Cas9 in Vitro Cleavage Assay.

To generate the DNA substrates for in vitro Cas9 cleavage assays, templates BL408, BL409, BL410, BL487, BL488, and BL489 (1 ng per 50-μL reaction) were PCR-amplified with primers BL415 (400 nM) and BL416 (400 nM), and OneTaq DNA polymerase in 1× OneTaq standard reaction buffer supplemented with dNaMTP (100 μM), dTPT3TP (100 μM), and MgCl₂ (1.5 mM), under the following thermocycling conditions: [25 × (95 °C 0:15 | 56 °C 0:15 | 68 °C 1:30)].

To generate the DNA templates for in vitro transcription of sgRNAs, templates BL318, BL484, BL485, and BL486 (1 ng per 50-μL reaction), which contain the T7 promoter and a CRISPR RNA (crRNA) spacer sequence, were PCR-amplified with primers BL472 (200 nM) and BL473 (200 nM), and OneTaq DNA polymerase in 1× OneTaq standard reaction buffer supplemented with MgCl₂ (6 mM), under the following thermocycling conditions: [20 × (95 °C 0:15 | 60 °C 0:15 | 68 °C 1:30)]. DNA from this first PCR (0.5 μL) was then transferred into a second PCR (100 μL) containing primers BL472 (400 nM), BL439 (500 nM), and BL440 (600 nM), and thermocycled under the following conditions: [4 × (95 °C 0:15 | 68 °C 0:15 | 68 °C 1:30) 20 × (95 °C 0:15 | 60 °C 0:15 | 68 °C 1:30)]. In vitro transcription of the PCR products with T7 RNA polymerase was performed according to the manufacturer’s protocol, and transcribed sgRNAs were purified by PAGE, band excision, and extraction (37 °C, overnight) into an aqueous solution of NaCl (200 mM) and EDTA (1 mM, pH 7), followed by concentration and purification by ethanol precipitation.

For in vitro cleavage reactions, Cas9 nuclease (125 nM) was incubated with each transcribed sgRNA (125 nM) in 1 × Cas9 nuclease reaction buffer for 5 min, then DNA substrate was added, and the reaction was incubated (37 °C, 10 min). The reaction was quenched with SDS/PAGE loading buffer [62 mM Tris⋅HCl, 2.5% (wt/vol) SDS, 0.002% bromophenol blue, 0.7 M β-mercaptoethanol, and 10% (vol/vol) glycerol], heat-denatured (95 °C, 10 min), and then loaded onto an SDS/PAGE gel. The resulting cleavage bands were quantified by densitometric analysis using ImageJ (40). For each sgRNA, raw cleavage efficiencies were divided by the maximum cleavage observed for that sgRNA across the set of the six DNA substrates, to account for differences in sgRNA activity and/or minor variations in preparation. Experiments were performed in technical triplicate, and averages represent an average of three in vitro cleavage reactions performed in parallel.