Multi-Input CRISPR-Based Kill-Switches for Biocontainment of Engineered Microbes

Probiotics are effective chassis for diagnostic and therapeutic applications. However, there are safety concerns associated with using genetically engineered organisms for medical applications. To address these issues, we engineered Escherichia coli Nissle to survive only when and where it is needed using CRISPR-based kill-switches (CRISPRks). We first designed a CRISPRks that induces cell death by expressing Cas9 and genome-targeting guide RNAs (gRNAs) in response to a chemical inducer. This design allows cell killing to occur while the microbe is in the gut in response to oral administration of the chemical. We optimized the efficiency and stability of the CRISPRks, achieved more than a 9-log reduction in cell number, and demonstrated genetic stability for up to 28 days of continuous growth. This high killing efficiency was maintained in vivo, where we achieved complete elimination of the probiotic after oral administration of the inducer. To our knowledge, this is the first time on-demand elimination of an engineered microbe that has been demonstrated in vivo. We next modified our chemically inducible-CRISPRks to also induce cell death in response to ambient temperatures below 33’C. This design induces cell killing either in response to oral administration of the chemical or when the microbe is excreted from the body in response to the reduced environmental temperature. Our CRISPRks strategy provides a benchmark for future microbial biocontainment circuits. The sensor and killing mechanism are well characterized and functional in many microbes, allowing the CRISPRks design to be broadly applicable in diverse microbiota.