Development of a CRISPR interference system for mycobacteria

分枝杆菌 CRISPR 干扰系统的开发

基本信息

批准号：
8765966
负责人：
ROBERT N HUSSON
金额：
$ 8.79万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8765966
关键词：
Address Amino Acid Substitution Antibiotics Antisense RNA Bacteria Bacterial Genes Bacteriophages Binding Sites Chromosomes, Human, Pair 6 Clustered Regularly Interspaced Short Palindromic Repeats Code DNA Binding DNA Sequence Development Disease Drug Resistant Tuberculosis Drug Targeting Drug resistance Escherichia coli Essential Genes Eukaryota Fluorescence Gene Expression Gene Proteins Gene Targeting Genes Genetic Genus Mycobacterium Goals Grant Growth Guide RNA Immunity Laboratories Location Messenger RNA Methods Mutagenesis Mycobacterium smegmatis Mycobacterium tuberculosis Operon Output Pathway interactions Pharmaceutical Preparations Physiology Plasmid Cloning Vector Process Prokaryotic Cells Promoter Regions Proteins RNA Binding RNA Interference Reporter Genes Repression Research Research Personnel Resources Shuttle Vectors Site Small RNA Specificity Streptococcus pyogenes System Technology Testing Translating Treatment Protocols Tuberculosis Variant Virulence base drug candidate endonuclease gene function gene repression inhibitor/antagonist insight interest meetings mycobacterial novel novel strategies nuclease null mutation pathogen promoter public health relevance research study resistant strain success tool tuberculosis treatment vector

项目摘要

DESCRIPTION (provided by applicant): There is an urgent need to develop new drugs for the treatment of tuberculosis, both to shorten therapy so that higher completion rates can be achieved using fewer resources, and to address the growing problem of drug-resistant tuberculosis. Nearly all current antibiotics target essential genes or processes. Though new approaches that inhibit genes important for virulence may provide an alternative, targeting essential genes is likely to remain the predominant approach to develop new M. tuberculosis inhibitors. Characterization of essential genes whose function is unknown or incompletely understood, however, is challenging because they cannot simply be deleted. In the absence of null mutations, regulated conditional gene expression can be informative, but is difficult to achieve in M. tuberculosis using current tools. Technology derived from the clustered, regularly interspaced, short palindromic repeat (CRISPR) system of Streptococcus pyogenes, using the Cas9 endonuclease together with small RNAs to provide sequence specificity, has rapidly become a method of choice for gene editing in eukaryotes. A recent variation of this system, in which amino acid substitutions in Cas9 result in a protein that lacks nuclease activity but retains RNA and DNA binding (dCas9), has been used to obtain specific repression of gene expression in both bacteria and eukaryotes. The goal of this small RO3 project is to adapt this system for use in mycobacteria, particularly in M. tuberculosis. This goal will be achieved through two specific aims. First we will construct E. coli-mycobacterial shuttle vectors that incorporate anyhydrotetracycline-inducible gene expression to express dCas9 and the required small RNA constructs, to target genes for repression. Second we will test this system, using 1) a gene that expresses the fluorescent mCherry protein, so that repression can be readily quantified, and 2) two native chromosomal loci so that we can examine the function of this system under conditions in which it would be used. By targeting different sites in these genes we will be able to assess the extent to which different target binding sites, e.g. promoter region versus coding sequence, leads to repression of the gene of interest. Once developed, this system will be useful for investigators to characterize essential genes whose function is not known, and will facilitate the development of inhibitors that target the protein products of these genes.

描述（由申请人提供）：迫切需要开发治疗结核病的新药，既可以缩短治疗时间，从而使用更少的资源实现更高的完成率，又可以解决日益严重的耐药结核病问题。目前几乎所有抗生素都针对重要基因或过程。尽管抑制毒力重要基因的新方法可能提供一种替代方案，但针对必需基因可能仍然是开发新结核分枝杆菌抑制剂的主要方法。然而，功能未知或不完全了解的必需基因的表征具有挑战性，因为它们不能简单地删除。在不存在无效突变的情况下，受调节的条件基因表达可以提供信息，但使用现有工具很难在结核分枝杆菌中实现。源于化脓性链球菌成簇、规则间隔、短回文重复 (CRISPR) 系统的技术，使用 Cas9 核酸内切酶与小 RNA 一起提供序列特异性，已迅速成为真核生物基因编辑的首选方法。该系统最近的一个变体，其中 Cas9 中的氨基酸替换导致蛋白质缺乏核酸酶活性，但保留了 RNA 和 DNA 结合 (dCas9) 已被用于获得细菌和真核生物中基因表达的特异性抑制。这个小型 RO3 项目的目标是使该系统适用于分枝杆菌，特别是结核分枝杆菌。这一目标将通过两个具体目标来实现。首先，我们将构建大肠杆菌-分枝杆菌穿梭载体，该载体掺入任何氢四环素诱导的基因表达以表达 dCas9 和所需的小 RNA 构建体，以靶向基因进行抑制。其次，我们将测试该系统，使用 1) 表达荧光 mCherry 蛋白的基因，以便可以轻松量化抑制，以及 2) 两个天然染色体位点，以便我们可以检查该系统在以下条件下的功能：被使用。通过针对这些基因中的不同位点，我们将能够评估不同目标结合位点的程度，例如启动子区域与编码序列相比，导致感兴趣基因的抑制。一旦开发出来，该系统将有助于研究人员表征功能未知的重要基因，并将促进针对这些基因的蛋白质产物的抑制剂的开发。