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

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 (protospacer adjacent motif) specificity, mismatch tolerance, and reaction kinetics and to evaluate the feasibility and inherent challenges of amplification-free DNA detection.