Multilayered genetic safeguards limit growth of microorganisms to defined environments

Genetically modified organisms (GMOs) are com-monly used to produce valuable compounds in closed industrial systems. However, their emerging applications in open clinical or environmental set-tings require enhanced safety and security mea-sures. Intrinsic biocontainment, the creation of bac-terial hosts unable to survive in natural environ-ments, remains a major unsolved biosafety prob-lem. We developed a new biocontainment strategy containing overlapping ‘safeguards’––engineered ri-boregulators that tightly control expression of es-sential genes, and an engineered addiction mod-ule based on nucleases that cleaves the host genome––to restrict viability of Escherichia coli cells to media containing exogenously supplied syn-thetic small molecules. These multilayered safe-guards maintain robust growth in permissive con-ditions, eliminate persistence and limit escape fre-quencies to <1.3 × 10−12. The staged approach to safeguard implementation revealed mechanisms of escape and enabled strategies to overcome them. Our safeguarding strategy is modular and employs conserved mechanisms that could be extended to clinically or industrially relevant organisms and un-domesticated species