Development of a Thermosensitive Endonuclease to Act as a Plasmid Kill-Switch

Biocontainment is an integral part of biomedical research that aims to protect the environment and human health by containing hazardous or invasive organisms in the laboratory. Containment systems often rely on elaborate genetic circuits; however, cells may escape containment by developing mutations that render the genetic circuits inviable or resistant to killing mechanisms. The aim of this thesis was to create a site-specific nuclease for biocontainment of plasmids in the mammalian gastrointestinal tract. LAGLIDADG homing endonucleases would be good candidate nucleases for a biocontainment system as they are resistant to mutations in their coding sequence and their target sequence in comparison to other nucleases, such as Cas9. Screening mutagenic libraries of the I-OnuI nuclease yielded a single variant that displays thermosensitive properties in vivo that may prove useful for biocontainment purposes. Using thermosensitive LAGLIDADG homing endonucleases for this purpose may prove to be a new, more robust approach to biocontainment