Structural basis of Cas12a R-loop propagation on pathway to DNA cleavage

Cas12a is a CRISPR RNA-guided nuclease that engages target DNA through protein-DNA and RNA-DNA contacts. Initial PAM binding by Cas12a leads to formation of a 20bp R-loop between the complementary crRNA guide and target strand. Following specificity-determining R-loop formation, both DNA strands undergo RuvC-mediated cleavage. Current structures of Cas12a bound to its target only show the R-loop after formation, leaving an important gap in knowledge as to how Cas12a accommodates the extending R-loop and coordinates R-loop formation with nuclease activation. Here, we use cryo-EM to capture a series of kinetically trapped Cas12a R-loop intermediates and observe how Cas12a delivers each DNA strand into the RuvC active site. We show that Cas12a first interrogates target DNA via a 5bp seed, followed by dramatic Rec domain conformational flexibility to accommodate R-loop extension. Only during formation of the final R-loop base pairs do the Rec and BH domains engage in the majority of contacts with the R-loop. R-loop completion leads the nontarget strand to displace the RuvC lid and kink into the active site via a base stacking interaction. Following nontarget strand cleavage, we observe substantial Rec2 and Nuc domain dynamics as the TS is brought to the RuvC active site. Our kinetics-guided structural snapshots provide a comprehensive model describing Cas12a DNA targeting and highlight mechanistic differences between Cas12a and Cas9.