Researchers developed a CRISPR-based kill switch for E. coli (EcN) that induces cell death in response to the chemical inducer anhydrotetracycline (aTc). The kill switch utilizes a CRISPR/Cas9 approach, where Cas9 and guide RNAs (gRNAs) are expressed from separate plasmids using aTc-inducible promoters. The system was optimized through functional redundancy, where multiple copies of the Cas9 expression cassette were integrated into the genome. The kill switch was also modified to eliminate reliance on antibiotics for efficient killing by expressing an essential gene, infA, on the gRNA plasmid.
To improve the kill switch’s efficacy in vivo, the researchers knocked out key components of the SOS response, which reduces DNA mutagenesis. They also introduced intra-niche competition by co-gavaging the kill switch strain with a control strain. This approach led to almost complete eradication of the engineered microbe from mice. Additionally, a 2-input CRISPRks was developed, which responds to both aTc and reduced temperatures, providing a secondary layer of biocontainment. The kill switches were shown to have minimal impact on protein expression in EcN, making them suitable for biocontainment of engineered therapeutic and diagnostic microbes.