(590a) Engineering Multilevel CRISPR-Based Kill-Switches for Probiotic Escherichia coli

Probiotic microbes have become an effective framework for diagnostic and therapeutic technologies [1, 2]. However, there are safety concerns associated with using genetically engineered organisms for medical applications. Probiotic microbes have the potential to evolve growth advantages over natural microbes and characteristics that are harmful to the host or to the outside environment. To mitigate these concerns, we engineered the probiotic Escherichia coli Nissle 1917 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 in response to the chemical inducer anhydrotetracycline. 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 by combining four genomic Cas9 expression cassettes with three plasmid-based guide RNA expression cassettes, removing the antibiotic dependence for maintenance of the guide RNA plasmid, and knocking out genes involved in DNA recombination and mutagenesis. Using this optimized circuit in vitro, we 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 24 hours after oral administration of the inducer. This is the first time on-demand elimination of an engineered microbe 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 two-input 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. This two-input circuit achieved more than a 9-log and 7-log reduction in cell number in vitro after exposure to the chemical inducer and temperature downshift, respectively.

Future directions will include incorporating the CRISPRks in microbes engineered to diagnose and treat diverse medical conditions [1, 2]. Our CRISPRks strategy provides a template for future microbial biocontainment circuits. The sensor and killing mechanism employed in the kill-switch are well characterized and functional in many microbes, allowing the CRISPRks design to be broadly utilized. In addition, the temperature-sensing module can be easily replaced with sensors that recognize alternative signals [3-9], allowing comparable kill-switches to be created for applications beyond medicine.

[1] Amrofell, M. B., Rottinghaus, A. G., Moon, T. S., Engineering microbial diagnostics and therapeutics with smart control. Current opinion in biotechnology 2020, 66, 11-17.

[2] Rottinghaus, A. G., Amrofell, M. B., Moon, T. S., Biosensing in Smart Engineered Probiotics. Biotechnology journal 2020, 15, 1900319.

[3] DeLorenzo, D. M., Henson, W. R., Moon, T. S., Development of Chemical and Metabolite Sensors for Rhodococcus opacus PD630. ACS Synthetic Biology 2017, 6, 1973–1978.

[4] DeLorenzo, D. M., Moon, T. S., Construction of Genetic Logic Gates Based on the T7 RNA Polymerase Expression System in Rhodococcus opacus PD630. ACS Synthetic Biology 2019, 8, 1921-1930.

[5] Hoynes-O'Connor, A., Hinman, K., Kirchner, L., Moon, T. S., De novo design of heat-repressible RNA thermosensors in E. coli. Nucleic acids research 2015, 43, 6166-6179.

[6] Hoynes-O'Connor, A., Moon, T. S., Programmable genetic circuits for pathway engineering. Current opinion in biotechnology 2015, 36, 115-121.

[7] Hoynes-O'Connor, A., Shopera, T., Hinman, K., Creamer, J. P., Moon, T. S., Enabling complex genetic circuits to respond to extrinsic environmental signals. Biotechnology and bioengineering 2017, 114, 1626-1631.

[8] Immethun, C. M., DeLorenzo, D. M., Focht, C. M., Gupta, D., et al., Physical, chemical, and metabolic state sensors expand the synthetic biology toolbox for Synechocystis sp. PCC 6803. Biotechnology and bioengineering 2017, 114, 1561-1569.

[9] Immethun, C. M., Ng, K. M., DeLorenzo, D. M., Waldron-Feinstein, B., et al., Oxygen-responsive genetic circuits constructed in synechocystis sp. PCC 6803. Biotechnology and bioengineering 2016, 113, 433-442.