Associate toxin–antitoxin with CRISPR-Cas to harness (ATTACH) engineered microbes

Robust biocontainment is essential for the safe use of engineered microbes, but existing strategies suffer from genetic instability and/or laborious construction. Here, we present ATTACH, a kill switch that associates toxin-antitoxin with CRISPR-Cas to harness engineered microbes. Our approach employs a CRISPR-repressed toxin-antitoxin (CreTA) module to make microbes addicted to the type I-F Cas effector proteins, and places both the Cas3 nuclease and the chromosome-targeting guide RNA under inducible promoters, thereby improving the genetic stability and stringency of the CRISPR-based suicidal program. Additionally, we have developed a single-plasmid, antibiotic-independent ATTACH device, which shows robust, stringent containment of a microbial chassis in murine gut, and negligible impacts on culture growth or lycopene production during batch fermentation. Our data highlight the potential of CreTA to stabilize CRISPR-based kill switches, advancing their development into more portable and reliable biocontainment tools for engineered microbes.