Collaborative Research: EDGE FGT: Advancing CRISPR approaches for bacterial functional genomics applied to marine Vibrio-host interactions

About This Grant

The project will develop tools to enable genetic manipulation of bacteria to facilitate an array of biotechnology applications. The tools build on established methods to influence the production of protein from individual bacterial genes, including scaling up to impact all the genes in a bacterium within a single test tube. Furthermore, advances to the technology will expand to enable more precise alterations to turn genes “down” or “up,” decreasing or increasing production of proteins encoded by those genes on a broad scale. Previously, such approaches were limited to a small number of targeted bacterial strains. This work seeks to expand the biotechnological reach of available bacteria with a generalized approach that will be tested in a group of marine bacteria called vibrios. Vibrios are important in our food supply, are prominent in causing animal and human disease, and are widely studied to understand animal-host interactions and basic microbiology. As such, they represent a valuable test bed in which to develop and deploy the new technology and assemble resources for laboratories to apply that technology in their own research and in classrooms worldwide. Sharing of the resources developed, including laboratory protocols, teaching materials, computer code, databases of DNA sequences to target bacterial strains, and the resulting bacterial strains themselves, will facilitate broadscale adoption and provide important tools toward growing our bioeconomy. The biotechnology tools developed will be made readily available to the research community, and the proof-of-concept experiments proposed here will be developed as part of a teaching curriculum that can be deployed in courses and workshops. Targeted gene perturbations using CRISPR technology have caused a paradigm shift in eukaryotic functional genomics, but comparable approaches for bacteria have lagged with a reliance on species and/or strain-specific genetic tools and a focus on laboratory strains. Thus, it is essential that bacteriologists extend their studies beyond lab strains and develop tools that can be readily deployed into environmental isolates. This project has two parallel goals: (1) to expand established tools for genome-wide gene knockdowns called CRISPR interference (CRISPRi), and (2) to develop novel bacterial CRISPR technologies for gene overexpression and gene interaction studies. This study focuses on the diverse bacterial group Vibrionaceae, which includes over 100 species that inhabit marine waters and can have significant effects on nutrient cycling and ecosystem health. Many bacterial species in the group cause vibriosis disease, leading to widespread mortality in marine organisms and impacting global aquaculture, disrupting the human food chain, and increasing risks to seafood consumption. The variety in this group is highlighted by the increasing application of Vibrio natriegens in biotechnology applications. Furthermore, Vibrio species serve as some of the most widely-studied model systems for understanding host-microbe interactions, in the context of both mutualism (e.g., V. fischeri) and pathogenesis (e.g., V. campbellii in marine animals, V. cholerae in humans). This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.