문태석 (Washington University in St. Louis)

한빛사논문

조회718

Washington University in St. Louis

CV File 516 kb

Nat. Commun., Published 03 February 2022 | https://doi.org/10.1038/s41467-022-28163-5 Genetically stable CRISPR-based kill switches for engineered microbes

Austin G. Rottinghaus^1,7, Aura Ferreiro^2,3,7, Skye R. S. Fishbein^2,4, Gautam Dantas^2,3,4,5,6,* & Tae Seok Moon^1,6,*

¹Department of Energy, Environmental and Chemical Engineering, Washington University in St. Louis, St. Louis, MO, USA. ²The Edison Family Center for Genome Sciences & Systems Biology, Washington University School of Medicine, St. Louis, MO, USA. ³Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO, USA. ⁴Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA. ⁵Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO, USA. ⁶Division of Biology and Biomedical Sciences, Washington University in St. Louis, St. Louis, MO, USA. ⁷These authors contributed equally: Austin G. Rottinghaus, Aura Ferreiro.

^*Corresponding author.

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.

논문정보

형식Research article
게재일2022년 02월 (BRIC 등록일 2022-04-27)
연구진국외 연구진
분야 생명과학 > 생물공학

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