Upon binding the crRNA:tracrRNA guide, the two structural lobes of Cas9 reorient such that the two nucleic acid binding clefts face each other. This generates a central DNA binding channel, which allows access to dsDNA. Target DNA binding in the central channel and PAM-dependent R-loop formation result in a further structural rearrangement. Here, the nuclease domain lobe undergoes further rotation relative to the α-helical lobe, fully enclosing the DNA target, and the two nuclease domains engage both DNA strands for cleavage.