For the bulk DNA cleavage assay, 1 μM wtCas9 was first complexed with sgRNA at a 1:5 ratio at room temperature for 10 min in reaction buffer. The complexed wtCas9 (100 nM) was incubated with annealed Cy5-DNA (1 nM) for the indicated time at room temperature (table S2). The reactions were stopped by adding loading dye containing 96% formamide and 40 mM EDTA and heating them to 95°C for 5 min, followed by slow cooling, and they were analyzed by 12% denaturing polyacrylamide gel electrophoresis and phosphorimaging.

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