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Cas12a is an RNA-guided endonuclease, which displays great potentials and several advantages over the well-known Cas9 in genome editing and engineering. Here, we established a quantitative kinetic scheme to describe the conformational dynamics of Cas12a/crRNA/dsDNA ternary complexes. The highly dynamic nature of Cas12a complexes, including their reversible formation, disassembly, and transition between different conformational states, is likely to be one of the key aspects contributing to their high specificity. The non-target strand is cleaved when its cleavage sites are released from DNA duplex after DNase activation of Cas12a. Cleaved non-target strand stabilizes target strand pre-cleavage states to permit subsequent cleavage and to ensure two DNA strands cleaved in a well-defined order. The extent of complementarity between crRNA and DNA modulates the relative stabilities of target strand pre-cleavage states targeting different cleavage sites. Our discoveries provide insights to fully elucidate the working mechanisms of Cas12a and to optimize it for genome engineering.

Conformational Dynamics and Cleavage Sites of Cas12a Are Modulated by Complementarity between crRNA and DNA