Cas9 RNP Preparation and Cell Lysis

The Cas9 RNP was prepared according the protocols described previously (42 (link)). The Cas9 RNP was made immediately before the experiment. To make the Cas9 RNP, purified Cas9 protein was incubated with sgRNA at a 1:1.2 molar ratio in 20 μM Hepes at pH 7.5, 150 mM KCl, 1 mM MgCl₂, 10% glycerol, and 1 mM TCEP at 37°C for 10 min. Cells were incubated at 37°C for 24 to 48 hours after Cas9 RNP delivery. To extract the genomic DNA, the cells were lysed by 20 to 100 μl of QuickExtract solution (Epicentre) at 65°C for 20 min and then at 95°C for 20 min and were stored at −20°C. The concentration of genomic DNA was measured by NanoDrop (Thermo Scientific).