CRISPR-Cas-directed transposition in Tn7-like elements

Project Summary / Abstract Transposons are mobile genetic elements that provide an important mechanism for the acquisition of pathogenesis functions and antibiotic resistance in bacteria. A family of these elements, Tn7 and Tn7- like elements, tightly control transposition allowing them to be particularly successful across diverse bacteria. On five distinct occasions discovered by the lab and collaborators these elements have coopted CRISPR-Cas systems. CRISPR-Cas systems typically function as adaptive immune systems in prokaryotes that utilize an RNA-based system to recognize and cleave viruses and other invading DNA elements. In coopting CRISPR-Cas systems they were naturally adapted for guide RNA-directed transposition suggesting promising new tools for programable editing. As an editing tool, CRISPR-Cas transposons (CASTs) direct a single cargo DNA into a pre-programmed position in one orientation without the negative side effects of inducing a double strand break in the target DNA. CAST enable genome editing of bacteria, individually and in communities. They also have future potential for human therapeutic gene editing. Despite the potential promise with the CAST systems, major questions remain about how they function, which limits their broad application. By advancing our understanding across diverse CAST systems we provide foundational knowledge to enable important genome and population editing applications. Each of the four projects focuses on CAST systems based on different families of Tn7-like elements that use different mechanisms of transposase assembly and activation. This mechanistic understanding will be critical for optimizing these systems and adapting them to new hosts. Understanding the basic features of all CAST systems will bring the field closer to the aspirational goal of setting up a system where associations between a transposon and any CRISPR- Cas system could be engineered de novo. Our work will additionally provide insight into canonical CRISPR-Cas systems and the unappreciated widespread use of atypical guides for gene regulation. The mechanistic understanding we gain with diverse CAST elements will allow also allow us to understand the outsized role of Tn7 and Tn7-like elements in pathogens for the acquisition of antibiotic resistance and virulence factors. Relevance to Public Health: Public health will be served because we will provide the framework for developing important new genome modification techniques that will be broadly applicable for gene editing, especially for future human therapeutic gene editing. Fundamental information about these systems will also help us understand molecular mechanisms that allow the evolution of pathogens and multidrug resistant bacteria though the transfer of genetic information.