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Genetically stable CRISPR-based kill switches for engineered microbes

Author

Listed:
  • Austin G. Rottinghaus

    (Environmental and Chemical Engineering, Washington University in St. Louis)

  • Aura Ferreiro

    (The Edison Family Center for Genome Sciences & Systems Biology, Washington University School of Medicine
    Washington University in St. Louis)

  • Skye R. S. Fishbein

    (The Edison Family Center for Genome Sciences & Systems Biology, Washington University School of Medicine
    Washington University School of Medicine)

  • Gautam Dantas

    (The Edison Family Center for Genome Sciences & Systems Biology, Washington University School of Medicine
    Washington University in St. Louis
    Washington University School of Medicine
    Washington University School of Medicine)

  • Tae Seok Moon

    (Environmental and Chemical Engineering, Washington University in St. Louis
    Washington University in St. Louis)

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.

Suggested Citation

  • Austin G. Rottinghaus & Aura Ferreiro & Skye R. S. Fishbein & Gautam Dantas & Tae Seok Moon, 2022. "Genetically stable CRISPR-based kill switches for engineered microbes," Nature Communications, Nature, vol. 13(1), pages 1-17, December.
    • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-28163-5
    DOI: 10.1038/s41467-022-28163-5
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    References listed on IDEAS

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    1. In Young Hwang & Elvin Koh & Adison Wong & John C. March & William E. Bentley & Yung Seng Lee & Matthew Wook Chang, 2017. "Engineered probiotic Escherichia coli can eliminate and prevent Pseudomonas aeruginosa gut infection in animal models," Nature Communications, Nature, vol. 8(1), pages 1-11, April.
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    Cited by:

    1. Xuan Zou & Xiaohong Xiao & Ziran Mo & Yashi Ge & Xing Jiang & Ruolin Huang & Mengxue Li & Zixin Deng & Shi Chen & Lianrong Wang & Sang Yup Lee, 2022. "Systematic strategies for developing phage resistant Escherichia coli strains," Nature Communications, Nature, vol. 13(1), pages 1-12, December.
    2. Dae-yeol Ye & Myung Hyun Noh & Jo Hyun Moon & Alfonsina Milito & Minsun Kim & Jeong Wook Lee & Jae-Seong Yang & Gyoo Yeol Jung, 2022. "Kinetic compartmentalization by unnatural reaction for itaconate production," Nature Communications, Nature, vol. 13(1), pages 1-10, December.
    3. Dalton R. George & Mark Danciu & Peter W. Davenport & Matthew R. Lakin & James Chappell & Emma K. Frow, 2024. "A bumpy road ahead for genetic biocontainment," Nature Communications, Nature, vol. 15(1), pages 1-5, December.
    4. Yu-Yu Cheng & Zhengyi Chen & Xinyun Cao & Tyler D. Ross & Tanya G. Falbel & Briana M. Burton & Ophelia S. Venturelli, 2023. "Programming bacteria for multiplexed DNA detection," Nature Communications, Nature, vol. 14(1), pages 1-11, December.

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