Synthetic Cassettes for pH-Mediated Sensing, Counting, and Containment

Abstract

Graphical Abstract Highlights d Containment system with an escape frequency below the detection limit of 10 À11 d Evolutionary stability achieved through toxin-antitoxin balancing d Pulse counter developed that responds to the falling edge of a signal d Counter will not advance unless 2 distinct signals are registered

Genetically stable CRISPR-based kill switches for engineered microbes

“…Biocontainment 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 . 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 – 23 .…”

“…Numerous genetic circuits have been developed that initiate cell death in response to a chemical inducer 25 – 28 . Similarly, 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 Cas3 proteins 28 .…”

“…Similarly, 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 Cas3 proteins 28 . Both temperature-sensitive circuits designed by Piraner et al and Stirling et al used the E. coli CcdB-CcdA toxin-antitoxin system to control cell survival.…”

“…The kill switch engineered by Piraner et al used a modified version of the Salmonella typhimurium -native P tlpA - tlpA sensor and achieved a 4-log reduction in fecal cell number 30 , while the kill switch engineered by Stirling et al used the E. coli- native P cspA promoter and achieved a 5-log reduction in fecal cell number 29 . Notably, functional redundancy offered by the combination of the P cspA -controlled temperature-sensitive kill switch with an orthogonal pH-sensitive kill switch mechanism synergistically improved in vitro killing efficacy such that surviving colony counts were below the 11-log limit of detection 18 . However, 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. The temperature-inducible toxin-antitoxin kill switch engineered by Stirling et al was shown to be stable over 140 generations of growth in vitro and at least 10 days of growth in the mouse gut 29 , while the combined temperature- and pH-inducible kill switch was stable over at least 100 generations in vitro 18 . In contrast, a CRISPR-Cas3-based system has been shown to be stable for 1700 generations when applied to plasmid removal 28 .…”