Bacterial Genetics Core D

核心D.细菌遗传学核心 核心领袖：杰里米·洛克 摘要 斯图尔特·科尔和他的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进入了一个新时代，包括 生物体范围的基因敲除和敲除技术的发展使其能够确定 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点，几乎没有新基因作为活跃临床的靶点 药物开发。这一不足的部分原因是，在系统地 在整个生物体的基础上询问结核分枝杆菌基因组。为了帮助克服这一限制，杰里米·洛克和 同事们开发了CRISPRi干扰(CRISPRi)技术，实现了强大的、可编程的基因 Mtb中的静音。Rock实验室现在已经验证了一个包含96,700个独立基因组规模的文库 CRISPRi突变体，它们构成了细菌遗传学核心D的核心技术。这些努力 最终改进了CRISPRi设计规则，允许生成高效和特定的CRISPRi 几乎所有结核分枝杆菌基因的敲除，包括可滴定敲除必要基因的方法。这个 细菌遗传学核心将通过设计和建造结核分枝杆菌个体和池来支持项目1 CRISPRi突变体识别毒力、屏障功能和致病相关基因下游的新脂质 人类患者中结核分枝杆菌菌株的变异。核心D将支持项目2，方法是在 MtrAB信号转导通路是结核分枝杆菌固有多药耐药的中枢调节因子。此外, CRISPRi将被用来沉默参与分枝杆菌药物反应的基因，内在的耐药性 利福平，以及结核分枝杆菌包膜成分。这些研究将确定新的遗传和生化 抗分枝杆菌新药的开发目标。此外，这些实验为以下策略提供了信息 通过靶向调节结核分枝杆菌敏感性的细菌基因来增强现有药物的疗效 到抗生素。

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.