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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.

Efficient inter-species conjugative transfer of a CRISPR nuclease for targeted bacterial killing