Genetically stable CRISPR-based kill switches for engineered microbes

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.

Comprehensive analysis of kill switch toxins in plant-beneficial Pseudomonas fluorescens reveals drivers of lethality, stability, and escape

“…Kill switches that respond to pH 11 , temperature 12,13 , and CO2 14 have been devised, and the additional innate modularity of transcription factors means DNA-binding and small moleculesensing domains can be shuffled to enable circuit customization 10 . Many well-characterized protein toxins also exist for driving containment, including toxin-antitoxin (TA) systems, proteases, polymorphic exotoxins, anti-phage restriction or CRISPR-associated endonucleases, and phage lysis genes 9,10,13,[15][16][17][18][19] . To date, however, the vast majority of kill switches have been developed in Escherichia coli 10,11,13,[18][19][20][21][22] , and tested under ideal laboratory conditions (i.e., in rich media).…”

“…Many well-characterized protein toxins also exist for driving containment, including toxin-antitoxin (TA) systems, proteases, polymorphic exotoxins, anti-phage restriction or CRISPR-associated endonucleases, and phage lysis genes 9,10,13,[15][16][17][18][19] . To date, however, the vast majority of kill switches have been developed in Escherichia coli 10,11,13,[18][19][20][21][22] , and tested under ideal laboratory conditions (i.e., in rich media). While kill switch designs have been developed in Pseudomonas putida and Saccharomyces cerevisiae 9,16,17,20,[23][24][25] , these studies reveal that porting of kill switch parts from E. coli into alternative chassis strains is challenging, resulting in unpredictable changes in expression and fitness 9,16 .…”

“…Another major challenge associated with kill switch deployment in GEMs is escape from containment via mutational inactivation of any circuit component imparting a fitness cost on the host (e.g., promoter, toxin, regulator) 10,15,18 . Different host-specific drivers of genetic escape would alter the spectrum of mutations and specific sequences that lead to circuit inactivation (e.g., mobile element insertions; recombination-mediated deletions; replication errors) 15,16,18,27,28 . Therefore, profiling escape mutants in specific chassis is important to understand these drivers of inactivation and ultimately identify strategies to prevent inactivation to achieve sufficiently long evolutionary stability 29 .…”

“…Previous efforts to minimize genetic escape have opted to include redundant or multiple toxins in the design, which reduces the probability of total circuit inactivation 15,18 . An alternative strategy to reduce mutational escape is to eliminate routes by which the host strain accrues inactivating mutations, such as stress-response genes and RecA 10,16,18 . Finally, elimination of mobile elements from the host genome can greatly reduce escape frequency in some hosts 27,30 .…”

“…Therefore, profiling escape mutants in specific chassis is important to understand these drivers of inactivation and ultimately identify strategies to prevent inactivation to achieve sufficiently long evolutionary stability 29 . Previous efforts to minimize genetic escape have opted to include redundant or multiple toxins in the design, which reduces the probability of total circuit inactivation 15,18 . An alternative strategy to reduce mutational escape is to eliminate routes by which the host strain accrues inactivating mutations, such as stress-response genes and RecA 10,16,18 .…”