Mechanisms of CRISPR Interference

CRISPR 干扰机制

基本信息

批准号：
8424275
负责人：
ERIK J. SONTHEIMER
金额：
$ 27.31万
依托单位：
NORTHWESTERN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-03-22 至 2015-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8424275
关键词：
Anatomy Antibiotic Resistance Archaea Area Bacteria Bacterial Infections Bacteriophages Base Pairing Base Sequence Binding Biochemical Biogenesis Biological Assay Biological Models Chromosome Deletion Clinical Cluster Analysis Communicable Diseases DNA Defect Dissection Double-Stranded RNA Eubacterium Eukaryota Genes Genetic Genome Genus staphylococcus Goals Guide RNA Health Horizontal Gene Transfer Human Immunity In Vitro Individual Length Mobile Genetic Elements Molecular Molecular Genetics Organism Pathway interactions Plasmids Process Property Proteins RNA RNA Interference RNA Interference Pathway RNA Processing Research Research Design Ribonucleoproteins Role Route Small RNA Specific qualifier value Specificity Staging Staphylococcus aureus Staphylococcus epidermidis Structure System Transcript Virus Work abstracting base fascinate gene function genetic analysis genetic element in vivo infancy mutant novel particle pathogen plasmid DNA prevent research study resistance mechanism tool

项目摘要

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.

项目摘要/摘要许多生物体利用RNA和DNA的碱基生产潜力来实现基于序列的抗药性机制，以对病毒和移动遗传元素进行抑制。这些机制中最著名的RNA干扰（RNAI）使用双链RNA来触发特定基因的沉默。但是，这种机制仅在真核生物中被记录。最近，在许多Eubacteria的基因组中存在的定期间隔，短，短，allindromic重复（CRISPR）基因座和几乎所有古细菌都显示出可赋予适应性的，可遗传的，基于序列的免疫力，以抗噬菌体。 CRISPR基因座中存在的重复和间隔者编码CRISPR RNA（CRRNA），它们是从较长的前体转录本处理的，并作为此干扰途径的指南。 CRISPR基因座伴随着一组CAS（CRISPR相关）基因，它们编码了基础酶促机制的蛋白质成分。然而，CRRNA指导干扰的分子机制几乎完全没有表征。我们旨在发现CRISPR干扰的机理基础。由于其临床重要性和实验性障碍性，我们将革兰氏阳性病原体葡萄球菌作为模型系统。我们的工作已经产生了三个主要进步：（i）CRISPR基因座可以限制赋予表皮链球菌和金黄色葡萄球菌抗生素耐药性的共轭质粒的扩散；（ii）表皮链球菌中的CRISPR途径直接靶向传入的DNA，因此从根本上与RNAi不同；（iii）CRRNA通过在间隔区域外部的差分基碱基对将非靶向的“自我” 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。我们的工作提高了我们对CRISPR干扰的理解，建议途径限制抗生素耐药性的传播，验证了我们将表皮链球菌作为模型系统的选择，并导致了许多菌株，质粒和测定方法，这些菌株，质粒和测定方法是对这项新颖分析的深入分析的理想之选。我们预计，在实用领域和应用领域中利用CRISPR途径的前景将与我们对基本机制的理解并行前进。因此，我们提出的研究旨在揭示CRISPR干扰链球菌的新和基本方面。重要的是，我们将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/间隔区域的功能解剖结构以及它们编码的CRRNA；（ii）识别并表征干扰所需的其他基因座（包括在CAS基因座之外的任何位置）；（iii）表征含CRRNA的核糖核蛋白（CRRNP），并定义其特性，成分，活动和前体产物关系。这项工作将阐明CRISPR干扰的分子基础，并阐明在与抗生素耐药性和细菌感染的重要战斗中挖掘其潜力的途径。