EMSA for Cas9 and Cas12a Binding

Electrophoretic mobility shift assays with Cas9 and Cas12a were done using catalytically dead versions of the proteins, dCas9 and dCas12a (12 (link),37 (link)). 1 μM dCas9 and dCas12a were incubated with 4 μM sgRNA and crRNA respectively in binding buffer (20 mM HEPES, pH 7.5, 250 mM KCl, 2 mM MgCl₂, 0.01% Triton X-100, 0.1 mg/ml bovine serum albumin, 10% glycerol) for 10 min at 25°C as described in (38 (link)). A dilution series was made with 0.1, 0.3, 1, 3, 10, 30, 100 and 300 nM final protein concentration using binding buffer. 3 nM 5′ ³²P-radiolabeled target DNA was added in a 30 μl reaction and incubated for 10 minutes at 37°C. 10 μl 6× loading dye (Thermo Scientific) was added and the complete samples were loaded on a 12% native acrylamide gel containing 1× TBE. The gel was run in 1× TBE at 15 mA for 2–3 h. Subsequently, the gel was exposed for 16–48 h in a phosphor imaging cassette (Molecular Dynamics) at –20°C. The phosphor imaging cassette was scanned using a Personal Molecular Imager (Biorad).