Figure 8.

Mechanism underlying the enhanced Cas12a editing specificity by using chimeric DNA–RNA guide. In this model, when the 3′-end DNA-substituted crRNA was applied to the Cas12a system, the principle that the off-target mutation was reduced compared to wild-type crRNA was explained by the change of the hybridization energy between crRNA and target DNA strand. When wild-type crRNA is applied, crRNA-target DNA hybridization energy can endure mismatch caused by off-target binding, but when chimeric crRNA is applied, induction of off-target mutation is reduced due to destabilization of hybridization energy. The small and large red arrowheads indicate the cleavage site and degree for double-strand break by Cas12a. Red color in the (cr)RNA indicates DNA substitution. PAM sequences are shown in blue color and mismatched sequence to target sequence are shown in orange color, respectively.