In Vitro Cas9 Cleavage Assay

The wtSaCas9/SaCas9 mutant proteins (~100 nM) were first mixed with sgRNA (~200 nM) in the reaction buffer (20 mM Hepes, 100 mM KCl, 1 mM DTT, 0.5 mM EDTA, 2 mM MgCl₂, 5% glycerol, pH 7.5) and incubated at 37 °C for 30 min. Then, the substrate DNA (~40 nM) was added to the Cas9-sgRNA solution and incubated at 37 °C for 1 h. Finally, the cleavage products were detected by gel electrophoresis on 1% agarose gel stained with GeneGreen nucleic acid dye (Tiangen Biotech Co. Ltd., Beijing, China). The activity was quantified using ImageJ, with the percentage of the cleavage rate being $100\times \left(1-\sqrt{1-fraction cleavaged}\right)$ [39 (link)].