Figure. 6.

Quantitative analysis of dsDNA and ssDNA cleavage by wtCas12a and its variants. (A) Force-extensions curves of λ-DNA tethers in the presence and Mg²⁺ and wtCas12a and its variants for dynamic dsDNA cleavage experiments. (B) Plots of unspecific ssDNA degradation in a static setting by wtCas12a and its variants as a distribution of time. All experiments in the static form were conducted at 20 pN. λ-DNA ssDNA tethers that remained present after 4 force–distance cycles (A, dynamic) or 200s (B, static) were considered non-cleaved (indicated by the dashed line). The static cleavage assays for variants Cas12a_KO and Cas12a_M3 were not performed, as Cas12a_KO does not display catalytic activity and Cas12a_M3 activity of can only be observed at high forces. In addition, neither of these variants display the indiscriminate ssDNase activity.