Mechanisms of CRISPR Interference

描述（由申请人提供）：项目总结/摘要：许多生物体利用RNA和DNA的碱基配对潜力来实现针对病毒和移动的遗传元件的基于序列的抗性机制。这些机制中最著名的是RNA干扰（RNAi），它使用双链RNA来触发特定基因的沉默。然而，这种机制仅在真核生物中有记载。最近，在许多真细菌和几乎所有古细菌的基因组中存在的成簇的规则间隔的短回文重复序列（CRISPR）基因座已被证明可赋予针对细菌的适应性、可遗传的、基于序列的免疫力。CRISPR基因座中存在的重复序列和间隔区编码CRISPR RNA（crRNA），其由较长的前体转录物加工而成，并充当该干扰途径的向导。CRISPR基因座伴随有一组cas（CRISPR相关）基因，其编码潜在酶机制的蛋白质组分。然而，crRNA定向干扰的分子机制几乎完全未被表征。 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，因为它的临床重要性和实验易处理性。我们的工作已经取得了三项重大进展：（i）CRISPR基因座可以发挥作用，限制在S.表皮葡萄球菌和金黄色葡萄球菌中的CRISPR途径;表皮病毒直接靶向进入的DNA，因此从根本上不同于RNAi;和（iii）crRNA通过间隔区外部的差异碱基配对将非靶向的“自身”DNA（CRISPR基因座）与靶向的“非自身”DNA（质粒和噬菌体基因组）区分开。我们的工作推进了我们对CRISPR干扰的理解，提出了限制抗生素耐药性传播的途径，验证了我们对S. epidermidis作为一个模型系统，并导致了许多菌株，质粒和检测，是理想的深入分析这一新的和迷人的途径。 我们预计，我们在实际和应用领域利用CRISPR途径的前景将与我们对潜在机制的理解同步推进。因此，我们提出的研究旨在揭示CRISPR干扰S.表皮重要的是，我们将联合收割机结合体内和体外方法，并利用它们之间的协同作用。特别是，我们将（i）定义重复/间隔区及其编码的crRNA的功能解剖学;（ii）鉴定和表征干扰所需的其他基因座（包括cas基因座以外的任何基因座）;（iii）表征含crRNA的核糖核蛋白（crRNP）并定义其性质，组分，活性和底物-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战役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.