Several such biocontainment strategies have been developed with varying degrees of efficacy and stability, including use of auxotrophy 11, 19, 20 and synthetic amino acids 21, 22, 23. To mitigate these concerns, engineered probiotics should possess biocontainment systems that enable both selective removal from the host and prevent their environmental dissemination 17.īiocontainment circuit designs are focused on preventing proliferation in the wild, and typically involve an input that is specific to the permissive environment and repressive to the killing circuit, such that upon exit of the permissive environment, the lethal components are expressed 18. Such adaptations can include loss of beneficial functions of the engineered system, gain of deleterious functions such as competitive exclusion of native microbes, pathogenic potential against the host, or environmental contamination if they spread outside the host 13, 14, 15, 16. Probiotics are living organisms that have the potential to mutate and evolve undesirable traits over the course of diagnosis or treatment. However, there are important safety concerns associated with organisms genetically engineered for medical applications. EcN strains engineered to treat metabolic disorders are being evaluated in human clinical trials with promising early-phase results 11, 12.
Engineered strains of EcN have been successfully used to diagnose and treat bacterial infections 1, 2, cancers 3, 4, 5, gastrointestinal bleeding 6, inflammatory disorders 7, 8, 9, and obesity 10 in a variety of animal models. One of the most commonly engineered probiotic strains is Escherichia coli Nissle 1917 (EcN). Probiotic microbes have become effective chasses for engineering diagnostic and therapeutic technologies. Leveraging redundant strategies, we demonstrate robust biocontainment of our kill switch strains and provide a template for future kill switch development. 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. 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. 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. 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. However, the genetic stability of biocontainment circuits, including kill switches, is a challenge that must be addressed.
Microbial biocontainment is an essential goal for engineering safe, next-generation living therapeutics.
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