Fig. 1: Kinetic analysis of Cas9 programmed with truncated sgRNAs. | Nature Communications

Fig. 1: Kinetic analysis of Cas9 programmed with truncated sgRNAs.

From: Visualization of a multi-turnover Cas9 after product release

Fig. 1

a Schematic of Cas9 reaction cycle. b Diagram of the experimental setup for Cas9 turnover assays where plasmid DNA was added to a pre-formed Cas9 RNP in 5-fold molar excess. c Cas9 turnover assays with 20-nt, 15-nt, and 2 mm sgRNAs. 20 nM active Cas9 RNP was added to 100 nM plasmid substrate and product generation was plotted over time in minutes. d Cleavage rate constants (k2) derived from global fitting of the data shown in panel c. The cleavage rate constant for the 20-nt sample was locked at 60 min−1 as it was not captured in the time-scale used for the turnover assays and has been well-characterized in previous studies. e Dissociation rate constants (k3) determined for the 20-nt, 15-nt, and 2 mm sgRNA samples. Source data are provided as a Source Data file.

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