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，即CRISPR（定期间隔短的短质体重复序列）。 CRISPR使用RNA介导的干扰（CRISPR干扰）机制适应有害入侵的核酸，例如共轭质粒，可转移元素和噬菌体。到目前为止，已经在88％的古细胞基因组和39％的细菌基因组中鉴定了CRISPR-CAS防御系统，包括重要的人类病原体，例如弯曲杆菌的人类空肠，肉毒杆菌，埃希氏梭菌，大肠杆菌，单细胞增生核细胞儿童，乳腺细胞增生，乳腺菌菌，结膜菌和yersiaia pestis。已显示它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，但确定了两个不同的阶段：（i）首次暴露于外国核酸后，从而将与CRISPR相关（CAS）蛋白的某种组合结合在一起，可以从病毒（噬菌体）的基因组中识别出可识别的特征（噬菌体）和质粒作为质体的质体，这些特征随后是Sppre eendeci的基因，并在5'eDnaCers中均融合了5'; （ii）干扰重新暴露于相同的核酸，从而由源自基因组CRISPR和不同CAS蛋白的小引导RNA（CRRNA）组成的核糖核蛋白复合物靶向外核酸来破坏。缺乏有关CAS蛋白和复合物的分子和结构特性的信息，严重阻碍了CRISPR介导的细菌免疫的研究。拟议的研究是基于几种重要CAS蛋白的成功结构确定，并成功地重建了B. halodurans的I型级联级别络合物。在此提案中，我们提出了实验，以了解I型I-C CRISPR-CAS系统中的CRIS PRTR干扰机制。我们以强大的初步数据为基础（1）表征了I-C级级联的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cascade-Intercade-Inclacteracting Protin cas3的结构功能，这是所有类型I型CRISPR-CAS系统中必不可少的因素。我们的发现将有助于揭示不同CRISPR-CAS系统之间的共同主题和机械多样性。