Fig. 4: Energy kinetic mechanism model of various crRNAs.
The model is divided into four parts from top to bottom. The first part represents the crRNA spacer region, where blue circles indicate matching bases and yellow circles indicate mismatched bases. The second part presents the two-step kinetic model of Cas12a/crRNA binding to the target substrate (black) and a mismatched off-target substrate (red) by using a free energy reaction diagram, where the wells represent states and the peaks represent the barriers for transitions between those states. The third part shows the amplification curves for the on-target and mismatched off-target substrates. The fourth part illustrates the sensitivity, specificity, and universality of Cas12a for various crRNAs using a triangular radar chart. a WT crRNA programmed with the Cas12a enzyme is unable to discriminate between on-target and a mismatched off-target substrate. b Truncated crRNA programmed with the Cas12a enzyme achieves specificity gains by increasing koff for both the on-target and a mismatched off-target substrate. c Truncated crRNA + single-base mismatch increases the specificity of Cas12a by greatly increasing koff for both the on-target and a mismatched off-target substrate. This promotes the dissociation of a mismatched off-target substrate but also reduces the cleavage of the on-target substrate, resulting in a decrease in sensitivity. d A truncated spacer + wobble base pairs at the spacer-TS DNA heteroduplex 14 position increases the specificity of Cas12a without sacrificing sensitivity by increasing koff and the mismatch penalty.
