Structure and mechanism of CRISPR interference.

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CRISPR（定期间隔的短回文重复序列簇）。CRISPR利用rna介导的干扰（CRISPR干扰）机制驱动对有害入侵的核酸（如共轭质粒、转座因子和噬菌体）的适应，这种机制与我们的先天和适应性免疫反应具有基本的相似性。到目前为止，已有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，包括重要的人类病原体，如人类空肠弯曲杆菌、肉毒梭菌、大肠杆菌、单核增生李斯特菌、结核分枝杆菌和鼠疫杆菌。它已被证明可以调节水平基因转移和生物膜的形成。尽管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，作为原间隔物，随后作为间隔物被纳入基因组CRISPR位点的5'端；（ii）再次暴露于同一核酸时的干扰，即由基因组crispr和不同Cas蛋白衍生的小向导rna （crRNA）组成的核糖核蛋白复合物靶向破坏外来核酸。缺乏关于Cas蛋白和复合物的分子和结构特性的信息严重阻碍了CRISPR介导的细菌免疫研究的进展。这项研究是基于成功地确定了几个重要的Cas蛋白的结构，并成功地重建了来自B. halodurans的I-C型级联复合物。在本提案中，我们提出了在I-C型CRISPR- cas系统中了解CRISPR干扰机制的实验。我们建立在强大的初步数据基础上，以(1)表征I- c级联各组分的结构-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，Cas3是所有I型CRISPR-Cas系统的重要因素。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.