Exogenous PAM richer resonator effect unlocks CRISPR/Cas12a biosensor-in-microdroplet for ultrasensitive and...

Accurate, sensitive, and multiplexed DNA detection is crucial for food safety. We describe a counterintuitive phenomenon in which the mere addition of exogenous protospacer adjacent motif richer double-stranded DNA (ePAM-R-dsDNA) boosts the trans-cleavage activity of Lachnospiraceae bacterium Cas12a (LbCas12a) for PAM-lacking dsDNA targets in microdroplets. We call this the exogenous PAM richer resonator effect. Leveraging this, we present a biosensor-in-microdroplet, named CC-Drop-OPT (CRISPR/Cas12a microDroplet Overcoming PAM restriction on Targets), breaking the PAM dependency and highly enhancing (10⁴ times) the sensitivity for PAM-lacking dsDNA targets. CC-Drop-OPT demonstrates a wide detection range and a low limit of detection of 10 CFU/ml for foodborne pathogens without DNA preamplification; it is also capable of detecting food authenticity and genetically modified foodstuffs. CC-Drop-OPT supports digital, multiplexed, and background-free identification of three different foodborne pathogens. CC-Drop-OPT introduces a molecular strategy for unamplified DNA detection in food safety and beyond.