Lba cas12a

For the CRISPR-based detection system, We used Lba Cas12a from New England Biolabs (EnGen® Lba Cas12a (Cpf1) #M0653T). Firstly, Cas12-gRNA complexes were generated by pre-incubating LbaCas12a (50 nM, final concentration) with the gRNA for each gene (50 nM final concentration, the gRNA targeting each gene—E/N/ORF1a—was added in equal proportion)) for 20 min at 37 °C in 1X NEBuffer 2.1 for a final volume of 20 µL. After the incubation, the fluorescent reporter molecule was added at a final concentration of 4 µM and placed on ice. After the desired target amplification by RT-LAMP, 2 µL of each reaction is added to 20 µL of the Cas12-gRNA mixture and incubated at 37 °C for 20 min. This RNP detects amplicons produced by the RT-LAMP reaction simultaneously for all the 3 different genes (E/N/ORF1ab). The real-time fluorescence acquisition was done using the Varioskan™ LUX multimode microplate reader using 20 cycles of 1 min. One pot RT-LAMP/Cas12a reactions were tested using 50 µL of Cas12a solution to 20 µL of RT-LAMP reaction. After 30 min of reaction time at 63 °C, the Eppendorf strip was briefly centrifuged (spin down) to mix the enzymatic solution located in the lid of the Eppendorf with the RT-LAMP reaction. This mixture was incubated at 37 °C for 10 min.

The endonuclease and collateral cleavage activity of LbaCas12a (New England Biolabs (Beijing) Ltd., China) were tested in 30 μL reaction mixtures according to the manufacturer’s instructions. The reaction system was comprised of 3 μL 10× NEBuffer 2.1 (New England Biolabs (Beijing) Ltd., China), 20 μL nuclease-free water, 1 μL Cas12a (final concentration: 33 nM), 3 μL crRNA (final concentration: 300 nM), and 3 μL DNA substrate from the previous section (final concentration: 10 nM). The complex of Cas12a and crRNA were pre-incubated at room temperature for 10 min, then the DNA substrate was added, and the mixture was incubated at 37 °C for 3 h. Finally, after inactivating Cas12 at 65 °C for 10 min, the mixture was checked by 2% (w/v) agarose gel electrophoresis at 120 V for 30 min. The test of collateral cleavage activity was performed according to the procedures provided by Andrea Bonini et al.^{16 (link)}, in which a 50 bp single-stranded DNA was synthesized (Fig. S1, Genscript Co. Ltd., China). 3 μL of this 50 bp ssDNA (final concentration: 1 μM) was added to above reaction mixture together with the DNA substrate. The mixture was incubated at 37 °C for 1 h. The inactivating of Cas12a and agarose gel electrophoresis were the same as described above.