Genetically stable CRISPR-based kill switches for engineered microbes

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…

• NS
  National Science Foundation

  Awards: CBET-1350498, 1350498
• UE
  U.S. Environmental Protection Agency

  Award: 84020501
• UD
  U.S. Department of Agriculture

  Award: 2020-33522-32319
• NI
  National Institutes of Health

  Award: R01 AT009741
• AR
  Agricultural Research Service

  Award: 2020-33522-32319
• OO
  Office of Naval Research

  Awards: N00014-17-1-2611, N00014-19-1-2357, N00014-17-1-2611 & N00014-19-1-2357, N00014-17-1, N00014
• DO
  Division of Chemical, Bioengineering, Environmental, and Transport Systems

  Award: CBET-1350498
• NC
  National Center for Complementary and Integrative Health

  Award: R01 AT009741