Figure 6.

Kinetics of Cas3-mediated cleavage of plasmid DNA bound by Cascade. (A) Target plasmids containing indicated g8 protospacer variants were preincubated with Cascade–g8–crRNA. DNA cleavage reaction was initiated by the addition of Cas3. At indicated times after Cas3 addition reactions were terminated. Products were separated by agarose gel electrophoresis. Sketches next to gel images illustrate positions of the DNA topoisomers (supercoiled, nicked and linear plasmid). (B) Kinetics of DNA cleavage quantified from the disappearance of the supercoiled DNA species in the agarose gels. Error bars represent standard deviations from two to three repeat measurements. (C and D) Initial cleavage rate and cleavage efficiency obtained from the fits of the data shown in B.