PlmCas12e Utilizes Glu662 to Prevent Cleavage Site Occupation by Positively Charged Residues Before Target Strand Cleavage

CRISPR-Cas12e is a recently identified gene-editing tool mainly known because its relatively small size benefits cell delivery. Drastically different from Cas9, it creates a blunt-end double-strand breakage of the DNA via two cleavage sites; Cas12e produces a sticky-end double-strand breakage of the DNA through only one cleavage site in its RuvC domain, meaning two consecutive cleavage events first on the non-target strand (ntsDNA) and then the target strand (tsDNA). Though crucial for Cas12e's cleavage efficiency, the mechanism by which Cas12e loads tsDNA for the second cleavage remains elusive. Through molecular dynamics simulations and our recently matured traveling-salesman-based automated path-searching (TAPS) algorithm, we identified a series of positively charged residues (Arg856^TSL, Arg768^RuvC, Lys898^TSL, Arg904^TSL, Arg764^RuvC) that guide the tsDNA backbone toward the cleavage site of wild-type <i>Plm</i>Cas12e. Further simulations of the R856L and R904L mutants supported such observations. More interestingly, we found the key role of Glu662^RuvC in coordinating Arg764^RuvC, preventing its occupation of the cleavage site, and facilitating tsDNA cleavage. Additional simulations confirmed that mutating Glu662^RuvC to valine disabled such coordination and created a stable intermediate state with Arg764^RuvC occupying the cleavage site before tsDNA loading. These insights, revealing an elaborate mechanism of cleavage facilitation, offer essential guiding principles for future rational engineering of Cas12e into more efficient gene-editing tools.