Torsional Constraints of DNA Substrates Impact Cas9 Cleavage.

To examine the effect of the torsional constraints imposed on DNA substrates on Cas9 cleavage, we prepared con-strained DNA substrates using a DNA origami frame. By fixing the dsDNA at the connectors of the DNA frame, we created four different dsDNA substrates containing torsion-ally constrained or relaxed strands. We quantified the cleav-age of constrained and relaxed substrates by Cas9 with quantitative PCR. Moreover, we observed the Cas9/sgRNA complex bound to the DNA substrates and characterized the dissociation of the complex with high-speed AFM. The results revealed that the constrained non-target strand re-duced the cleavage efficiency of Cas9 drastically, whereas torsional constraints on the target strand had little effect on the cleavage. The present study suggests that highly ordered and constrained DNA structures could be obstacles for Cas9 and additionally provides insights in Cas9 dissociation at a single molecule level.