Rcutsmart buffer

Cas13a:crRNA complexes were individually preassembled by incubating 1 µL of 500 nM of LbuCas13a protein with 1 µL of 500 nM of crRNA for 5 min at room temperature, followed by 1 µL of rCutSmart™ Buffer (NEB #B6004V), pure water and 1 µL of 20 µM FQ5U reporter. Then, 1 µL of contiguous target RNAs (T₂₀, T₁₀–T₁₅, and T_10’–T_15’) or 11 groups of noncontiguous target RNAs were added to the above premix to form the 10 μL reaction system (contiguous target RNAs or noncontiguous target RNAs with 1 nM final concentration). The mix were reacted in a fluorescence plate reader (ABI Thermo Fisher Scientific) at 25 °C for 60 min with fluorescence measurements taken every 1 min (λ_ex: 485 nm; λ_em: 535 nm).

45 μl of 100 μM FragA1 oligo (Supplementary Table S1), 45 μl of 100 μM FragA2 oligo (Supplementary Table S1), and 10 μl of 10× Annealing Buffer (1 M potassium acetate; 300 mM HEPES–KOH, pH7.5) were mixed followed by 95°C denaturation for 3 min. The reaction was slowly cooled down to anneal FragA1 and FragA2, and generate double-stranded Frag1. Double-stranded Frag2, Frag_me, and Frag_hemi were generated by annealing FragB1 and FragB2, Frag_mC1 and Frag_mC2, and Frag_C1 and Frag_mC2 (Supplementary Table S1), respectively. A ligation reaction was prepared with 1 μl of Frag1, 1 μl of Frag2, 1 μl of Frag_mC1 or Frag_mC2, 2 μl of 10× NEB T4 DNA ligase buffer, 1 μl of NEB T4 ligase (M0202L, NEB) and 14 μl of ddH₂O. This reaction was incubated at 20–25°C for 2 h, and the 146 bp ligation product was then purified by Ampure beads (A63880, Beckman). The ligation product was digested in a reaction containing 100 ng of this ligation product, 2 μl of 10x rCutSmart buffer (B6004S, NEB), 0.7 μl of 30× Enzyme Activator, 0.3 μl of MspJI (R0661S, NEB), and 15 μl of ddH₂O at 37°C for 2 h. The digestion products were then assessed using a 20% polyacrylamide gel.