Rnase alert

For the Cas13a RNA detection assays crRNA was first diluted with processing buffer (1x is 20 mM HEPES pH 6.8, 50 mM KCl, 5 mM MgCl₂ and 5% glycerol) to a final assay concentration of 150 nM crRNA in 1x processing buffer, and incubated for 5 min at 65°C to ensure complete folding of the crRNA. Then, Cas13a was added to a final assay concentration of 20 nM and the sample was incubated for 10 min at 37°C to mediate binding between Cas13a and crRNA. Finally, 200 nM of RNase Alert (Thermo Fischer Scientific), 1.5 U/μl of RNase Inhibitor, Murine (NEB, No. M0314) and 100 nM of target RNA (all final concentrations in the assay) were added on ice. The solution was mixed by pipetting up and down, and immediately applied to the filter paper and measured.

The CRISPR-MTB assay incorporated a PCR amplification step and a subsequent Cas13a detection step. The PCR mixture contained 25 μl 2× MightyAmp® Buffer Ver.3, 0.3 μM IS1081 sense (5′-ACAAAGCTTTCCAAGTCGCA-3′) and 0.3 μM IS1081 antisense (5′-AATTCTAATACGACTCACTATAGGGCCCA GGATCTCTCGGTAGC-3′) primers, 1 μl MightyAmp® DNA Polymerase Ver.3 and 2 μl of DNA sample in a total volume of 50 μl (adjusted with ddH₂O). The PCR amplification programme consisted of denaturation at 95°C for 5 min, followed by 36 cycles (98°C for 10 s, 68°C for 20 s and 1 cycle at 68°C for 5 min) and generated a 237-base-pair amplicon. Cas13a detection was conducted using a reaction mixture containing the following constituents: 0.5 μl of murine RNase inhibitor (New England Biolabs), 45 nM purified LwCas13a, 45 nM crRNA, 125 nM quenched fluorescent RNA reporter (RNAse Alert, Thermo Scientific, Waltham, MA, United States), 0.5 μl of T7 polymerase mix, 1 mM ATP, 1 mM GTP, 1 mM UTP, 1 mM CTP and 2 μl of PCR product in nuclease assay buffer (40 mM Tris–HCl, 60 mM NaCl, 6 mM MgCl2, pH 7.3). Assays were carried out at 37°C for 30 min and monitored for a fluorescent signal using an ABI 7500 instrument (Thermo Fisher, MA, United States). Fluorescein (FAM) fluorescence values were read every 1 min.