Cas6-mediated RNA cleavage assay

Two micromolars purified recombinant Cas6 was incubated with 1–5 nM [γ-³²P] ATP-labelled RNA (CD, AB or ncRNA60) at 60°C in nuclease reaction (NR) buffer (20 mM NaH₂PO₄/Na₂HPO_4­ pH 7.5, 100 mM NaCl, 5 mM EDTA, 0.5 mM dithiothreitol). This buffer supported a higher Cas6 activity than that reported previously (14 (link)). To stop the reaction, aliquots of 10 μl were quenched by addition to 30 μl acid phenol/chloroform (Ambion), vortexed briefly and centrifuged at 15 000 × g for 1 min. Five microlitres of the upper aqueous phase was removed and mixed 1:1 with formamide. Samples were heated at 95°C for 2 min and loaded onto a pre-run 20% denaturing polyacrylamide gel (20% acrylamide, 8 M urea, 1× Tris-borate-EDTA (TBE)) then electrophoresed at 80 W for 90 min in 1× TBE running buffer. Following electrophoresis, gels were scanned by phosphorimaging and analysed using Fuji Imagegauge software as described previously (24 (link)).