Purification and Labeling of Cas9 Variants

WT Cas9, dCas9 (D10A/H840A), Cas9_HNH-1 (C80S/S355C/C574S/S867C), dCas9_HNH-1 (D10A/C80S/S355C/C574S/H840A/S867C), and Cas9_HNH-2 (C80S/C574S/S867C/N1054C) were purified as described (12 (link)). Dye-labeled Cas9 was prepared as previously described (12 (link)), with the following modifications: Labeling reactions contained 10 μM Cas9, 200 μM Cy3-maleimide, and 400 μM Cy5-maleimide (GE Healthcare). For steady-state measurements, regular cyanine dyes (GE Healthcare) were used. Free Cy3- and Cy5-maleimeide dyes were separated from labeled Cas9 through Micro Bio-Spin 6 columns (Bio-Rad) that were buffer-exchanged into Cas9 gel filtration buffer [20 mM tris-HCl (pH 7.5), 200 mM KCl, 5% glycerol, and 1 mM tris(2-carboxyethyl)phosphine (TCEP)]. To enhance the photostability in dynamic FRET measurements, Cas9 was labeled with Cy3 and Cy5 derivatives, LD550-MAL and LD650-MAL (Lumidyne Technologies). Free dyes were separated from labeled Cas9 by gel filtration on a Superdex 200 Increase 10/300 GL column [20 mM tris-HCl (pH 7.5), 200 mM KCl, 5% glycerol, and 1 mM TCEP].