Structure and mechanism of CRISPR interference.

描述(由申请人提供)：本项目旨在提供对最近发现的原核生物适应性免疫系统CRISPR(规则间隔的短回文重复序列簇)的详细了解。CRISPR使用一种RNA介导的干扰(CRISPR干扰)机制，与我们的先天性和获得性免疫反应有根本的相似之处，从而推动对有害入侵核酸的适应--如接合质粒、可转座元件和噬菌体。到目前为止，已在88%的古菌基因组和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，如人类空肠弯曲菌、肉毒梭菌、大肠杆菌、单核细胞增生性李斯特菌、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的基因转移和生物膜的形成。虽然这一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)首次接触外源核酸时的适应，其中CRISPR相关(Cas)蛋白的某种组合从病毒(噬菌体)和质粒的基因组中提取可识别的特征，这些原始体随后作为间隔物并入基因组CRISPR基因座的5‘端；(Ii)再次暴露于同一核酸时的干扰，由来自基因组CRISPR的小引导RNAs(CrRNA)和不同的Cas蛋白组成的核糖核蛋白复合体靶向破坏外源核酸。由于缺乏对Cas蛋白和复合体的分子和结构性质的了解，严重阻碍了CRISPR介导的细菌免疫的研究进展。这项研究是在成功确定了几种重要的CaS蛋白的结构和成功地重组了盐生双歧杆菌的I-C型级联复合体的基础上进行的。在该方案中，我们建议通过实验来理解I-C CRISPR-CAS系统中的CRISPR干扰机制。我们基于强大的初步数据来(1)表征I-C级联单个组件的结构功能，(2)建立功能分析并确定完整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，它是所有I型CRISPR-CAS系统中的一个基本因子。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制多样性。

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.