Figure 5.

Cas12a variants biochemical cleavage assay and binding on dsDNA and ssDNA. (A) 15% urea gel showing enzymatic activity of Cas12a with crRNA-1 (See Supplementary Table S1) with the annealed dsDNA formed by TS-1 and NTS-1 (see Supplementary Table S1 and Materials and Methods). (n = 3). (B) 15% urea gel showing indiscriminate ssDNA activity of Cas12a variants using crRNA-1, and activated by Activator TS (See Supplementary Table S1), with Unspecific ssDNA (See Supplementary Table S1) as the substrate (n = 3). (C) Binding profiles of wtCas12a and its variants at increasing forces binned at 120 s (n = 5, at each force for all the samples). The red line indicates the target site centered at 29.720 kb, which is located at the middle of the target sequence including a TTTN PAM. The black line indicates the prominent off-target binding site at 13.539 kb. (D) Representative 60 s-long kymographs showing binding of wtCas12a and its variants to ssDNA in the presence and absence of the activator at a force of 20 pN. All kymographs were obtained in the presence of Mg²⁺. In addition, in experiment the ssDNA tether was first exposed to a buffer without the activator, then, the same tether was moved to the buffer with the activator. In the case of wtCas12a and Cas12a-M1, the kymographs show a section of the data prior to the breaking of the tether.