A novel photo-controlled CRISPR-Cas12a system based on split crRNA for one-pot detection of multiple viral aerosols

CRISPR-Cas12 technology has revolutionized the field of nucleic acid detection. Although aerosols are a critical transmission route for epidemic viruses, most current CRISPR-based detection technologies focus on liquid samples. Direct detection of viruses in aerosols remains uncommon. In this study, we designed a novel photo-controlled CRISPR-Cas12a detection system based on split crRNA and integrated it with reverse transcription-recombinase polymerase amplification (RT-RPA) technology to develop a “one-pot” detection protocol for viruses in aerosols. By integrating the processes of viral aerosol capture and nucleic acid enrichment, the system can enable the process from aerosol sample input to detection signal output within 1 h. The modular photo-activated CRISPR-Cas12a system constructed in this study, based on split crRNA, addresses the issues in traditional photo-controlled strategies, such as the need to customize blocking sequences for different targets and the cumbersome system optimization process. It provides a universal molecular tool for the detection of various viruses, significantly reducing design costs and enables the simultaneous detection and early warning of H7N9 influenza virus and SARS-CoV-2 in aerosols. This study marks the first application of photo-controlled CRISPR technology in the field of viral aerosol detection. It establishes an integrated platform for viral aerosol detection, enabling both fluorescent detection and LFA-based visual detection of low-abundance viruses in aerosols. The platform provides an accurate, sensitive, rapid, and easy-to-operate universal technical solution for diverse detection needs, and holds practical value for the prevention and control of viral transmission.