Figure 4.

LbCas12a cleaves the non-target strand faster than the target strand. (A) Linear DNA substrates were ³²P-labelled on either the TS (upper panel) or NTS (lower panel). The PAM, approximate positions of NTS and TS cleavage, and sizes of the intact and cut labelled-strands are indicated. DNA species were separated by alkaline denaturing agarose gel electrophoresis. Each lane represents the reaction following LbCas12a addition (left to right): 0, 0.5, 1, 2, 3, 5, 10, 20, 30, 40, 50 and 60 min. (B) Diagrams showing overlap between substrate, intermediates and products depending on the labelled strand. Dotted lines enclose those species which will have the same labelled strand identity on a denaturing gel (C) Ordered kinetic model and (D) random kinetic model, simulated (solid and dashed lines, respectively) using values in Table 2. NTS and TS data points in both panels came from separate gels, as in panel (A), and are the average from 3 repeat experiments, with error bars as SD.