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

Thomas A. Hamilton, Gregory M. Pellegrino, Jasmine A. Therrien, Dalton T. Ham, Peter C. Bartlett, Bogumil J. Karas, Gregory B. Gloor () and David R. Edgell ()
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Thomas A. Hamilton: Schulich School of Medicine and Dentistry
Gregory M. Pellegrino: Schulich School of Medicine and Dentistry
Jasmine A. Therrien: Schulich School of Medicine and Dentistry
Dalton T. Ham: Schulich School of Medicine and Dentistry
Peter C. Bartlett: Schulich School of Medicine and Dentistry
Bogumil J. Karas: Schulich School of Medicine and Dentistry
Gregory B. Gloor: Schulich School of Medicine and Dentistry
David R. Edgell: Schulich School of Medicine and Dentistry

Nature Communications, 2019, vol. 10, issue 1, 1-9

Abstract: Abstract 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.

Date: 2019
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DOI: 10.1038/s41467-019-12448-3

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