RESUMEN

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.

Asunto(s)

Antiinfecciosos/administración & dosificación , Proteína 9 Asociada a CRISPR/administración & dosificación , Conjugación Genética , Sistemas de Liberación de Medicamentos/métodos , Técnicas de Transferencia de Gen , Biopelículas/efectos de los fármacos , Proteína 9 Asociada a CRISPR/genética , Técnicas de Cocultivo , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Pruebas de Sensibilidad Microbiana , Plásmidos/genética , ARN Guía de Kinetoplastida/genética , Saccharomyces cerevisiae , Salmonella enterica/efectos de los fármacos , Salmonella enterica/genética