Multi-toolkit CRISPR/Cas platform for portable electrochemical biosensing in One Health settings

The CRISPER project aims to develop a portable diagnostic platform that integrates CRISPR/Cas technology integrated into a battery-powered electrochemical biosensor. The proposal is framed within the One Health approach, addressing the early detection of pathogens and biomarkers in human health, animal health, and water and food safety. The central idea is to provide a versatile system that enables DNA and RNA quantification directly at the point of need, without relying on central laboratories or complex instrumentation. The method combines magnetic sample preparation, nucleic acid amplification, CRISPR/Cas enzymatic treatment, and electrochemical readout within a streamlined, modular workflow. To generate the analytical signal, CRISPER employs isothermal preamplification techniques (LAMP and RPA) and PCR. Amplification is carried out in a handheld, battery-powered thermocycler. After amplification, the product is treated with CRISPR/Cas enzymes and the corresponding lyophilized reporting reagents in a single step and, finally, this mixture is introduced into a disposable cartridge coupled to an electrochemical biosensor for signal readout using a portable, battery-operated reader. The platform is organized into three complementary toolkits: a Cas9-based module for the detection of double- stranded DNA amplicons; a Cas12a module that exploits its collateral activity on single-stranded DNA probes; and a Cas13a module designed for RNA detection, including transcripts present in exosomes. In all cases, nuclease activity is converted into an ON/OFF electrical signal that is easy to interpret in field settings. The system will be validated in three representative One Health domains: detection of coliforms and Legionella in environmental water and food matrices; surveillance of avian respiratory viruses in poultry farms; and analysis of inflammatory transcripts in extracellular vesicles to support infection severity stratification in patients. Building on previous laboratory-scale projects, CRISPER moves toward a modular prototype with lyophilized reagents and autonomous operation, narrowing the gap between proof of concept and real-world field use. Supported by a strong network of academic, clinical, and industrial partners, the project aims to pave the way for future pathways for transfer, standardization, and adoption of portable CRISPR tools in decentralized diagnostic settings.