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

Biocontainment strategies, such as kill switches, have been developed to avoid the unintended proliferation of genetically engineered microbes (GEMs) intended for open-release environmental applications. However, the presence of GEM DNA after successful biocontainment presents new environmental risks and challenges for monitoring. In this study, we investigated whether biocontainment using a CRISPR-Cas9 kill switch, which causes double-strand breaks in target genes essential for GEM growth, could resolve this challenge in a model <i>Escherichia coli</i> GEM. Surprisingly, the escape rates of the GEM as determined by CRISPR-targeted gene abundances were as high as 10^-1.6 to 10^-1.0 in LB media, despite the escape rates measured by colony forming units (cfu) being only 10^-6.2 under the same condition. This discrepancy suggested that the CRISPR-Cas9 kill switch prevents colony growth while still leaving a large fraction of target genes intact for detection by molecular methods. Within 1 h after biocontainment, these target genes remained predominantly inside an intact cell membrane and were resistant to degradation by DNase, though degradation was observed in river water over multiple days. Overall, a detailed understanding of the impact of the biocontainment mechanism on both the GEM and its DNA is needed to minimize unintended environmental risks.