Bacterial Genetics Core D

核心D。细菌遗传学核心 核心领导人：Jeremy Rock 摘要 斯图尔特科尔和同事确定了结核分枝杆菌（Mtb）的完整基因组序列， 1998年（1）。这一具有里程碑意义的成就预示着分枝杆菌研究的新时代， 生物体范围内基因敲除和敲低技术的发展，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 尽管研究了数千个新的潜在靶点，但很少有新基因成为积极临床研究的靶点 药物开发这一不足的部分原因是，在系统地 在生物体范围内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 他的同事开发了CRISPRi干扰（CRISPRi）技术， Mtb. Rock实验室现在已经验证了一个包含96，700个独立基因的基因组规模的文库。 CRISPRi突变体，构成细菌遗传学核心D的核心技术。这些努力 产生了精细的CRISPRi设计规则，允许产生高效和特异性的CRISPRi 敲除几乎所有的Mtb基因，包括用于可滴定敲除必需基因的方法。的 细菌遗传学核心将通过设计和构建结核分枝杆菌的个体和池来支持项目1 CRISPRi突变体，以鉴定参与毒力、屏障功能和代谢的基因下游的新脂质。 人类患者的结核分枝杆菌菌株变异。核心D将通过提供基因突变体来支持项目2。 MtrAB信号转导通路是结核分枝杆菌内源性多药耐药的中心介质。此外，本发明还提供了一种方法， CRISPRi将用于沉默参与分枝杆菌药物反应的基因， 利福平以及结核分枝杆菌包膜组合物。这些研究将确定新的遗传和生化 新的抗分枝杆菌药物的开发目标。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功效 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.