Figure 6.

(A) The HNH and RuvC time dependence experiments in Figure 5 were repeated over a shorter time scale. Cas9.gRNA was mixed simultaneously with DNA and Mg²⁺ to initiate the cleavage reaction. The scheme is shown above the gel image. (B) The plot of product formation as function of time was fit by a single-exponential equation to define the cleavage rates of 1 s⁻¹ and 0.2 s⁻¹ for the HNH and RuvC domains, respectively. (C) The experiments in (A) were repeated; however, Cas9.gRNA was preincubated with DNA for 30 min in the absence of Mg²⁺ followed by addition of Mg²⁺ to initiate the reaction. The scheme is shown above the gel image. (D) The plot of product formation as function of time was fit by a single-exponential equation to define the cleavage rates of 4.3 s⁻¹ and 3.5 s⁻¹ for the HNH and RuvC domains, respectively. These figures are adapted from Gong et al. 2018.