FIG. 3.
DNA binding competition assay results. The DNA oligo substrates used are schematically shown as wild-type (WT), T(-1)G/A(+1)C (dsDNA with mismatch to sgRNA at position +1), T(-1)T/A(+1)C (bulged DNA oligo at position +1 that mismatches with sgRNA), and non-target strand bulge (bulged DNA oligo at position at +1 that matches sgRNA). For each competition binding experiment, the cleavage gel image and fraction of cleavage versus competitor concentration at logarithmic scale are shown. Solid curves are fitted theoretical curves and fitted KI values are indicated. (A) Competition binding result for the wild-type or T(-1)G oligo with AceCas9 (WT) and the V709A variant. (B) Competition binding result for the bulged oligos with AceCas9 (WT) and the V709A variant. Experiments performed in triplicate (Supplementary Fig. S5). Color images are available online.