To experimentally test the ONERING system, we chose four targets: Salmonella enterica serovar Typhimurium, Helicobacter pylori, pESBL-12 E. coli, and E. coli carrying regulatory elements and target sequences from Vibrio cholerae (the toxin-antitoxin system). After identifying the target sequences and strain-specifc promoters, we engineered the plasmid.

To investigate its efficiency, we allow the bacterial ONERING-bearer to conjugate with target strains expressing fuorescent markers, enabling us to trace and evaluate the rates of bacterial killing using fluorescence microscopy.

Additionally, to demonstrate that the ONERING plasmid is safe for use within a larger microbiome, we hope to soon conjugate it into non-pathogenic commensal bacteria and record any changes in the bacterial populations.

We also assess the stability and persistence of the ONERING plasmid in various environmental conditions to evaluate its potential for real-world applications. To determine whether it requires additional layers of control such as programmed or inducible plasmid self-destruction.

In our design, we emphasize an optimal pipeline that simplifes the generation of targets for any pathogenic strain. We propose that strain-specifc targeting of the plasmid can be achieved by exploiting virulence factors and pathogenicity islands present in bacteria, which are responsible for their pathogenicity. These sequences are typically distinct compared to non-pathogenic genes, reducing the off-taregt effects in non-target bacteria.

We explore a variety of potential targets that can be disrupted using Cas12a. First, Cas12a can create double-strand breaks in bacterial chromosomes, leading to the collapse of the replication fork, thereby inhibiting bacterial growth and causing imminent death. To avoid of-target efects, we focused on target sequences in genes associated with pathogenicity, supported by inter-species sequence comparisons. Second, Cas12a can target genes such as betalactamase, which are responsible for antibiotic resistance. Although these genes are commonly found in plasmids rather than the nucleoid, their disruption restores susceptibility to common antibiotics. Finally, we can also disrupt toxin-antitoxin systems in pathogens by cleaving genes encoding antitoxins, resulting in cell death.

We integrate target sequences into the CRISPR array responsible for directing AsCas12a to specific sequences in the genomes of pathogens – all in one high-copy conjugative plasmid (ONERING), making it capable of sequence-specifc targeting and bacterial killing.