A genetic safeguard for eliminating target genes in synthetic probiotics in response to a loss of the permissive signal in a gut environment

Following the development of therapeutic probiotics, there is an emerging demand for constraining engineered microbial activities to ensure biosafety. Many biocontainment studies developed genetic devices that involve cell death and growth inhibition on the engineered microbes, which often create basal levels of cytotoxicity that hamper cell fitness and performance on therapeutic functions; furthermore, these toxic pathways may promote genetic instability that leads to mutations and breakdown of biocontainment circuit. To address this issue, here we explore a circuit design that destroys the engineered genetic materials in a probiotic strain, instead of killing these cells, under non-permissive conditions. Our safeguard circuit involves a two-layered transcriptional regulatory circuit to control the expression of a CRISPR system that targets the engineered genes for degradation. In Escherichia coli Nissle 1917 ( EcN ), the biocontainment system continuously scavenged and destroyed the target until no engineered cellular function could be detected, suggesting this strategy has the potential to avoid escapee formation. Additionally, this safeguard circuit did not affect EcN cell fitness. We further demonstrated that the engineered probiotics inhabited in mouse guts and continued the engineered activities for at least 7 days when the permissive signal was supplied constantly; when the permissive signal was not provided, the engineered activities became undetectable within two days. Together, these studies support that our safeguard design is feasible for synthetic probiotic applications.