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Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Edgell, David R.

2nd Supervisor

Gloor, Gregory B.

Co-Supervisor

Abstract

Changes to the human microbiome’s composition and metabolome are associated with numerous diseases and alterations to xenobiotic metabolism. As such, targeting the human microbiome is an increasingly popular option for therapeutic interventions. However, traditional therapies that target the microbiome such as antibiotics lack specificity, which can affect the beneficial species of the microbiome and cause adverse health outcomes such as the rise of antimicrobial-resistant bacteria. Therefore, the research and development of specific, targeted antimicrobial therapies is crucial to effectively treating microbiome dysbioses.
CRISPR and CRISPRi provide easily modifiable, RNA-guided mechanisms mediated by the Cas9 or dCas9 enzymes to induce sequence-specific bacterial killing or transcriptional regulation, respectively. However, their inherent toxicity in bacteria is an obstacle to utilizing them in complex bacterial ecosystems. Here, I demonstrate design considerations for effectively implementing CRISPR systems in clinically relevant bacterial species to effect either targeted cell death or transcriptional repression of harmful genes. First, I showed that plasmid-encoded nuclease-active Cas9 delivered by conjugation can effectively kill targeted bacteria, but different sgRNAs exhibit highly variable target killing efficiency and can induce inactivating plasmid mutations when in donor species. Next, I developed a plasmid with dCas9 expression regulated by the inducer of the clinically relevant glucuronide metabolic pathway intended for down-regulation. I demonstrate that this regulation system restricts dCas9 expression to bacteria with the glucuronide metabolic pathway and efficiently represses expression of the glucuronide metabolizer GusA when the plasmid is conjugated to relevant enteric bacteria. Finally, I show that overexpression of dCas9 negatively impacts plasmid maintenance in certain bacterial strains and that my glucuronide-regulated dCas9 plasmid is more resistant to dCas9-induced plasmid loss. The research presented here shows the necessity of precise CRISPR regulation and generally informs of a strategy to repurpose bacterial transcription factors for ligand-dependent expression of genetic tools in diverse bacterial species. I hope this work will one day assist the implementation of CRISPR-based microbiome therapies for specific, targeted treatment of microbiome-related diseases.

Summary for Lay Audience

Improved research technologies have recently expanded the knowledge of how the collection of microorganisms that live in the human body, called the human microbiome, can impact and affect human health. This has increased the demand for new therapies that target the human microbiome, as traditional antibiotics often act nonspecifically, which can kill beneficial microorganisms and has led to the rise of antimicrobial-resistant species. One potential solution is the CRISPR system, which uses a protein called Cas9 that can be targeted to specific sequences of bacterial DNA to induce cell death. A modified form of the protein called dCas9 can instead target specific DNA sequences to turn off harmful bacterial genes. CRISPR-based therapies could provide a specific and targeted method of controlling the human microbiome for the benefit of human health, but for successful implementation, research and development of strategies to limit their potential off-target toxicity is required.
Here, I develop CRISPR-based tools encoded on circular pieces of DNA called plasmids that can be delivered to bacteria to exert the desired therapeutic effect. First, I showed that plasmid-encoded Cas9 could be delivered to targeted bacteria and subsequently induce cell death. However, some target sequences resulted in mutations in the Cas9 plasmid, highlighting the need for strict regulation of the CRISPR system. Next, I used dCas9 to target and repress a harmful bacterial gene, gusA, which can cause toxicity in patients treated with the chemotherapeutic drug irinotecan. I developed a regulation system that limited dCas9 expression to bacterial species with the gusA gene and only expressed dCas9 when a molecule that turns on the gusA gene was present. Finally, I showed that this new regulation system improved plasmid integrity when dCas9 was turned on compared to other regulation systems. This research demonstrates the need for strict CRISPR regulation systems to avoid the problems associated with nonspecific therapies such as antibiotics and provides a framework for successfully implementing CRISPR-based antimicrobials in diverse bacterial species.

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

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