In the past few decades, metagenomics has allowed researchers to study the role of the microbial community living in the intestinal tract, the gut microbiota, in human health. The gut microbiome, the collection of genes belonging to the gut microbiota, has since been linked to a variety of physiological processes. When gut community structure is disturbed, a state known as “dysbiosis”, it can contribute to the development of diseases, such as cardiovascular disease, colorectal cancer, and type 2 diabetes, suggesting that a modulatory role for the microbiome in disease is pervasive. Therefore, manipulation of the microbiome, otherwise known as ‘microbiome engineering’, has risen as a potential therapeutic option. Microbiome engineering approaches can vary in their modulation, ranging from the addition of a single molecule to the community to a total microbiome transplantation. However, it is difficult to determine the mechanism by which methods that cause community-wide changes in the gut modulate disease. Therefore, precise alterations that do not affect community structure in a significant manner are desirable for microbiome engineering. One rising potential option is genome editing. However, current microbiome-focused genome editing technologies are limited in either their delivery to a broad range of microbes or their efficiency in vivo. Therefore, in this dissertation, I discuss the development of broad-host plasmid tools for engineering microbial communities, using gut microbial species as a proof-of-concept. Chapter 2 discusses a computational analysis of sequences obtained from antibiotic-resistant bacteria to characterize broad host-range plasmids that could be used as delivery vectors for microbiome engineering purposes. The results of this analysis uncovered 22 potential broad-host candidate plasmid sequence types that could be further engineered for use as delivery vectors. Chapter 3 discusses the creation and testing of a broad-host base editing tool, termed SCALPEL (Specific CRISPR-guided Alteration of Loci for Pathofunction ELimination). However, we observed that expression of the base editing tool from a broad host-range plasmid led to a decrease in editing efficiency, suggesting a potential interaction between the vector and base editor. I conclude with a brief remark on the contributions of this work to further optimization of microbiome engineering technologies.

Loyola Irizarry HG, Brito IL. Characterizing conjugative plasmids from an antibiotic-resistant dataset for use as broad-host delivery vectors. Front Microbiol. 2023 Jun 14;14:1199640. doi: 10.3389/fmicb.2023.1199640. PMID: 37389338; PMCID: PMC10301749.