Genetic Markers Remain Detectable in Genetically Engineered Microbes Biocontained with a CRISPR Kill Switch

Area of Science:

• Environmental microbiology
• Synthetic biology
• Molecular biology

Background:

• Biocontainment strategies are crucial for genetically engineered microbes (GEMs) in environmental applications.
• CRISPR-Cas9 kill switches aim to prevent unintended proliferation of GEMs.
• Residual GEM DNA post-biocontainment presents environmental monitoring challenges.

Purpose of the Study:

• To assess the effectiveness of a CRISPR-Cas9 kill switch in preventing GEM proliferation and DNA release.
• To investigate the fate of GEM DNA after biocontainment using a CRISPR-Cas9 system.
• To evaluate the environmental persistence and detectability of GEM DNA.

Main Methods:

• Utilized a model Escherichia coli GEM with a CRISPR-Cas9 kill switch.
• Quantified GEM escape rates using colony forming units (cfu) and CRISPR-targeted gene abundances.
• Assessed DNA integrity and DNase resistance within 1 hour and over multiple days in different environments.

Main Results:

• CRISPR-Cas9 kill switch significantly reduced viable GEMs (cfu) but showed high target gene abundance, indicating intact DNA.
• High escape rates (10^-1.6 to 10^-1.0) were observed via gene abundance, contrasting with low cfu rates (10^-6.2).
• GEM DNA remained largely intact and DNase-resistant within cells for at least 1 hour, degrading over days in river water.

Conclusions:

• CRISPR-Cas9 kill switches effectively inhibit GEM growth but do not eliminate detectable DNA.
• Intact GEM DNA persists after biocontainment, posing risks and complicating monitoring.
• Further research is needed to understand the full impact of biocontainment on GEMs and their DNA to mitigate environmental risks.