Toward a comprehensive genetic interaction network in bacteria

The National Institute of General Medical Sciences awarded Icahn School of Medicine at Mount Sinai $420,000 on August 1, 2026, under the Biomedical Research and Research Training program (CFDA 93.859) to develop and deploy genetic interaction mapping tools in bacteria.

The project will construct a comprehensive genetic interaction network in Escherichia coli by developing a barcoded CRISPR strategy to systematically profile all pairwise genetic interactions (approximately 10 million) and generate a gene similarity network defining functional relationships among genes and biological processes. The recipient will perform mechanistic characterization of newly identified genes and interactions, and will profile genetic interactions in E. coli in the context of recombinant protein production to identify engineering principles for enhanced protein yields. In parallel, the recipient will extend the CALM-CRISPRi functional genomics tool to bacteria with low transformability and limited systems genomics infrastructure, including Bacteroides and Bifidobacteria species.

Performance occurs in New York, NY 10029. The period of performance runs from August 1, 2026, through March 31, 2031. The award is classified as a Project Grant.

Abstract

Rationale: genetic interaction (GI) networks reveal fundamental principles of cellular biology, including gene functions, rules governing cellular organization, and synthetic lethality landscapes. However, a comprehensive GI network is lacking in bacteria. To date, only ~10% of all possible digenetic interactions have been mapped in escherichia coli, one of the best-studied bacteria. Moreover, the systems genomics infrastructure required to construct GI networks is absent in many bacterial species due to low transformability (IE, genetic recalcitrance). These limitations have severely hindered our ability to investigate gene function and genetic architecture across diverse bacteria. As a result, at least 30% of prokaryotic genes still have unknown functions. Objective: (1) in the first part of this R35 project, we will develop a barcoded crispr strategy to systematically profile, for the first time, all pairwise GIS (~10 million) in e. Coli. This effort will generate the first comprehensive bacterial GI network that captures both synergistic and antagonistic interactions. In parallel, we will construct a gene similarity network that defines functional relationships among genes and biological processes while enabling the discovery of new gene functions. We will then perform mechanistic characterization of both newly identified genes and GIS. Because e. Coli is a widely used synthetic biology chassis for recombinant protein production, we will also profile GIS in this context, potentially uncovering generalizable rules to guide the engineering of microbial systems with enhanced protein yields. (2) in the second part of this project, we will extend the calm-crispri functional genomics tool we previously developed to bacteria with low transformability and limited systems genomics infrastructure, such as bacteroides and bifidobacteria. A unique advantage of calm is its ability to bypass restriction-modification barriers by "manufacturing" libraries in situ, thereby enabling the generation of highly diverse crispr libraries in genetically recalcitrant bacteria. Using diverse engineering and systems biology approaches, we will construct gene similarity networks in bacteroides and bifidobacteria. These abundant GUT microbes currently lack robust crispr-based functional genomics tools, and our approach will accelerate the functional characterization of their genes. Impact: successful completion of this project will significantly advance our understanding of gene function and genetic architecture in broad prokaryotic organisms, identify vulnerable nodes within GI networks to inform new antimicrobial strategies, and facilitate the engineering of microbial systems with enhanced synthetic capabilities.