Bacterial Genetics Core D

细菌遗传学核心 D

基本信息

批准号：
10612029
负责人：
Jeremy Michael Rock
金额：
$ 41.07万
依托单位：
BRIGHAM AND WOMEN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-01 至 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10612029
关键词：
Achievement Age Antibiotics Antimycobacterial Agents Bacterial Genes Base Pairing Biochemical Biological CRISPR interference Clinical Drug Development Development Drug Targeting Drug resistance Drug resistant Mycobacteria Tuberculosis Essential Genes Gene Silencing Gene Targeting Generations Genes Genetic Genetic Transcription Genome Growth Guide RNA Human Immune response Immune system Individual Laboratories Length Libraries Lipids M. tuberculosis genome Mediator Metabolic Methods Microbiology Multi-Drug Resistance Mutation Mycobacterium tuberculosis Nature Nucleotides Open Reading Frames Organism Patients Pharmaceutical Preparations Phenotype Production Proteins RNA Research Rifampicin resistance Role Signal Transduction Pathway Technology Variant Virulence Virulence Factors Work bacterial genetics design design and construction experimental study genetic manipulation genome-wide in vivo interest knock-down knockout gene lipidomics mutant mycobacterial novel novel strategies nuclease response screening small molecule stem

项目摘要

Core D. Bacterial Genetics Core Core Leader: Jeremy Rock ABSTRACT Stewart Cole and colleagues determined the complete genome sequence of Mycobacterium tuberculosis (Mtb) in 1998 (1). This landmark achievement heralded a new age in mycobacterial research, including the development of organism-wide gene knockout and knockdown technologies that made it possible to determine the roles of specific mycobacterial genes in survival and host response. However, despite the ability to interrogate thousands of new potential targets, few new genes have advanced as targets for active clinical drug development. This shortfall stems, in part, from key technical limitations in the ability to systematically interrogate the Mtb genome on an organism-wide basis. To help overcome this limitation, Jeremy Rock and colleagues developed CRISPRi interference (CRISPRi) technologies that achieve robust, programmable gene silencing in Mtb. The Rock laboratory has now validated a genome-scale library of 96,700 independent CRISPRi mutants, which comprise the central technology of the Bacterial Genetics Core D. These efforts have resulted refined CRISPRi design rules, allowing the generation of highly efficacious and specific CRISPRi knockdown for nearly all Mtb genes, including methods for titratable knockdown of essential genes. The Bacterial Genetics Core will support Project 1 by designing and constructing individual and pools of Mtb CRISPRi mutants to identify new lipids that are downstream of genes involved in virulence, barrier function and Mtb strain variations in human patients. Core D will support Project 2 by providing genetic mutants within the MtrAB signal transduction pathway, a central mediator of intrinsic multi-drug resistance in Mtb. In addition, CRISPRi will be used to silence genes involved in the mycobacterial drug response, intrinsic drug resistance to rifampicin, as well as Mtb envelope composition. These studies will identify novel genetic and biochemical targets for development of new anti-mycobacterial drugs. Further, these experiments inform strategies for augmenting the efficacy of existing drugs through targeting bacterial genes that modulate the sensitivity of Mtb to antibiotics.

核心 D. 细菌遗传学核心核心领导者：杰里米·洛克抽象的 Stewart Cole 及其同事确定了结核分枝杆菌 (Mtb) 的完整基因组序列 1998年（1）。这一里程碑式的成就预示着分枝杆菌研究的新时代，包括开发全生物体基因敲除和敲除技术，使确定特定分枝杆菌基因在生存和宿主反应中的作用。然而，尽管有能力询问数千个新的潜在靶标，很少有新基因已成为活跃临床的靶标药物开发。这种不足部分源于系统性能力的关键技术限制。在整个生物体的基础上询问 Mtb 基因组。为了帮助克服这一限制，Jeremy Rock 和同事开发了 CRISPRi 干扰 (CRISPRi) 技术，可实现稳健的可编程基因山地沉默。 Rock 实验室现已验证了包含 96,700 个独立样本的基因组规模文库。 CRISPRi 突变体，构成细菌遗传学核心 D 的核心技术。这些努力已经产生了完善的 CRISPRi 设计规则，从而可以生成高效且特异的 CRISPRi 几乎所有 Mtb 基因的敲除，包括必需基因的可滴定敲除方法。这细菌遗传学核心将通过设计和构建 Mtb 个体和池来支持项目 1 CRISPRi 突变体可识别位于毒力、屏障功能和相关基因下游的新脂质人类患者的 Mtb 菌株变异。 Core D 将通过提供基因突变体来支持项目 2 MtrAB 信号转导通路，Mtb 内在多药耐药性的中心介质。此外， CRISPRi将用于沉默参与分枝杆菌药物反应、内在耐药性的基因利福平，以及结核分枝杆菌包膜成分。这些研究将鉴定新的遗传和生物化学开发新的抗分枝杆菌药物的目标。此外，这些实验还为以下策略提供了信息：通过靶向调节 Mtb 敏感性的细菌基因来增强现有药物的功效抗生素。