Figure 3.

Apparent DNA cleavage rates are affected by substrate topology. Cleavage of 3 nM supercoiled (A), pre-nicked circular (B) and linear (C) substrates by 50 nM LbCas12a-crRNA. Diagrams represent the substrate, intermediate and product states: supercoiled (SC); nicked (open circle, OC); full length linear (FLL); linear substrate (LIN1); and products of linear DNA cleavage (LIN2). DNA species were separated by native agarose gel electrophoresis. (The OC and LIN1 data shown are from the same gel and thus share the same marker lane). The kinetic models were simultaneously fitted (solid lines) to each of three repeats and the rate constants averaged (Table 2, with SD errors). For panel A, k_rloop was fixed as infinitely fast. For panels (B) and (C), the k₁ and k₂ values were fixed using the fits in panel (A). Plotted points are the average of the 3 repeats, errors bars as SD.