Supplementary Figure 2: Inhibition of MbCas12a by AcrVA4 and AcrVA5.

a, Inhibition assays for MbCas12a by AcrVA4 and AcrVA5. Left panel: crRNA was added to buffer containing MbCas12a following addition of AcrVA4 or AcrVA5. Right panel: MbCas12a was pre-incubated with crRNA before AcrVA4 or AcrVA5 was added. After reaction, the reaction mixtures were loaded onto 8% denature polyacrylamide gels. Molar ratios of an anti-CRISPR protein and MbCas12a are shown at the top of each lane. b, Electrophoretic mobility shift assay (EMSA) was used to test the binding of MbCas12a with crRNA or dsDNA in the presence of anti-CRISPR protein AcrVA4. MbCas12a or MbCas12a (D874A/E968A)-crRNA complex was first incubated with increasing amounts of AcrVA4, then crRNA (upper panel) or fluorophore-labelled dsDNA (middle panel) was added and incubated. Nucleic acid-bound MbCas12a complexes were resolved from free nucleic acid by native gel electrophoresis. CrRNA was visualized by ethidium bromide staining (upper panel), and dsDNA was visualized by a fluorescence imaging machine (600 nm) (middle panel). Coomassie blue staining of AcrVA4 at different molar ratios was shown in lower panel. Molar ratios of MbCas12a:AcrVA4 are shown at the top of each lane. Data shown is representative of three independent experiments.