Figure 5.

Characterization of target activated Cas12a trans-nuclease

(A–C) Cas12a, crRNA-1, and varied Target DNA-1 tested with trans-substrates consisting of (A) single or multiple repeats of TTATT, (B) differing sequence, or (C) varied concentrations of trans-substrate C₁₀ where linear fit was used to estimate kinetic constants (Inset). Data are represented as mean ± SD. (D–G) (Top) Cas12a RNP was pre-reacted with target, then reacted with varied trans-substrate used in each panel. (Middle) Linear fit of time courses to determine V₀. Examples shown: (D) Cas12a-crRNA-1, 10 pM Target DNA-1, substrate C₁₀; (E) Cas12a-crRNA-2, 10 pM Target DNA-2, DNaseAlert substrate; (F) Cas12a-crRNA-2, 1.0 nM Target DNA-2, non-target dsDNA substrate (Fluorescent Target DNA-1); (G) Cas12a-crRNA-1, 1.0 nM Target DNA-1, RNA substrate U₂₀. (Bottom) Hyperbolic fit of V₀ (symbols) to yield kinetic constants (Table 1); data are represented as value ±SE. See also Figures S4 and S5.