Functionalizing conjugative systems to deliver CRISPR nucleases for targeted bacterial killing
Abstract
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.