PAM-Free Programmable Enzymatic Cascade Design Enabling Highly Sensitive CRISPR/Cas12a Lateral Flow Assay for Foodborne Pathogen Detection

Abstract CRISPR/Cas12a-assisted nucleic acid assays have shown great potential for rapid pathogen detection. However, their implementation in lateral flow assays remains constrained by strict protospacer adjacent motif (PAM) requirements and low-contrast, signal-off readouts. To effectively address these limitations, we present a new PAM-free programmable enzymatic cascade-enabled CRISPR/Cas12a lateral flow assay (PAEC-LFA) for highly sensitive foodborne pathogen detection. In this strategy, target amplification products are enzymatically digested to generate programmable single-stranded DNA activators, enabling PAM-independent Cas12a activation and flexible target recognition. Upon activation, Cas12a exhibits efficient trans-cleavage activity, which subsequently triggers terminal deoxynucleotidyl transferase (TdT)-mediated extension, forming an enzymatic cascade that amplifies signal output and generates a high-contrast signal-on readout on lateral flow assays. Integrating RPA and enzymatic digestion with PAM-free Cas12a-TdT signal amplification into two sequential one-pot modules simplifies the workflow, minimizes contamination risk, and maintains effective amplification performance. Under optimized conditions, PAEC-LFA achieved detection limits of 1.05 CFU/mL for Salmonella and Staphylococcus aureus, and 3.13 CFU/mL for Pseudomonas aeruginosa. Visual detection limits in spiked food samples reached 10 CFU/mL for all three pathogens. This work establishes a generalizable PAM-free enzymatic cascade framework for highly sensitive CRISPR/Cas12a lateral flow assays, advancing practical pathogen detection in food safety applications.