Mechanisms of CRISPR Interference

描述(由申请人提供)：项目摘要/摘要：许多生物利用RNA和DNA的碱基配对潜力来实现对病毒和可移动遗传元件的基于序列的抗性机制。其中最著名的机制是RNA干扰(RNAi)，它使用双链RNA来触发特定基因的沉默。然而，这种机制只在真核生物中有文献记载。最近，在许多真细菌和几乎所有古生菌的基因组中存在的规则间隔短回文重复(CRISPR)基因座，已被证明具有针对噬菌体的适应性、可遗传的、基于序列的免疫。CRISPR基因座中存在的重复序列和间隔区编码CRISPR RNAs(CrRNAs)，该RNAs是从较长的前体转录本加工而来的，并作为这一干扰途径的指南。CRISPR基因座伴随着一组cas(CRISPR相关)基因，这些基因编码潜在的酶机制的蛋白质成分。然而，crRNA定向干扰的分子机制几乎完全未知。我们的目标是揭示CRISPR干扰的机制基础。我们使用革兰氏阳性病原体表皮葡萄球菌作为模型系统，因为它具有临床重要性和实验可操作性。我们的工作已经取得了三大进展：(I)CRISPR基因座可以限制接合质粒在表皮葡萄球菌和金黄色葡萄球菌中的传播；(Ii)表皮葡萄球菌中的CRISPR途径直接针对传入的DNA，因此与RNAi根本不同；(Iii)crRNAs通过间隔区外的差异碱基配对区分非靶标的“自我”DNA(CRISPR基因座)和靶标的“非自我”DNA(质粒和噬菌体基因组)。我们的工作提高了我们对CRISPR干扰的理解，提出了限制抗生素耐药性传播的途径，验证了我们选择的表皮葡萄球菌作为模型系统，并产生了许多菌株、质粒和分析方法，这些都是深入分析这一新颖而迷人的途径的理想选择。我们预计，我们在实际和应用领域探索CRISPR途径的前景将与我们对基本机制的理解同步推进。因此，我们建议的研究旨在揭示表皮葡萄球菌CRISPR干扰的新的和基本的方面。重要的是，我们将结合体内和体外的方法，并利用它们之间的协同效应。特别是，我们将(I)定义重复/间隔区及其编码的crRNAs的功能解剖学；(Ii)识别和鉴定干扰所需的其他基因座(包括位于cas基因座之外的任何基因座)；以及(Iii)鉴定含有crRNA的核糖核蛋白(CrRNPs)，并确定它们的性质、成分、活性和前体-产物关系。这项工作将阐明CRISPR干扰的分子基础，并阐明在对抗抗生素耐药性和细菌感染的关键斗争中挖掘其潜力的途径。

DESCRIPTION (provided by applicant): Project Summary/Abstract: Many organisms exploit the base-pairing potential of RNA and DNA to enable sequence-based resistance mechanisms against viruses and mobile genetic elements. The best known of these mechanisms, RNA interference (RNAi), uses double-stranded RNA to trigger the silencing of specific genes. However, this mechanism has only been documented in eukaryotes. More recently, clustered regularly interspaced, short, palindromic repeat (CRISPR) loci, present in the genomes of many eubacteria and nearly all archaea, have been shown to confer adaptive, heritable, sequence-based immunity against phages. The repeats and spacers present in CRISPR loci encode CRISPR RNAs (crRNAs) that are processed from longer precursor transcripts and serve as guides for this interference pathway. CRISPR loci are accompanied by a set of cas (CRISPR-associated) genes that encode protein components of the underlying enzymatic machinery. However, the molecular mechanisms of crRNA-directed interference are almost completely uncharacterized. We aim to uncover the mechanistic basis for CRISPR interference. We are using the gram-positive pathogen Staphylococcus epidermidis as a model system because of its clinical importance and experimental tractability. Already our work has yielded three major advances: (i) CRISPR loci can function to limit the spread of conjugative plasmids that confer antibiotic resistance in S. epidermidis and Staphylococcus aureus; (ii) the CRISPR pathway in S. epidermidis directly targets incoming DNA and is therefore fundamentally distinct from RNAi; and (iii) crRNAs distinguish untargeted "self" DNA (the CRISPR locus) from targeted "non- self" DNA (plasmids and phage genomes) by differential base pairing outside of the spacer region. Our work has advanced our understanding of CRISPR interference, suggested routes towards limiting the spread of antibiotic resistance, validated our selection of S. epidermidis as a model system, and resulted in many strains, plasmids, and assays that are ideal for in-depth analyses of this novel and fascinating pathway. We anticipate that our prospects for exploiting the CRISPR pathway in practical and applied realms will advance in parallel with our understanding of the underlying mechanisms. Accordingly, our proposed studies are designed to uncover new and fundamental aspects of CRISPR interference in S. epidermidis. Importantly, we will combine in vivo and in vitro approaches and capitalize on the synergies between them. In particular, we will (i) define the functional anatomy of the repeat/spacer region and the crRNAs that they encode; (ii) identify and characterize other loci (including any that lie outside of the cas locus) that are required for interference; and (iii) characterize crRNA-containing ribonucleoproteins (crRNPs) and define their properties, components, activities, and precursor-product relationships. This work will clarify the molecular basis of CRISPR interference and illuminate routes toward tapping its potential in the critical battle against antibiotic resistance and bacterial infection.