We are interested in the molecular mechanisms that lead to the acquisition of pathogenesis functions and antibiotic resistance in bacteria. We are investigating one of the ways genomes can be altered in a process called transposition. Transposons are mobile DNA elements that can move within a cell and are found in nearly every type of organism and are abundant in the human genome. The specific transposon we are working with also has important practical applications for genome modification using CRISPR/Cas in bacteria (and potentially beyond). CRISPR-Cas systems are revolutionizing our ability to genetically modify a wide array of organisms. However, these systems suffer from a fundamental limitation when it comes to inserting new genetic information; these systems make a precise break in the DNA, but rely on endogenous host repair mechanisms to insert the desired new sequence information (a second DNA strand transformed into the cell at the same time). CRISPR- Cas systems are found naturally in bacteria and archaea for protection from viruses. While all CRISPR-Cas systems utilize a guide RNA to target a virus for destruction they are extremely diverse. Our preliminary work indicated the existence of a novel minimal CRISPR-Cas system of unusual function in transposons. In this grant, we will establish the CRISPR/Cas transposon targeting system in E. coli, a type of bacteria that is easy to manipulate in the lab to study its basic functioning. We will also modify the system to work in different types of bacteria. We will study the basic functioning of the system and isolate mutant components that will allow us to make the system more efficient and more specific. Relevance to Public Health: We will determine the molecular mechanisms that allow the evolution of emerging pathogens and multi drug resistant bacteria though the transfer of genetic information. This will lead to better treatments and strategies to limit the evolution of more serious pathogens. Public health will also be served because we will be developing an important new genome modification technique that will be broadly applicable in bacteria and possibly other kinds of organisms.
This project involves understanding the basic functioning of genetic elements that play a critical role in making some bacteria pathogenic or difficult or impossible to treat because they allow the development of drug resistance. It will also provide an important new CRISPR/Cas tool for manipulating bacteria genomes.