For the dsDNA target cleavage assay of AapCas12b, 1.2 μM AapCas12b protein, 1.2 μM sgRNA, 300 ng dsDNA substrate and 0.8 unit/μL Murine RNase Inhibitor (Vazyme, China) were incubated for 1 h at 60°C in cleavage buffer (NEBuffer 2.1, NEB, USA) as discribed.54 (link)
For the ssRNA target cleavage assay of LwaCas13a, 0.6 μM LwaCas13a protein, 0.6 μM crRNA, 1300 ng ssRNA substrate and 0.8 unit/μL Murine RNase Inhibitor were incubated for 1 h at 37°C in NEBuffer 1.1 as discribed.97 (link)
To test the orthogonal collateral cleavage of AapCas12b and LwaCas13a, 50 nM AapCas12b protein, 150 nM sgRNA, 10 nM dsDNA substrate, 0.8 unit/μL Murine RNase Inhibitor, 500 nM quenched ssDNA fluorescent probes and 500 nM quenched ssRNA fluorescent probes were incubated in NEBuffer 1.1 for 15 min at 60°C as discribed.50 For LwaCas13a, an assay was performed with 50 nM LwaCas13a protein, 50 nM crRNA, 10 nM ssRNA substrate, 0.8 unit/μL Murine RNase Inhibitor, 500 nM quenched ssDNA fluorescent probes, 500 nM quenched ssRNA fluorescent probes and NEBuffer 1.1 for 15 min at 37°C as discribed.50 The fluorescence signals of these reactions were recorded every minute by a LightCycler 96 (Roche, Swiss) in the FAM channel (for ssDNA probe) and ROX channel (for ssRNA probe). Detailed probe sequences are listed in Table S3.
Wang Z., Wang Y., Zhang Y., Qin G., Sun W., Wang A., Wang Y., Zhang G, & Zhao J. (2024). On-site detection and differentiation of African swine fever virus variants using an orthogonal CRISPR-Cas12b/Cas13a-based assay. iScience, 27(4), 109050.
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