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，称为CRISPR（规则间隔短回文重复序列簇）。CRISPR利用RNA介导的干扰（CRISPR干扰）机制驱动对有害入侵核酸的适应-例如接合质粒，转座因子和DNA-与我们的先天性和适应性免疫反应基本相似。CRISPR-Cas防御系统已经在88%的古细菌基因组和39%的细菌基因组中被鉴定，包括重要的人类病原体，如空肠弯曲杆菌、肉毒梭菌、大肠杆菌、单核细胞增生李斯特菌、结核分枝杆菌和鼠疫耶尔森菌。已显示其调节水平基因转移和生物膜形成。虽然这种防御机制的细节仍有待确定，但认识到两个不同的阶段：（i）首次暴露于外源核酸后的适应，其中CRISPR相关（Cas）蛋白的一些组合从病毒的基因组中提取可识别的特征（噬菌体）和质粒作为原型间隔区，所述原型间隔区随后作为间隔区掺入基因组CRISPR基因座的5'端;和（ii）在再次暴露于相同核酸时的干扰，由此由源自基因组CRISPR的小向导RNA（crRNA）和不同Cas蛋白组成的核糖核蛋白复合物靶向外源核酸以进行破坏。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。所提出的研究是基于几种重要Cas蛋白的成功结构测定和来自B的I-C型级联复合物的成功重构。耐盐生物。在该提案中，我们提出了一些实验来理解I-C型CRISPR-Cas系统中的CRISPR干扰机制。我们建立在强有力的初步数据基础上，以（1）表征I-C型级联的各个组分的结构-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Cas 3的结构-功能，Cas 3是所有I型CRISPR-Cas系统中的一个重要因子。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。

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.