CRISPR-Cas systems employ three general steps in invader defense: adaptation, crRNA, biogenesis, and invader silencing. The initial step results in integration of a fragment of invader DNA sequence into the CRISPR locus to provide a heritable record of the invasion and source of targeting information (adaptation). The CRISPR locus is transcribed and the transcript is processed to produce mature crRNA species (crRNA biogenesis). The crRNA and Cas proteins form an effector complex that seeks out and destroys the invading nucleic acid through base-pairing of the crRNA and destruction by an associated Cas protein nuclease (invader silencing). Translational research on CRISPR-Cas effector complexes has yielded the gene editing powerhouse CRISPR-Cas9 from the Type II-A system. The focus of my research is to understand invader silencing by the less well characterized Type III-A or Csm effector complex. Type III systems are unique in that they target RNA as well as DNA. The overall objective of this proposal is to obtain a detailed mechanistic understanding of how the RNA and DNA targeting activities of the Type II-A Csm system are achieved through the concerted interactions of the associated crRNA and six Cas proteins. Aim 1 is to elucidate the molecular basis of transcription-dependent DNA targeting by the Csm system. Aim 2 is to delineate the functional relationship of Csm6 with the Csm complex and its role in invader elimination. A powerful combination of mostly established in vitro and in vivo approaches will be employed to address the proposed specific aims. The studies will contribute to our fundamental understanding of CRISPR-Cas biology. In addition, the knowledge obtained will afford opportunities for the development of novel Type III CRISPR- Cas-based technologies with far reaching potential biotechnological and biomedical applications.

项目摘要 CRISPR-Cas（重复的规则间隔短回文重复序列-CRISPR 相关）系统是基于RNA的免疫系统，其在原核生物中起保护作用， 抵抗病毒和其他侵入性的移动的遗传因素。我们对这些的理解 适应性免疫系统的功能在过去十年中呈指数增长。 重要的是，这些信息已经被利用和利用， 用于基因组编辑和可预测控制基因表达的新研究工具， 广泛的工业和医疗应用。有多种多样的CRISPR-Cas 通过不同的机制和不同的机器对抗病毒的系统， 提供独特的生物学和潜在的应用探索。CRISPR-Cas系统采用 入侵防御的三个一般步骤：适应，crRNA，生物发生和入侵沉默。 初始步骤导致入侵DNA序列的片段整合到CRISPR中， 基因座提供入侵的可遗传记录和靶向信息的来源 （适应）。CRISPR基因座被转录并且转录物被加工以产生 成熟crRNA种类（crRNA生物发生）。crRNA和Cas蛋白形成效应子 复合物，通过碱基配对寻找并破坏入侵的核酸。 crRNA和相关Cas蛋白核酸酶的破坏（入侵沉默）。平移 对CRISPR-Cas效应复合物的研究产生了基因编辑的动力 来自II-A型系统的CRISPR-Cas9。我研究的重点是了解入侵者 通过不太好表征的III-A型或Csm效应复合物沉默。第三类系统是 它们的独特之处在于它们靶向RNA和DNA。本提案的总体目标是， 详细了解RNA和DNA靶向活性的类型 II-A Csm系统是通过相关crRNA的协同相互作用和 六种Cas蛋白。目的1是阐明转录依赖性DNA的分子基础 CSM系统的定位目的二是阐明Csm 6与细胞凋亡的功能关系。 csm复合物及其在入侵者清除中的作用。一个强大的组合，主要建立在 将采用体外和体内方法来实现所提出的具体目标。的 这些研究将有助于我们对CRISPR-Cas生物学的基本理解。此外，本发明还提供了一种方法， 所获得的知识将为开发新型III型CRISPR提供机会- 具有深远潜力的基于cas的生物技术和生物医学技术 应用.