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Genetically stable CRISPR-based kill switches for engineered microbes
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Journal Article
Genetically stable CRISPR-based kill switches for engineered microbes
2022
Overview
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.
Biocontainment is a key to developing safe genetically-engineered microbes (GEMs). Here the authors demonstrate genetically stable CRISPR-based kill switches that control GEMs’ viability in animal hosts, enabling their safe biomedical applications.
Publisher
Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
Subject
/ 38/44
/ 38/47
/ 38/77
/ 64/60
/ 9/10
/ 96/31
/ Animals
/ Anti-Bacterial Agents - pharmacology
/ Circuits
/ CRISPR
/ CRISPR-Cas Systems - genetics
/ E coli
/ Escherichia coli - metabolism
/ Escherichia coli - physiology
/ Female
/ Gene Expression Regulation - drug effects
/ Genetic Engineering - methods
/ Humanities and Social Sciences
/ Mice
/ Microbial Viability - drug effects
/ Microbial Viability - genetics
/ Science
/ SOS Response, Genetics - drug effects
/ SOS Response, Genetics - genetics
/ Switches
