Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article


Doctor of Philosophy




Edgell, David R.

2nd Supervisor

Gloor, Gregory B.



The interactions between humans and microbes are intimately important to human health, with both commensal and pathogenic bacteria affecting homeostasis and disease. Increasing concern over antibiotic resistance in bacterial pathogens represents a significant threat to human health, and use of traditional antibiotics to treat infections can be detrimental to commensal bacteria as well as pathogens, demonstrating a need for more specific antibacterial reagents. RNA-guided CRISPR nucleases, which can target and cleave genomes of interest, are a potential tool for specific bacterial targeting. A key limitation to the use of CRISPR antimicrobials is effective and robust delivery to the target bacteria. My thesis addresses this key issue by functionalizing conjugative systems to deliver a CRISPR nuclease for bacterial killing. First, a plasmid containing an arabinose-inducible TevCas9 nuclease that is mobilizable in-trans by an RK2-based conjugative system was constructed. Inclusion of the RK2-based conjugative system in-cis on the same plasmid was shown to greatly increase the conjugation frequency over time. Furthermore, when conjugating in liquid we observed that providing glass beads to increase surface area for biofilm development and cell-cell contact significantly improved conjugation frequency. Crucially, conjugated TevCas9 was able to kill Salmonella enterica with up to 99% efficiency, depending on the sgRNA provided. Next, to explore the importance of conjugative systems for delivery, a database containing thousands of conjugative systems identified from gut metagenomic data was constructed. From this database, a conjugative system of 54 kb native to the Citrobacter genus was constructed de novo. This conjugative plasmid, p20298-15a, showed 30-fold increased conjugation frequency to Citrobacter rodentium than to Escherichia coli, and was capable of conjugation to several additional Citrobacter strains. The p20298-15a plasmid was then functionalized to clone the arabinose-inducible CRISPR-TevCas9 system, which was able to target and kill C. rodentium. Importantly, the construction and engineering of p20298 shows that large genetic systems found in metagenomic data sets can be synthesized and functionalized. Overall, this thesis demonstrates the effective use of conjugative systems as a delivery mechanism for CRISPR-based antimicrobials for the targeted killing of bacteria.

Summary for Lay Audience

Bacteria are present in ecosystems throughout the world and are intimately linked to human health. Many bacteria in the human body are beneficial and required for health, whereas other bacteria are pathogenic. To conventionally treat infections of pathogenic bacteria, patients are typically given antibiotics, however, a growing concern in medicine is the rise of antibiotic resistant bacteria. Furthermore, these same antibiotics can have a detrimental impact on healthy, commensal bacteria. To continue to treat pathogenic bacteria, novel methods of specific bacteria killing are required. To address this problem, this thesis utilizes two key technologies. The first is bacterial conjugation, which is a naturally occurring mechanism that bacteria use to share genetic information. The second is CRISPR (clustered regularly interspaced short palindromic repeats) technology, which allows for the targeting of genomes at specific DNA sequences. Importantly, CRISPR nucleases are proteins that are guided towards the bacterial genome and cleave the DNA leading to cell death. First a plasmid was constructed that contained a CRISPR nuclease that could specifically target bacterial DNA for cleavage. This plasmid was capable of being mobilized from a donor bacterial strain to a recipient bacterial strain. Importantly, including the conjugative machinery on the same plasmid allowed for better spread to recipient bacteria. A variety of plasmids were then constructed to target Salmonella enterica and were capable of killing up to 99% of the recipient cells. Next, new conjugative systems were identified computationally in a gut metagenomic data set. These data represent the cumulative microbial DNA that is found in the human gut, from which thousands of different conjugative systems were identified. I proceeded to construct one of these conjugative systems synthetically to target a bacterium that the system was native to and showed that it could be transferred between bacteria. The CRISPR nuclease was then added to the new functional plasmid, and it was delivered to kill bacteria from the genus the original conjugative system was identified from. Overall, this thesis describes an approach for the delivery of CRISPR nucleases via bacterial conjugation to target and kill specific bacteria of interest.