Mapping genetic interactions in mycobacteria

The National Institute of Allergy and Infectious Diseases awarded Weill Medical College of Cornell University $110,228 on February 14, 2027, under the Allergy and Infectious Diseases Research program (CFDA 93.855) to map genetic interactions in mycobacteria and apply those maps to tuberculosis drug discovery and regimen design.

The award funds large-scale genetic interaction mapping in Mycobacterium tuberculosis and the model organism M. smegmatis using a CRISPR interference platform for inducible, tunable gene knockdown. Aim 1 generates genetic interaction maps among essential genes in both organisms. Aim 2 applies those maps to investigate predicted gene function using ribosome profiling and tRNA charging state analysis. Aim 3 demonstrates translational applications to TB drug regimen optimization. The work addresses two persistent barriers to TB treatment: over one-third of MTB genes lack functional annotation, limiting drug target discovery, and current TB regimens rely on trial-and-error design of multi-drug combinations rather than systematic exploration of the combinatorial space.

Performance occurs in New York, New York. The period of performance runs through August 16, 2030.

Abstract

Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), remains the leading bacterial cause of mortality worldwide, with two key scientific barriers contributing to this persistent status. First, over a third of MTB genes lack functional annotation. Since unknown function is a non-starter in drug discovery, many vulnerable and selective targets have been neglected, representing lost opportunities for the development of effective, safe, and mechanistically novel antituberculars. Second, TB is treated with a combination of three or more drugs, producing a vast combinatorial space that is difficult to explore systematically with current tools. Despite some progress, regimen design still relies heavily on trial and error, an inefficient method liable to miss optimal solutions. To address these two barriers, the present proposal aims to generate large-scale maps of genetic interactions (GIS), pairs of genes whose concurrent disruption has an unexpected effect on bacterial fitness. In yeast, GI mapping has enabled the functional annotation of uncharacterized genes; in oncology, IT has successfully guided the rational design of combination chemotherapies. This proposal's central hypothesis is that GI mapping in mycobacteria will have similar effects, revealing gene function and aiding the design of optimized TB drug regimens. An innovative crispr interference platform for inducible, tunable gene knockdown in mycobacteria provides the necessary technical foundation for this work. The objective of aim 1 is to map GIS among all essential genes in MTB and the model mycobacterium M. Smegmatis. These maps form the basis for aims 2 and 3, which seek to demonstrate their applications in fundamental and translational biology. Specifically, aim 2 will investigate the predicted function of an uncharacterized gene, using ribosome profiling and an analysis of trna charging states to establish a specific role in translation. Aim 3 will assess the validity of predicted drug-drug interactions, using checkerboard assays to measure drug synergy and antagonism. The successful completion of these aims will revolutionize TB research and drug discovery, resulting in: (1) accelerated functional annotation of MTB genes, expanding the drug target space; (2) identification of drug target interactions, enabling biology-inspired regimen design; and (3) characterization of a novel factor essential for protein synthesis, opening new avenues for a proven drug target. Additionally, this project will foster the professional development of its principal investigator, an aspiring clinician-scientist in the TRI- institutional MD-PHD program. With close mentorship from her sponsor, DR. Jeremy rock, and the benefit of a supportive and resource-rich environment, she will emerge well-prepared for an impactful independent investigative career in biomedicine.