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.

项目概要/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遗传元件的基于序列的抵抗机制。这些机制中最著名的是 RNA 干扰 (RNAi)，它使用双链 RNA 触发特定基因的沉默。然而，这种机制仅在真核生物中被记录。最近，许多真细菌和几乎所有古细菌基因组中存在的成簇规则间隔、短回文重复（CRISPR）基因座已被证明能够赋予针对噬菌体的适应性、可遗传、基于序列的免疫力。 CRISPR 位点中存在的重复序列和间隔区编码 CRISPR RNA (crRNA)，这些 RNA 由较长的前体转录物加工而成，并充当该干扰途径的指导。 CRISPR 位点伴随着一组 cas（CRISPR 相关）基因，这些基因编码潜在酶促机制的蛋白质成分。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易处理性。我们的工作已经取得了三项重大进展：(i) CRISPR 位点可以限制接合质粒的传播，从而赋予表皮葡萄球菌和金黄色葡萄球菌抗生素抗性； (ii) 表皮葡萄球菌中的 CRISPR 途径直接靶向传入的 DNA，因此与 RNAi 根本不同； (iii) crRNA 通过间隔区外的差异碱基配对区分非靶向“自身”DNA（CRISPR 位点）与靶向“非自身”DNA（质粒和噬菌体基因组）。我们的工作增进了我们对 CRISPR 干扰的理解，提出了限制抗生素耐药性传播的途径，验证了我们对表皮葡萄球菌作为模型系统的选择，并产生了许多适合深入分析的菌株、质粒和检测方法这条新颖而迷人的道路。我们预计，随着我们对潜在机制的了解，我们在实际和应用领域中利用 CRISPR 途径的前景将得到进一步发展。因此，我们提出的研究旨在揭示表皮葡萄球菌中 CRISPR 干扰的新的基本方面。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/间隔区及其编码的 crRNA 的功能解剖结构； (ii) 识别和表征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）； (iii) 表征含 crRNA 的核糖核蛋白 (crRNP) 并定义其特性、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。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis.

使用来自脑膜炎奈瑟菌的 Cas9 对人类多能干细胞进行有效的基因组工程。

DOI：
发表时间：
2013-09-24
期刊：
影响因子：
11.1
作者：
Hou, Zhonggang;Zhang, Yan;Propson, Nicholas E;Howden, Sara E;Chu, Li;Sontheimer, Erik J;Thomson, James A
通讯作者：
Thomson, James A

SPO24 is a transcriptionally dynamic, small ORF-encoding locus required for efficient sporulation in Saccharomyces cerevisiae.

SPO24 是酿酒酵母中高效孢子形成所需的转录动态、小型 ORF 编码位点。