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）：迫切需要开发新药物来治疗结核病，既要缩短治疗疗法，以便可以使用更少的资源来实现更高的完成率，并解决耐药性结核病的日益增长的问题。几乎所有当前的抗生素靶向基本基因或过程。尽管抑制对毒力重要的基因的新方法可能会提供替代方法，但靶向基因可能仍然是发展新的结核分枝杆菌抑制剂的主要方法。但是，其功能未知或未完全理解的基本基因的表征是具有挑战性的，因为它们不能简单地被删除。在没有零突变的情况下，调节的条件基因表达可能是有益的，但是在结核分枝杆菌中很难使用当前工具实现。使用CAS9核酸链球菌链球菌的簇，短期间隔，短的，短的回文重复（CRISPR）系统的技术得出的技术，使用CAS9核酸内切酶和小RNA提供了序列特异性，已迅速成为真核生物中基因编辑的一种选择方法。该系统的最新变化，其中CAS9中的氨基酸取代导致缺乏核酸酶活性但保留的蛋白质 RNA和DNA结合（DCAS9）已用于在细菌和真核生物中获得特定的基因表达抑制。这个小型RO3项目的目的是使该系统适应分枝杆菌，尤其是在结核分枝杆菌中。这个目标将通过两个具体目标来实现。首先，我们将构造大肠杆菌型穿梭载体，该载体结合了任何hydrototetrycline诱导的基因表达以表达DCAS9和所需的小RNA构建体，以靶向抑制基因。其次，我们将使用1）表达荧光麦克环蛋白的基因测试该系统，以便可以轻松量化抑制作用，以及2）两个天然染色体基因座，以便我们可以在使用的条件下检查该系统的功能。通过靶向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，例如启动子区域与编码序列可导致对感兴趣基因的抑制。一旦开发，该系统将对研究人员表征其功能尚不清楚的必需基因，并将促进针对这些基因蛋白质产物的抑制剂的发展。