Structure and mechanism of CRISPR interference.

CRISPR干扰的结构和机制。

• 批准号: 8883207
• 负责人: Ailong Ke
• 金额: $ 28.96万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8883207
• 关键词: Address; Antibiotic Resistance; Archaeal Genome; Bacillus (bacterium); Bacteria; Bacterial Genome; Bacteriophages; Biochemical; Biological Assay; Campylobacter; Campylobacter jejuni; Clostridium botulinum; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; DNA Transposable Elements; Data; Defense Mechanisms; Development; Digestion; Dissection; Docking; Enzymes; Escherichia coli; Exposure to; Genome; Genomics; Genus staphylococcus; Goals; Guide RNA; Health; Horizontal Gene Transfer; Human; Immune system; Immunity; Individual; Invaded; Listeria monocytogenes; Maps; Mediating; Metals; Microbial Biofilms; Modeling; Molecular; Molecular Conformation; Mutagenesis; Mycobacterium tuberculosis; Nucleic Acids; Operon; Pathway interactions; Plasmids; Process; Property; Proteins; RNA; RNA Binding; RNA Processing; Recruitment Activity; Research; Ribonucleoproteins; Staging; Structure; System; Transcript; Viral Genome; Virulence; Yersinia pestis; adaptive immunity; base; ds-DNA; genome sequencing; helicase; macromolecular assembly; microbial; nuclease; pathogen; pathogenic bacteria; prevent; protein complex; public health relevance; reconstitution; research study

DESCRIPTION (provided by applicant): This project is intended to provide a detailed understanding of a recently discovered prokaryotic adaptive immune system known as CRISPR (clusters of regularly interspaced short palindromic repeats). CRISPR drives adaptation to harmful invading nucleic acids - such as conjugative plasmids, transposable elements and phages - using an RNA-mediated interference (CRISPR interference) mechanism with fundamental similarities to our innate and adaptive immune responses. CRISPR-Cas defense systems have been identified in 88% of archaeal genomes and 39% of bacterial genomes thus far sequenced, including important human pathogens such as Campylobacter human jejuni, Clostridium botulinum, Escherichia coli, Listeria monocytogenes, Mycobacterium tuberculosis and Yersinia pestis. It has been shown to modulate the horizontal gene transfer and biofilm formation. Although the details of this defense mechanism remain to be determined, two distinct stages are recognized: (i) adaptation upon first exposure to the foreign nucleic acid whereby some combination of CRISPR-associated (Cas) proteins extracts recognizable features from the genomes of viruses (bacteriophages) and plasmids as protospacers that are subsequently incorporated as spacers at the 5' end of genomic CRISPR loci; and (ii) interference upon re-exposure to the same nucleic acid whereby a ribonucleoprotein complex comprised of small guide RNAs (crRNA) derived from genomic CRISPRs and different Cas proteins targets foreign nucleic acids for destruction. The lack of information on the molecular and structural properties of the Cas proteins and complexes severely impedes progress in the study of CRISPR mediated bacterial immunity. The proposed research is based on the successful structure determination of several important Cas proteins and the successful reconstitution of the Type I-C Cascade complex from B. halodurans. In this proposal, we propose experiments to understand the CRISPR interference mechanism in Type I-C CRISPR-Cas system. We build upon strong preliminary data to (1) characterize the structure-function of individual components of the Type I-C Cascade, (2) establish function assays and determine the EM and crystal structure of the intact I-C Cascade, and (3) characterize the structure-function of the Cascade-interacting protein Cas3, an essential factor in all Type I CRISPR-Cas systems. Our findings will serve to reveal the common theme and mechanistic diversity among different CRISPR-Cas systems.

描述（由申请人提供）：该项目旨在详细了解最近发现的原核适应性免疫系统，称为 CRISPR（规则间隔的短回文重复序列）。 CRISPR 使用与我们的先天性和适应性免疫反应基本相似的 RNA 介导的干扰（CRISPR 干扰）机制来驱动对有害入侵核酸（例如接合质粒、转座元件和噬菌体）的适应。迄今为止，已在 88% 的古细菌基因组和 39% 的细菌基因组中发现了 CRISPR-Cas 防御系统，其中包括重要的人类病原体，如人类空肠弯曲杆菌、肉毒杆菌、大肠杆菌、单核细胞增生李斯特氏菌、结核分枝杆菌和鼠疫耶尔森氏菌。它已被证明可以调节水平基因转移和生物膜形成。尽管这种防御机制的细节仍有待确定，但已认识到两个不同的阶段：(i) 首次暴露于外源核酸时的适应，其中 CRISPR 相关 (Cas) 蛋白的某些组合从病毒（噬菌体）和质粒的基因组中提取可识别的特征作为原型​​间隔区，随后将其作为间隔区掺入基因组 CRISPR 位点的 5' 端； (ii) 再次暴露于相同核酸时的干扰，其中由源自基因组 CRISPR 的小引导 RNA (crRNA) 和不同 Cas 蛋白组成的核糖核蛋白复合物靶向外来核酸进行破坏。 Cas 蛋白和复合物的分子和结构特性信息的缺乏严重阻碍了 CRISPR 介导的细菌免疫研究的进展。拟议的研究基于几种重要 Cas 蛋白的成功结构测定以及 B. halodurans 的 I-C 型级联复合物的成功重建。在本提案中，我们提出了实验来了解 I-C 型 CRISPR-Cas 系统中的 CRISPR 干扰机制。我们基于强有力的初步数据来 (1) 表征 I-C 型级联各个组件的结构-功能，(2) 建立功能测定并确定完整 I-C 级联的 EM 和晶体结构，以及 (3) 表征级联相互作用蛋白 Cas3 的结构-功能，Cas3 是所有 I 型 CRISPR-Cas 系统中的重要因素。我们的研究结果将有助于揭示不同 CRISPR-Cas 系统之间的共同主题和机制多样性。