Associate toxin-antitoxin with CRISPR-Cas to harness (ATTACH) engineered microbes

Area of Science:

• Synthetic Biology
• Microbial Engineering
• Genetic Engineering

Background:

• Robust biocontainment is crucial for safe engineered microbe applications.
• Current biocontainment methods face challenges with genetic instability and complex construction.

Purpose of the Study:

• To develop a stable and stringent CRISPR-Cas-based kill switch for engineered microbes.
• To improve biocontainment strategies using a novel toxin-antitoxin association.

Main Methods:

• Developed the ATTACH system, integrating a CRISPR-repressed toxin-antitoxin (CreTA) module with CRISPR-Cas.
• Engineered inducible promoters for Cas3 nuclease and guide RNA expression.
• Created a single-plasmid, antibiotic-independent ATTACH device.

Main Results:

• The ATTACH system demonstrated improved genetic stability and stringency of the suicidal program.
• Achieved robust and stringent containment of a microbial chassis in a murine gut model.
• Observed negligible impacts on microbial growth and product formation (lycopene) during fermentation.

Conclusions:

• The CreTA module effectively stabilizes CRISPR-based kill switches.
• ATTACH represents a portable and reliable biocontainment tool for engineered microbes.
• This approach advances the safety and applicability of synthetic biology.