Engineering Tools to Control Individual Microbes and Microbiota without Antibiotic Resistance Genes at a Single Strain Level | AIChE

Engineering Tools to Control Individual Microbes and Microbiota without Antibiotic Resistance Genes at a Single Strain Level

Authors 

Moon, T. S. - Presenter, Washington University in St Louis
Rottinghaus, A., Washington University In St. Louis
Amrofell, M., Washington University in St. Louis
Vo, S., Washington University in St. Louis
Microbial biocontainment is essential for engineering safe living therapeutics [1, 2]. However, the genetic stability of biocontainment circuits is a challenge. Kill switches are among the most difficult circuits to maintain due to the evolution of escape mutants. We engineered two CRISPR-based, chemical- or temperature-inducible kill switches in the probiotic Escherichia coli Nissle and demonstrated mutationally robust biocontainment [3]. In this presentation, we will discuss our machine learning-based microbiota engineering tools that are useful to manipulate microbiota and kill pathogens at a single strain level [4]. Specifically, we will discuss the development and validation of a novel computational program, ssCRISPR, which designs strain-specific CRISPR guide RNAs (gRNAs) that can be utilized to modify complex consortia. As a proof of concept, we applied the program to two novel applications: the isolation of specific microbes from consortia through plasmid transformations and the removal of specific microbes from consortia through liposome-packaged CRISPR antimicrobials. Additionally, we will discuss antibiotic resistance gene-free plasmid systems that prevent antibiotic resistance spread via horizontal gene transfer [5]. This new technology has vast implications in designing strain-specific antimicrobials and combating the growing concern of antibiotic- and bacteriocide-resistant microbes.

  1. Curr Opin Biotechnol, 2020. 66, 11-17.
  2. Biotechnology Journal, 2020. 15, 1900319.
  3. Nature Communications, 2022. 13, 672
  4. PNAS. 2023. 120, e2213154120
  5. Trends. Biotechnol, 2022, 40, 1405-1414