(70c) Engineering Genetic Tools to Control Individual Microbes and Microbiota without Antibiotic Resistance Genes at a Single Strain Level

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 [5-8]. This new technology has vast implications in designing strain-specific antimicrobials and combating the growing concern of antibiotic- and bacteriocide-resistant microbes.

[1] TS Moon, Probiotic and microbiota engineering for practical applications, Current Opinion in Food Science. https://doi.org/10.1016/j.cofs.2024.101130 (2024)

[2] Y Ma, A Manna and TS Moon. Advances in engineering genetic circuits for microbial biocontainment. Current Opinion in Systems Biology. doi.org/10.1016/j.coisb.2023.100483 (2023)

[3] AG Rottinghaus, A Ferreiro, SRS Fishbein, G Dantas and TS Moon. Genetically stable CRISPR-based kill switches for engineered microbes. Nature Communications. 13, 672 (2022)

[4] AG Rottinghaus, S. Vo and TS Moon. Computational design of CRISPR guide RNAs to enable strain-specific control of microbial consortia. PNAS. 120, e2213154120 (2023)

[5] TS Moon. SynMADE: Synthetic Microbiota Across Diverse Ecosystems. Trends in Biotechnology. 40, 1405-1414 (2022)

[6] TS Moon. EBRC: Enhancing Bioeconomy through Research and Communication. New Biotechnology. 78, 150–152 (2023)

[7] TS Moon. SynHEAL: Synthesis of Health Equity, Advancement, and Leadership. ACS Synth. Biol. 12, 1583–1585 (2023)

[8] MB Amrofell, S Rengarajan, S Vo, ESR Tovar, L LoBello, G Dantas and TS Moon. Engineering E. coli strains using antibiotic resistance gene-free plasmids. Cell Reports Methods. https://www.cell.com/cell-reports-methods/fulltext/S2667-2375(23)00348-X (2024)