biostudies-literatureFoo GWCanadian Institutes of Health Research3696-3708https://www.ebi.ac.uk/biostudies/studies/S-EPMC12455656biostudies-literatureUnknown14(9)Imbalances in the mammalian gut are associated with acute and chronic conditions, and using engineered probiotic strains to deliver synthetic constructs to treat them is a promising strategy. However, high rates of mutational escape and genetic instability <i>in vivo</i> limit the effectiveness of biocontainment circuits needed for safe and effective use. Here, we describe STALEMATE (<b>S</b>equence en<b>TA</b>ng<b>LE</b>d <b>M</b>ulti l<b>A</b>yered gene<b>T</b>ic buff<b>E</b>ring), a dual-layered failsafe biocontainment strategy that entangles genetic sequences to create pseudoessentiality and buffer against mutations. We entangled the colicin E9 immunity protein (Im9) with a thermoregulated meganuclease (TSM) by overlapping the reading frames. Mutations that disrupted this entanglement ACS synthetic biologyGenetic Entanglement Enables Ultrastable Biocontainment in the Mammalian Gut.PMC12455656PJT 191939Uruthirapathy ASHeinrichs DEZhang CQEdgell DRFoo GWBatko IZfalseGenetic Entanglement Enables Ultrastable Biocontainment in the Mammalian Gut.Imbalances in the mammalian gut are associated with acute and chronic conditions, and using engineered probiotic strains to deliver synthetic constructs to treat them is a promising strategy. However, high rates of mutational escape and genetic instability <i>in vivo</i> limit the effectiveness of biocontainment circuits needed for safe and effective use. Here, we describe STALEMATE (<b>S</b>equence en<b>TA</b>ng<b>LE</b>d <b>M</b>ulti l<b>A</b>yered gene<b>T</b>ic buff<b>E</b>ring), a dual-layered failsafe biocontainment strategy that entangles genetic sequences to create pseudoessentiality and buffer against mutations. We entangled the colicin E9 immunity protein (Im9) with a thermoregulated meganuclease (TSM) by overlapping the reading frames. Mutations that disrupted this entanglement 2025-01-01T00:00:00Z2025 Sep2026-07-15T05:51:43.016Z2026-06-30T03:16:03.472ZS-EPMC124556564091494910.1021/acssynbio.5c00412