Extended Data Figure 1: Biochemical preparation and DNA cleavage activity of dye-labelled Cas9.
From: Conformational control of DNA target cleavage by CRISPR–Cas9
a, Size-exclusion chromatograms of Cy3/Cy5-labelling reactions with cysteine-free Cas9 (C80S/C574S) or the two double-cysteine Cas9 variants used to generate Cas9hinge and Cas9HNH-1. Reactions contained 10 μM Cas9 and 200 μM Cy3- and Cy5-maleimide, and were separated on a Superdex 200 10/300 column (GE Healthcare). Cysteine-free Cas9 was unreactive. b, Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of unlabelled and dye-labelled Cas9 variants. The gel was scanned for Cy3 and Cy5 fluorescence (right) before being stained with Coomassie blue (left). For gel source data, see Supplementary Fig. 1. c, Representative radiolabelled DNA cleavage assay with wild-type (WT) Cas9 and doubly labelled Cas9 variants used in this study, resolved by denaturing PAGE (left); quantified data and exponential fits are shown on the right. S, substrate; NT, cleaved non-target strand; T, cleaved target strand. Error bars, s.d.; n = 3.
