A Looming Global Health Threat

So how serious is the AMR crisis? Currently, up to 5 million people die annually from AMR-related diseases each year, increasing to a shocking projected 10 million deaths by 2050¹. To put that into perspective, AMR will cause more deaths than cancer or diabetes in the future.

Source: WHO¹, American Heart Association²

Evidently, alternative treatments for AMR and MDR infections need to be developed. Otherwise, we risk entering a post-antibiotic era where minor infections could once again become fatal, and routine surgeries and treatments could become exceedingly risky.

OneRing: A Novel Solution to AMR

Learning From Nature: Bacterial Conjugation and Defence Systems

Considering the challenges facing current antimicrobial strategies, we came up with an alternative solution utilising bacterial conjugation – a naturally occurring gene transfer mechanism in bacteria. Only bacteria with a certain factor - the F+ plasmid - can transfer genes to other bacteria. This F+ plasmid allows the donor bacteria to extend a tube (called the pili) towards the recipient bacteria. Once the two are linked, the genes can then be transferred through this “bridge” from donor to recipient.

Additionally, we took inspiration from part of bacteria’s defence system. CRISPR-Cas occurs as part of the bacterial adaptive immune system that targets and cleaves invading exogenous DNA, such as DNA from viral infections. Using synthetic biology, the guide RNA – a component that “tells” the Cas protein where to cut the DNA - can be adjusted using a trans-activating small RNA (tracrRNA). This allows Cas to act like a pair of programmable molecular scissors, cutting at almost any desired site. The CRISPR-Cas system is especially efficient for elimination of bacteria because it generates double-stranded breaks in the bacterial chromosome, causing cell death.

Repurposing Conjugation and CRISPR-Cas Systems for Targeted Bacterial Killing

In our system, we use a conjugative RP4 plasmid as a delivery system. A unique feature of the RP4 conjugation apparatus is that it is agnostic to cell type - it does not require a surface membrane protein for docking or membrane penetration. This guarantees a high conjugation rates for effective plasmid transfer, across both Gram positive and negative bacteria. This RP4 system is used to transfer the OneRing plasmid into the recipient (AMR resistant) bacteria.

OneRing contains two components – the CRISPR-Cas12a-encoding gene and a crRNA array. A key factor determining the success of this system is its specificity. We wouldn’t want CRISPR-Cas12a to be cutting up the genomes of all bacteria (some bacteria - such as those in our guts - are actually required for good health!). At the same time, we want the system to effectively kill AMR-resistant bacteria. As such, OneRing was designed with specificity in mind. Firstly, CRISPR-Cas12a itself has a higher fidelity than the more widely explored CRISPR-Cas9, reducing off-target effects. Secondly, CRIPSR-Cas12a makes cuts only at specific, programmable sites. Through proper programming of the gRNA array, only pathogenic bacteria genomes will be targeted by OneRing. In addition, Cas12a has the unique ability to process its own gRNA array, which means that the genes encoding the gRNA(s) and the Cas12a can be encoded by a single plasmid. This allows the OneRing system to be used to eliminate multiple pathogens or families of pathogens using this single plasmid.

One Powerful Ring: Targeted but Robust

The number of AMR bacteria is constantly on the rise, so AMR solutions need to be targeted, yet easily adaptable to target emerging AMR bacteria. OneRing utilises synthetic target arrays for Cas12a, which can be rationally designed, produced, and integrated into a modular Cas12a plasmid. Additionally, OneRing is designed to encode the origins of replications for both Gram positive and negative bacteria, further increasing its therapeutic potential. This makes OneRing a highly flexible and robust solution to AMR.

OneRing in Human Health and Beyond

Given its targeted yet robust nature, we envision that OneRing will find applications across many fields – human health and beyond.

With its current design, OneRing is best suited for addressing gut, skin and wound infections rather than internal bacterial infections. This is because its highly specific nature allows for targeted killing of the pathogenic bacteria even within complex and varied microbiomes. Further development of delivery systems that allow tissue or cell specific-targeting might enable the treatment of internal bacterial infections in future.

One crucial area beyond human health in which AMR therapeutics can be applied would be the agriculture and farming industry. Shockingly, 70% of antibiotics produced globally are used in farm animals. In these animals, antibiotics are mainly used to prevent post-weaning diarrhoea in pigs, respiratory diseases in poultry, and dry cow therapy. But why are antibiotics so widely used in livestock? Antibiotics not only improve the animals’ health but also increase the output of these animals – in the form of meat or animal products. Consequently, if antibiotics are phased out without an appropriate substitution, the supply of animal products will decrease significantly, leading to potential shortages and economic problems for major producers of these products. Furthermore, if livestock farming is intensified in response to these shortages, the climate change crisis will be further exacerbated by the increase in greenhouse gas emissions.

To address this issue, the OneRing system can be employed in livestock and companion animals. Specifically, it can be produced as a food supplement (similar to probiotics), and customised for each geographical region, farm, or even individual animal depending on the dominant strains of bacterial pathogens. This is easily achievable by adjusting the Cas12a target array in our modular plasmid construct.