Intein-based thermoregulated meganucleases for containment of genetic material

• Published in: Nucleic acids research 2024-02, Vol.52 (4), p.2066-2077
• Main Authors: Foo, Gary W, Leichthammer, Christopher D, Saita, Ibrahim M, Lukas, Nicholas D, Batko, Izabela Z, Heinrichs, David E, Edgell, David R
• Format: Article
• Language: English
• Subjects: Animals; DNA - metabolism; Escherichia coli - genetics; Escherichia coli - metabolism; Inteins - genetics; Mammals - genetics; Mice; Plasmids - genetics; Protein Splicing; Proton-Translocating ATPases; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - genetics; Synthetic Biology and Bioengineering
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkad1247

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°C and not at higher temperatures. We constructed an orthogonal set of temperature-sensitive meganucleases (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°C but not at higher restrictive temperatures. Plasmid elimination is dependent on both TSM endonuclease activity and intein splicing. TSMs eliminated plasmids from E. coli Nissle 1917 after passage through the mouse gut when fecal resuspensions were incubated at 18°C but not at 37°C. Collectively, our data demonstrates the potential of thermoregulated meganucleases as a means of restricting engineered plasmids and probiotics to the mammalian gut. Graphical Abstract Graphical Abstract