Developing Regulated CRISPR Systems to Control Bacterial Microbiomes
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.