Sybr gold 2

The RNA cleavage assays were performed in a cleavage buffer containing 33 mM Tris-acetate pH 7.6 (at 32 °C), 66 mM potassium acetate, 10 mM MnCl₂. The reactions were performed at 37 °C for 20–150 min or as indicated and contained 100 nM LlCsm complex or 1 μM ΔCsm2-LlCsm complex and target RNA at 500 nM concentration or that linked to 5′ Cy3 fluorophore at 100 nM concentration (Table S3). The reactions were quenched using 2× formamide dye (95% formamide, 0.025% SDS, 0.025% bromophenol blue, 0.025% xylene cyanol FF, 0.5 mM EDTA). The reaction products were heated at 70 °C for 3 min and separated by 7 M Urea, 15% polyacrylamide gel electrophoresis (PAGE) gels in 1× Tris Borate EDTA (TBE) running buffer and were visualized by staining with SYBR Gold II (Invitrogen) stain or fluorescence imager.

The in vitro target RNA cleavage assays were performed in a cleavage buffer containing 40 mM Tris pH 8.0, 70 mM sodium chloride, 10 mM MgCl₂. A binary complex was first prepared by incubating 400 nM Cas7-11 with 500 nM crRNA for 30 min at 37°C. The resulting samples were then incubated with 500 nM target RNA (5Cy3 labeled or non-labeled) for 1 hr at 37°C. To test the metal dependence cleavage activity, reaction was supplemented with 16 mM EDTA. The reactions were stopped by using ×2 formamide dye (95% formamide, 0.025% SDS, 0.025% xylene cyanol FF, 0.5 mM EDTA). The samples were heated at 95°C for 5 min and run on 15% TBE-UREA gels (EC6885BOX, Thermo Fisher Scientific) at RT at 180 V in ×1 TBE running buffer. The gels were stained with SYBR Gold II (Invitrogen) stain and scanned by Bio-Rad ChemiDoc MP scanner. When Cy3-labeled RNA was used, gels were directly scanned by Bio-Rad ChemiDoc MP scanner.

For electrophoretic mobility shift assay, 1 µM fC7L.1 was separately added with 150 nM of polyA pre-crRNA (pA3) and non-specific RNA in the buffer containing 20 mM Tris pH 7.5, 70 mM NaCl, 5% glycerol, and 25 µg/mL heparin. The reaction was incubated for 30 min at 37°C. After the reaction was complete, the samples were mixed with ×6 loading dye (B7025S, New England Biolabs) and run on a 10% TBE gel (EC62752BOX, Thermo Fisher Scientific) at 4°C at 150 V. Gels were stained with SYBR Gold II (Invitrogen) and scanned by Bio-Rad ChemiDoc MP scanner.