Structural Insights into CRISPR-Cas12a Target Recognition and Cleavage Mechanisms
DOI:
https://doi.org/10.5281/zenodo.22688152Keywords:
CRISPR-Cas12a, cryo-EM, R-loop formation, RuvC nuclease, PAM recognition, genome editing specificityAbstract
Cas12a nucleases offer a single-crRNA-guided, T-rich PAM-dependent alternative to Cas9 with intrinsic
pre-crRNA processing activity and staggered double-strand break generation, but the structural basis for how
target-DNA mismatches are tolerated or rejected at different positions along the guide-target duplex remains
incompletely resolved for the Lachnospiraceae bacterium orthologue (LbCas12a) most widely used in genome
editing applications. We determined cryo-EM structures of LbCas12a at four discrete stages of target engagement
-- PAM-bound pre-R-loop, partial R-loop (10 bp), complete R-loop with un-activated RuvC domain, and the
catalytically engaged cleavage-competent state -- at resolutions between 2.9 and 3.4 angstrom, complemented by
single-molecule FRET measurements of R-loop propagation kinetics and cleavage assays across a panel of 32
systematically positioned single-mismatch target variants. The structures show that R-loop propagation proceeds
through a previously unresolved kinked intermediate at position 10-12 of the protospacer, coinciding with a
conformational checkpoint at which the WED and PI domains must complete a rigid-body rotation before
propagation can continue; mismatches placed within this checkpoint region reduced cleavage rate 40- to 90-fold
relative to matched targets, far more than equivalently positioned mismatches either proximal or distal to the
checkpoint. Single-molecule FRET confirmed that checkpoint-region mismatches stall R-loop propagation at the
partial state for a median of 340-fold longer than matched targets, rarely proceeding to complete R-loop formation
within the experimental observation window. Mutation of two checkpoint-proximal WED domain residues
(R306A/K318A) identified from the structures reduced off-target cleavage at three of four tested genomic off-target
sites by 60-85% while retaining greater than 90% on-target activity, without a corresponding rational design being
derivable from primary sequence or prior lower-resolution structural data alone. These results identify a discrete
structural checkpoint governing Cas12a target discrimination and demonstrate that this checkpoint is a tractable
engineering target for improving genome-editing specificity.
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