A gate and clamp regulate sequential DNA strand cleavage by CRISPR-Cas12a

CRISPR-Cas12a has been widely used for genome editing and diagnostic applications, yet it is not fully understood how RNA-guided DNA recognition activates the sequential cleavage of the non-target strand (NTS) followed by the target strand (TS). Here we used single-molecule magnetic tweezers microscopy, ensemble gel-based assays and nanopore sequencing to explore the coupling of DNA unwinding and cleavage. In addition to dynamic R-loop formation, we also directly observed transient dsDNA unwinding downstream of the 20 bp DNA:RNA hybrid and, following NTS cleavage and prior to TS cleavage, formation of a hyperstable “clamped” Cas12a-DNA intermediate resistant to DNA twisting. Alanine substitution of a conserved aromatic amino acid “gate” in the REC2 domain that normally caps the heteroduplex produced more frequent and extended downstream DNA breathing, a longer-lived twist-resistant state, and a 16-fold faster rate of TS cleavage. We suggest that both breathing and clamping events, regulated by the gate and by NTS cleavage, deliver the unwound TS to the RuvC nuclease and result from previously described REC2 and NUC domain motions. ### Competing Interest Statement The authors have declared no competing interest.