Kinetic basis for Cas12a off-target discrimination.

CRISPR-Cas12a proteins are RNA-guided endonucleases classified as type V-A effectors that function similarly to Cas9, but possess distinct biochemical features. Previous studies have reported that compared to Cas9, Cas12a exhibits reduced off-target activity, yet the mechanistic origin of this high specificity remains unclear. In this study, we used singlemolecule fluorescence assays to investigate the kinetic basis for the reduced off-target effects of Cas12a. Introducing double mismatches at various positions within the target DNA enabled systematic analysis of the off-target effects on individual reaction steps in the Cas12a-mediated DNA cleavage reaction: seeding, stable R-loop formation, and DNA cleavage. Our results show that mismatches within a 17 bp PAM-proximal seed region significantly impair stable R-loop formation and subsequent cleavage, whereas mismatches in the PAM-distal region exert minimal or negligible effects. These results suggest that the low off-target tolerance of Cas12a and the resulting high on-target selectivity arise from the high sensitivity of the R-loop formation rate to DNA mismatches in the PAM-proximal region, which strongly correlates with cleavage efficiency. This work establishes R-loop formation as a conformational checkpoint for specific target cleavage, and provides a mechanistic framework to improve the fidelity of genome editing. [BMB Reports 2025; 58(8): 364-368].