The selective regulation of bacteria in complex microbial populations is key to controlling pathogenic bacteria. CRISPR nucleases can be programmed to kill bacteria, but require an efficient and broad-host range delivery system to be effective. Here, using an Escherichia coli and Salmonella enterica co-culture system, we show that plasmids based on the IncP RK2 conjugative system can be used as delivery vectors for a TevSpCas9 dual nuclease. Notably, a cis-acting plasmid that encodes the conjugation and CRISPR machinery conjugates from E. coli to S. enterica with high frequency compared to a trans system that separates conjugation and CRISPR machinery. In culture conditions that enhance cell-to-cell contact, conjugation rates approach 100% with the cis-acting plasmid. Targeting of single or multiplexed sgRNAs to non-essential genes results in high S. enterica killing efficiencies. Our data highlight the potential of cis-acting conjugative plasmids as a delivery system for CRISPR nucleases or other microbial-altering agents for targeted bacterial killing.

The ability to restrict gene expression to a relevant bacterial species in a complex microbiome is an unsolved problem. In the context of the human microbiome, one desirable target metabolic activity are glucuronide-utilization enzymes (GUS) that are implicated in the toxic re-activation of glucuronidated compounds in the human gastrointestinal (GI) tract, including the chemotherapeutic drug irinotecan. Here, we take advantage of the variable distribution of GUS enzymes in bacteria as a means to distinguish between bacteria with GUS activity, and re-purpose the glucuronide-responsive GusR transcription factor as a biosensor to regulate dCas9 expression in response to glucuronide inducers. We fused the Escherichia coli gusA regulatory region to the dCas9 gene to create pGreg-dCas9, and showed that dCas9 expression is induced by glucuronides, but not other carbon sources. When conjugated from E. coli to Gammaproteobacteria derived from human stool, dCas9 expression from pGreg-dCas9 was restricted to GUS-positive bacteria. dCas9-sgRNAs targeted to gusA specifically down-regulated gus operon transcription in Gammaproteobacteria, with a resulting ∼100-fold decrease in GusA activity. Our data outline a general strategy to re-purpose bacterial transcription factors responsive to exogenous metabolites for precise ligand-dependent expression of genetic tools such as dCas9 in diverse bacterial species.