Fluorescence Encoding-Enabled Multiplexed Droplet Digital Recombinase Polymerase Amplification-CRISPR/Cas12a Assay for Rapid Screening of Lower Respiratory Tract Infection-Associated Bacterial Pathogens

Digital RPA-CRISPR/Cas12a assays enable accurate nucleic acid quantification; however, their multiplexing capacity is inherently limited by signal confounding arising from the promiscuous trans-cleavage activity of Cas12a effectors. To overcome this limitation, we developed a fluorescence encoding-enabled multiplexed droplet digital RPA-CRISPR/Cas12a (FLEMD-CRISPR) assay that integrates combinatorial fluorescence encoding, deterministic droplet pairing within a calabash-shaped microwell array, and a machine learning-assisted decoding pipeline. In FLEMD-CRISPR, small-volume fluorescence-encoded CRISPR/Cas12a droplets, each assigned a unique spectral signature corresponding to a specific crRNA, are one-to-one paired with large-volume RPA droplets. Following RPA amplification and droplet merging, Cas12a-mediated reporter cleavage occurs only in droplets containing target amplicons. The decoding pipeline then localizes droplets, classifies their colors to assign crRNA identities, and finally detects positive signals, enabling high-throughput, hands-off analysis of the entire droplet array. FLEMD-CRISPR enabled simultaneous quantification of nucleic acid targets from eight major lower respiratory tract infection-associated bacterial pathogens, showing strong linear correlation (R2 = 0.9987–0.9998) and consistent detection efficiency (slopes = 0.9849–1.0198) relative to single-plex digital PCR. The complete workflow, from sample preparation to final readout, can be completed within 2 h. Clinical validation using 30 bronchoalveolar lavage fluid samples showed that the bacterial detection outcomes of FLEMD-CRISPR were highly concordant with standard-of-care culture, achieving 100% (20/20) positive percent agreement and 99.1% (218/220) negative percent agreement. By enabling panel reconfiguration through simple modifications of the droplet encoding library, FLEMD-CRISPR provides a flexible platform for the development of highly multiplexed digital CRISPR assays.