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

Thomas A Hamilton,Bogumil J Karas,Dalton T Ham,Peter C Bartlett,Gregory M Pellegrino,Jasmine A Therrien,David R Edgell,Gregory B Gloor

doi:10.1038/s41467-019-12448-3

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.

Highlights

The selective regulation of bacteria in complex microbial populations is key to controlling pathogenic bacteria
We develop a cis-conjugative system where the plasmid encodes both the conjugative machinery and CRISPR nuclease[33], as opposed to previously tested trans setups where the conjugative machinery and nuclease were encoded on separate DNA molecules[20] (Fig. 1)
The pNuc plasmid encoded the TevSpCas[9] nuclease (I-TevI nuclease domain fused to Streptococcus pyogenes Cas933) controlled by an arabinose-inducible pBAD promoter[35], and a single-guide RNA cassette driven by a constitutive promoter derived from the tetracycline resistance gene into which we cloned oligonucleotides corresponding to predicted target sites in the S. enterica genome (Fig. 1b, Supplementary Data 1)

Summary

Introduction

The selective regulation of bacteria in complex microbial populations is key to controlling pathogenic bacteria. 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. A central problem is the lack of specific tools to selectively control pathogenic species, or to otherwise alter the composition of the human microbiome and other microbial communities Traditional methods such as antibiotic treatment suffer from a number of limitations that preclude selective control in a defined and efficient manner, and are becoming less effective because of overuse and the development of multidrug resistant bacteria[9]. The pNuc plasmid encoded the TevSpCas[9] nuclease (I-TevI nuclease domain fused to Streptococcus pyogenes Cas933) controlled by an arabinose-inducible pBAD promoter[35], and a single-guide RNA (sgRNA) cassette driven by a constitutive promoter derived from the tetracycline resistance gene (pTet) into which we cloned oligonucleotides corresponding to predicted target sites in the S. enterica genome (Fig. 1b, Supplementary Data 1). The pNuc-trans setup mimics the plasmids used in previous studies that examined conjugative delivery of CRISPR nucleases in an E. coli donor/recipient system[20,21,22]

Methods

Results

Conclusion