Abstract
Microbial biocontainment is an essential goal for engineering safe, next-generation living therapeutics. However, the genetic stability of biocontainment circuits, including kill switches, is a challenge that must be addressed. Kill switches are among the most difficult circuits to maintain due to the strong selection pressure they impart, leading to high potential for evolution of escape mutant populations. Here we engineer two CRISPR-based kill switches in the probiotic Escherichia coli Nissle 1917, a single-input chemical-responsive switch and a 2-input chemical- and temperature-responsive switch. We employ parallel strategies to address kill switch stability, including functional redundancy within the circuit, modulation of the SOS response, antibiotic-independent plasmid maintenance, and provision of intra-niche competition by a closely related strain. We demonstrate that strains harboring either kill switch can be selectively and efficiently killed inside the murine gut, while strains harboring the 2-input switch are additionally killed upon excretion. Leveraging redundant strategies, we demonstrate robust biocontainment of our kill switch strains and provide a template for future kill switch development.
Highlights
IntroductionMicrobial biocontainment is an essential goal for engineering safe, next-generation living therapeutics
Withholding these molecules in the gut, for example by administering an auxotrophic strain without the essential compound, effectively limits probiotic lifespan in vivo[11], but it may limit therapeutic potential depending on the rate of probiotic cell death in the absence of the permissive molecules
The aTconly and 2-input CRISPRks strains developed here allow the growth of Escherichia coli Nissle 1917 (EcN) to be tightly controlled during in vivo applications
Summary
Microbial biocontainment is an essential goal for engineering safe, next-generation living therapeutics. Biocontainment circuits have been developed in E. coli using temperature sensors tuned to differentiate physiological and environmental temperatures[18,29,30] These kill switches control cell survival using a variety of mechanisms, including expression of toxins and lysis proteins[18,25–27], degradation of essential proteins[26], and cleavage and degradation of the genome by Cas[3] proteins[28]. Kill switches that induce cell death by expressing toxins, lysis proteins, and proteases are prone to mutational inactivation, often leading to population dominance of non-functional variants, or have not been characterized for genetic stability[26] To overcome this stringent evolutionary selection, such kill switch systems must be designed to be highly stable. It is unclear whether the same stability would persist if the system was applied to cell death
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