Cas13a

The Cas13a cleavage reaction consisted of 25 mM Tris HCl (PH 7.4), 9 mM MgCl₂, 200 ng Cas13a (Genscript, Cat# Z03486), 200 ng crRNA, 500 ng target RNA, and DEPC water added to the total mixture volume of 20 μL. The mixture was incubated at 37 °C for 30 min and heated at 70 °C for 10 min to denature RNA. The cleavage mixture was loaded onto 12% TBE-Urea Gels with 120 V for 60 min and analyzed on a gel imager system (Thermo Fisher Scientific). As the fluorescent cleavage assay, the Cas13a/crRNA reaction included 25 mM Tris HCl, 9 mM MgCl₂, 100 ng Cas13a, 20 ng crRNA, 10 mM rNTP (NEB, Shanghai, China, Cat# N0466S), 15 U T7 RNA polymerase (NEB, Cat# M0251S), 8 U RNA inhibitor (NEB, Cat# M0314S), 0.25 μM FAM-labeled ssRNA reporters, 20 ng DNA template, 50 ng total human RNA (Thermo Fisher Scientific, Cat# 4307281), and DEPC water added to the total mixture volume of 20 μL. The fluorescent signal of the Cas13a reaction was detected by the 7500 fast Real-Time PCR Systems (Thermo Fisher Scientific, USA) at 37 °C for 40 min and analyzed with the 7500 Fast Software v2.3. By observing the TBE-Urea gels and fluorescence values, we determined the activity and specificity of the mutant-crRNA.

The specificity of each designed crRNA targeting RNA sample was validated by a CRISPR-Cas13a diagnosis assay based on fluorescence signals. The reaction mix contained 50 nM Cas13a (NEB), 50 nM crRNA, 1× Cas13a reaction buffer, 500 nM of single-stranded RNA (ssRNA) reporter (5′ 6-FAM/UUUUU/BHQ-1 3′, Sangon), 0.1 U/µL murine RNase inhibitor, RNA templates from pseudovirus of wild-type, BA.2, or BA.5 gene sequences, and DNA sequences from plasmids containing influenza virus, MERS, and HRSV sequences dispersed in pure water. During the incubation at 37 °C for 30 min, real-time fluorescence was measured with an excitation wavelength of 485 nm and emission wavelength of 528 nm. The trans- and cis-cleavage were further verified by electrophoresis (2.5% agarose gel, 100 V at 4 ℃ for 60 min) and observed using an electrophoresis analyzer.