In 2021, an estimated 4.71 million deaths were associated with bacterial AMR, including 1.14 million directly attributable deaths. Without urgent intervention, this burden will escalate dramatically.

By 2050, forecasts indicate AMR could cause 1.91 million attributable deaths and 8.22 million associated deaths annually, with projections suggesting a cumulative toll of up to 40 million lives lost. This crisis demands a paradigm shift in both diagnostics and therapeutics.

The system uses the AsCas12a enzyme, programmed with target-specific guide RNAs from our GHOST software, to identify unique pathogen DNA. Upon binding, its collateral cleavage activity is unlocked, which in turn cleaves an inhibitory aptamer bound to β-lactamase. This reactivates the enzyme, enabling it to hydrolyze the chromogenic substrate nitrocefin, producing a visible color change from yellow to red.

The Multiple Drop Design

This design is for parallel, multiplexed detection using a simple drop-based format. The device consists of a paper with multiple unique immobilised Cas12a probes and a cover featuring aligned drop points. Samples are added directly through these openings, followed by the chromogenic reagent. Each drop point corresponds to a distinct probe area, allowing simultaneous testing of multiple targets.

Our therapeutic agents are designed to be highly specific, targeting only the pathogenic bacteria and leaving the beneficial microbiome unharmed. This approach minimizes side effects and reduces the evolutionary pressure that leads to resistance.

The modular nature of our system allows for rapid adaptation to new threats, making it a sustainable and long-term solution to the growing crisis of antimicrobial resistance.

Safe Delivery & Enhanced Killing

A key advantage of using Cas12a is the ability to multiplex, programming it with multiple targets to target a broad range of bacterial species. We coupled this system with safe, acellular delivery systems like Minicells and bacterial Extracellular Vesicles (bEVs), which transport our plasmid into microbiome environments such as the gut, skin, and lung without the risks associated with live donor bacteria.

However, relying solely on Cas12a-induced DNA breaks is not enough, as bacteria can use repair pathways. To circumvent this, pCASPER also expresses DNA-repair inhibitors, preventing bacteria from fixing the damage and ensuring cell death.

Ensuring Plasmid Stability

Plasmids must compete to establish themselves. To ensure our therapeutic plasmid can stably replicate, we designed pCASPER with two broad-range replication origins. Additionally, we introduced a specialised 'Anti-defence island' which expresses proteins that inhibit bacterial defenses against our plasmid.

Combining all of these components gives us our modular therapeutic plasmid, pCASPER. It can be easily adapted to swap Cas12a targets, allowing us to target a single specific strain or an entire family of pathogenic bacteria.