Abstract CRISPR-associated proteins such as Cas9 and Cas12a are programable RNA-guided nucleases that have emerged as powerful tools for genome manipulation and molecular diagnostics. However, these enzymes are prone to cleaving off-target sequences that contain mismatches between the RNA guide and DNA protospacer. In comparison to Cas9, Cas12a has demonstrated distinct sensitivity to protospacer-adjacent-motif (PAM) distal mismatches, and the molecular basis of Cas12a's enhanced target discrimination is of great interest. In this study, we investigated the mechanism of Cas12a target recognition using a combination of site-directed spin labeling, fluorescent spectroscopy, and enzyme kinetics. With a fully matched RNA guide, the data revealed an inherent equilibrium between a DNA unwound state and a DNA-paired duplex-like state. Experiments with off-target RNA guides and pre-nicked DNA substrates identified the PAM-distal DNA unwinding equilibrium as a mismatch sensing checkpoint prior to the first step of DNA cleavage. The finding sheds light on the distinct targeting mechanism of Cas12a and may better inform CRISPR based biotechnology developments.