Intern-based thermoregulated meganucleases for biocontainment of genetic material

Abstract

Limiting the spread of synthetic genetic information outside of the intended use is essential for applications where biocontainment is critical. In particular, biocontainment of engineered probiotics and plasmids that are excreted from the mammalian gastrointestinal tract is needed to prevent escape and acquisition of genetic material that could confer a selective advantage to microbial communities. Here, we built a simple and lightweight biocontainment system that post-translationally activates a site-specific DNA endonuclease to degrade DNA at 18{degrees}C and not at higher temperatures. We constructed an orthogonal set of temperature sensitive-meganucleases, or TSMs, by inserting the yeast VMA1 L212P temperature-sensitive intein into the coding regions of LAGLIDADG homing endonucleases. We showed that the TSMs eliminated plasmids carrying the cognate TSM target site from laboratory strains of Escherichia coli at the permissive 18{degrees}C but not at higher restrictive temperatures. Plasmid elimination is dependent on both TSM endonuclease activity and intein splicing. We demonstrated that TSMs eliminated plasmids from the E. coli Nissle 1917 strain after passage through the mouse gut when fecal resuspensions were incubated at 18{degrees}C but not at 37{degrees}C. Collectively, our data demonstrates the potential of thermoregulated meganucleases as a means of restricting engineered plasmids and probiotics to the mammalian gut.