Rna inhibitor

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.

Template DNAs were amplified for 30 min in a Genie-II at 39°C according to the TwistAmp^™ Basic Kit (Cambridge, United Kingdom) Quick Guide to obtain RPA amplification products. The following CRISPR reaction system was composed of 40 μL reaction solution: 0.3 μL Cas12a (75 nM), 2 μL Flu-ssDNA (500 nM), 0.5 μL RNase inhibitor, 3 μL NEBuffer3.1, 10 μL crRNA (500 nM) and 4.2 μL double-distilled water (ddH₂O). LbCas12a protein, NEBuffer 3.1, and RNA inhibitor were provided by NEW ENGLAND BioLabs, Inc. (United States). DNase/RNase-free distilled, deionized water (ddH₂O) was provided by Tiangen Biochemical Co., Ltd. Positive reference plasmids for B. pseudomallei detection (pEASY-T1-LC1 and pEASY-T1-LC2) were constructed by our lab. Finally, we took 20 μL of the RPA-amplified product as a template, and then ran the CRISPR/Cas12a reaction system for 10 min at 37°C in the Genie-II.

The following bacterial strains were available in our lab: F. tularensis, Brucella melitensis, Brucella abortus, Burkholderia pseudomallei, Burkholderia mallei, Bacillus anthracis, Staphylococcus aureus, Bacillus thuringiensis, Yersinia pestis, Salmonella typhi, Bacillus subtilis, Escherichia coli, Vibrio vulnificus, Staphylococcus epidermidis, Vibrio parahaemolyticus, Bacillus cereus, and Vibrio cholerae. RPA primers, RPA probes, crRNA, and fluorescent single-stranded DNA reporter (Flu-ssDNA) were synthesized by Shanghai Sangon Biotech Co., Ltd. (China). LbCas12a protein, NEBuffer 3.1, and RNA inhibitor were provided by New England BioLabs, Inc. (USA). TwistAmp™ Exo Kit and TwistAmp™ basic Kit were provided by TwistDx (Cambridge, UK). A positive reference plasmid for F. tularensis detection (pEASY-T1-TUL4) was constructed by our lab. DNase/RNase-free distilled, deionized water (DDH₂O) was provided by Tiangen Biochemical Co., Ltd. A QIAamp™ DNA Mini Kit (Qiagen, Germany) was used to extract bacterial strain genomes, followed by the user manual's protocol.