Single-Molecule Characterization of CRISPR–Cas12a for Amplification-Free Genetic Testing

CRISPR-Cas-based genetic testing has gained considerable attention for rapid and accurate diagnosis of infectious and genetic diseases. Cas12a, an RNA-guided nuclease that targets DNA, has been widely applied for DNA detection; however, its detailed enzymatic properties have not been fully elucidated. Here, we performed a systematic single-molecule analysis of Cas12a using microchamber arrays to biophysically characterize its catalytic behavior including protospacer adjacent motif (PAM) specificity, mismatch tolerance, and reaction kinetics and to evaluate the feasibility and inherent challenges of amplification-free DNA detection. Cas12a exhibited high activation efficiency toward target DNA, demonstrating the feasibility of amplification-free DNA detection with a detection limit of 104 aM within 15 min. However, Cas12a also exhibited substantial nonspecific cross-reactivity with genome-length targets, particularly mammalian genomic DNA, identifying off-target activation as a major challenge for reliable diagnostic application. Together, these findings provide a quantitative biophysical characterization of Cas12a-based amplification-free DNA detection and highlight the need to improve its target specificity for future reliable application of Cas12a in amplification-free molecular diagnostics.