Bacterial Genetics Core D

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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.