Abstract

Genetically engineered microbes (GEMs) are poised to solve a myriad of societal challenges; among the many potential GEM applications, some include remediating environmental contaminants, increasing nutrient availability for crops, and sequestering carbon dioxide to mitigate climate change. While only a few GEM products have been approved for commercial use, advancements in genetic engineering are enabling the development of new and complex GEMs that are better suited for environmental applications. This recent increase in “scale, scope, and complexity” of GEMs has also coincided with a renewed interest in the promise of GEMs; researchers are actively working on strategies to support the scale-up of GEM deployment and policymakers are prioritizing processes that reduce burdensome regulation. Deployment of GEMs in applications that require open release of the microbe into the environment are contingent on our ability to minimize unintended risk. One tool to control GEM persistence is called biocontainment, which uses genetic engineering to render the GEM non-viable or in some instances, breakdown GEM DNA to prevent its horizontal gene transfer to native microbial communities – a prevalent hazard during the release of GEMs. One type of biocontainment utilizes CRISPR to cause double-strand breaks in the GEM DNA, in principle, preventing both GEM and DNA escape. Despite the potential for biocontainment to enable the safe deployment of GEMs in open-release applications, our understanding of the function of these strategies in the environment is limited. The first objective of this dissertation was to establish the historical and current state of GEM applications and regulations, specifically to perform an in-depth analysis of biocontainment strategies as they relate to the scale-up of GEM deployment in environmental systems. While the context in which biocontainment strategies are evaluated heavily influences its ability to function, prior assessments have only considered biocontainment success within controlled laboratory systems. Even when strategies are tested within environmental media, I found that GEM escape was higher and required a longer amount of time relative to laboratory media. Why escape rates were elevated within environmental conditions remains an open question that I explored in the next objective. The second objective of this dissertation was to investigate the effectiveness of a CRISPR kill switch in environmental media (i.e., surface waters), specifically by using culture-based methods to calculate escape rates, i.e., the ratio of the number of viable microbes when the kill switch is activated relative to those when the kill switch is not activated. I found that escape rates were higher by 3-4 orders of magnitude in all three surface waters tested relative to the LB media often used in prior biocontainment evaluations. I identified key environmental conditions (e.g., pH, nutrient levels) that potentially contributed to the elevated escape rates; these environmental conditions influenced critical functions of the kill switch such as the pH altering the chemical speciation of the compound used to activate the kill switch, reducing its uptake, and low nutrient levels likely hindering metabolic processes necessary for CRISPR cleavage. The third objective of this dissertation was to determine how the CRISPR kill switch, also explored in objective 2, influences the detection and persistence of the GEM DNA after cleavage. I found that escape rates calculated by CRISPR-targeted gene abundances using qPCR were at least 4 orders of magnitude higher relative to escape rates calculated from culture-based methods, indicating that the genes targeted by the kill switch remained detectable. Even after additional interventions such as isolating intracellular DNA, consolidating to a kill switch with only one target gene – as prior findings were performed with a kill switch targeting multiple genes – and removing a DNA repair gene, were unable to reduce abundances of the target genes shortly after biocontainment. Lastly, I observed DNA degradation in surface water over multiple days, suggesting that excess genes present after biocontainment may decrease within environmental conditions, potentially enabling more accurate detection of GEM viability with qPCR. In summary, this dissertation contributed to the understanding of biocontainment function in environmental systems to further support the scale-up of GEM deployment. Specifically, I detailed the state of open-release GEM applications, regulations, and biocontainment strategies to further identify oversights in prior biocontainment evaluations. I then utilized a CRISPR kill switch as a case analysis to assess biocontainment and its expected function in environmental systems; in doing so, I identified key environmental conditions that must be considered during the implementation of biocontained GEMs and determined the expected persistence of GEM DNA after biocontainment. Overall, my work will inform evidence-based decision making for regulatory agencies and will enable safe and effective GEM deployment.

Committee Chair

Kimberly Parker

Committee Members

Daniel Giammar; Natalie Farny; Yinjie Tang; Zhen (Jason) He

Degree

Doctor of Philosophy (PhD)

Author's Department

Energy, Environmental & Chemical Engineering

Author's School

McKelvey School of Engineering

Document Type

Dissertation

Date of Award

8-1-2026

Language

English (en)

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Available for download on Friday, July 30, 2027

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