Phosphor imaging cassette

Cleavage assays were done by pre-incubating 100 nM Cas12a with 400 nM crRNA in 1× Cas9 buffer (NEB) for 20 min at 37°C. Thereafter 8 nM 5′ ³²P radiolabeled target DNA was added in a 30 μl reaction and incubation was done at 37°C for 2 h. 30 μl 2× formamide loading dye was added and the complete samples were heated to 95°C for 5 min and loaded on an 8% acrylamide gel (with 7 M urea and 1× TBE). The gel was run in 1× TBE for ∼4 h at 15 mA and subsequently 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).

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).

Cascade purification was performed as described in (10 (link)). Cas3 was purified as described in (35 (link)). Target DNA constructs were incubated with 100 μM Cascade and 10 μM Cas3 in reaction buffer (10 mM HEPES pH 7.5, 60 mM KCl, 50 μM CoCl₂, 10 mM MgCl₂ and 2 mM ATP) at 37°C for 2 h. Reactions were stopped by transferring the tubes to ice and addition of 4 μl 6× loading dye (Thermo Scientific). Reaction products were run on a 6% acrylamide gel (with 7 M urea and 1× TBE). The gel was run in 1× TBE for ∼4 h at 15 mA and subsequently exposed for 48 hours in a phosphor imaging cassette (Molecular Dynamics) at –20°C. The phosphor imaging cassette was scanned using a Personal Molecular Imager (Bio-Rad).