Figure 6.

A Kinetic Scheme Describing Reaction Pathways of Cas12a/crRNA/dsDNA Ternary Complexes from Formation Until DNA Cleavage

Cas12a proteins are shown in gray, crRNAs are shown in black, and dsDNAs are shown in purple. Cy3 and Cy5 are labeled at crRNAs and TSs of dsDNAs, respectively. Two-way and one-way arrows indicate reversible and irreversible reactions, respectively. Once Cas12a/crRNA binary complexes recognize their target sites and initiate the R-loop formation, ternary complexes are formed in the S1 state. Cas12a complexes transit into the S2 state when the R-loop is extended to contain 14 or more base pairs. Cas12a complexes containing intact NTS can only transiently sample S3 and S4 states from S2 without carrying out TS cleavage. After irreversible cleavage of NTS, S3 and S4 states are stabilized, in which the nuclease activation centers target different cleavage sites on TS. Cleavage of TS occurring from S3 and S4 generates cleaved TS fragments of different lengths, and PAM-distal fragments are released from the complexes.