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

Nat Commun. 2019 Oct 4;10(1):4544. doi: 10.1038/s41467-019-12448-3.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anti-Infective Agents / administration & dosage*
  • Biofilms / drug effects
  • CRISPR-Associated Protein 9 / administration & dosage*
  • CRISPR-Associated Protein 9 / genetics
  • Coculture Techniques
  • Conjugation, Genetic*
  • Drug Delivery Systems / methods*
  • Escherichia coli / drug effects
  • Escherichia coli / genetics
  • Gene Transfer Techniques*
  • Microbial Sensitivity Tests
  • Plasmids / genetics
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Saccharomyces cerevisiae
  • Salmonella enterica / drug effects
  • Salmonella enterica / genetics


  • Anti-Infective Agents
  • RNA, Guide, CRISPR-Cas Systems
  • CRISPR-Associated Protein 9

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