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

Abstract

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 singlemolecule 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.

Probing the stability of the SpCas9–DNA complex after cleavage

“…Possibly, this not only inactivates the invader DNA but may generally slow down transcription and/or replication of the foreign DNA. Notably, a post-cleavage stabilization of the R-loop and the trapping of supercoiling has recently been observed for the Type V-A CRISPR–Cas effector complex Cas12a ( 48 ). Contrarily, a Cas9 variant from Staphylococcus aureus has been observed to be a multiple turnover enzyme indicating that diverse strategies may be used to impede foreign DNA invasion by regulating the post-cleavage behavior of the effector complexes ( 49 ).…”