Mg²⁺-dependent conformational rearrangements of CRISPR-Cas12a R-loop complex are mandatory for complete double-stranded DNA cleavage

Abstract

CRISPR-Cas12a, an RNA-guided DNA targeting endonuclease, has been widely used for genome editing and nucleic acid detection. As part of the essential processes for both of these applications, the two strands of double-stranded DNA are sequentially cleaved by a single catalytic site of Cas12a, but the mechanistic details that govern the generation of complete breaks in double-stranded DNA remain to be elucidated. Here, using single-molecule fluorescence resonance energy transfer assay, we identified two conformational intermediates that form consecutively following the initial cleavage of the nontarget strand. Specifically, these two intermediates are the result of further unwinding of the target DNA in the protospacer-adjacent motif (PAM)–distal region and the subsequent binding of the target strand to the catalytic site. Notably, the PAM-distal DNA unwound conformation was stabilized by Mg2+ ions, thereby significantly promoting the binding and cleavage of the target strand. These findings enabled us to propose a Mg2+-dependent kinetic model for the mechanism whereby Cas12a achieves cleavage of the target DNA, highlighting the presence of conformational rearrangements for the complete cleavage of the double-stranded DNA target.

Deciphering the QR Code of the CRISPR-Cas9 System: Synergy between Gln768 (Q) and Arg976 (R)

“…The MD method has been widely used to study biomolecular interactions, as in the case of Cas9. ,− To the best of our knowledge, most of the computational studies in the literature have focused so far on the wild-type (wt) Cas9, or the effect of base pair (bp) mismatches between sgRNA and tDNA. Similar to the study by Nierzwicki et al, our working hypothesis is that mutations in the bridge domain of Cas9, e.g., R63A, R66A, R70A, R69A, R71A, R74A, R78A (Figure C), along with different concentrations of Mg 2+ should induce conformational changes in the Cas9 protein scaffold that affect its mechanism of action, cleavage rate, and specificity but more importantly the allosteric communication toward the catalytic domains. , Thus, such external stimuli to the protein have been chosen to sample part of the configurational space of Cas9 within the MD method. Three Cas9–sgRNA–dsDNA variants have been prepared; the wt from S.…”

“…In the absence of Mg 2+ , the Cas9–sgRNA–dsDNA system is locked into the inactive conformation, as the Mg 2+ ions are necessary to lower the energy barriers for HNH movement into the active conformation for the tsDNA cleavage. , Mg 2+ ions have also been implicated in the unwinding of the PAM-distal dsDNA region in an allosteric manner by increasing the energy barrier for dsDNA rewinding . In general, the Mg 2+ ions are administered commonly in concentrations ∼10 mM along the CRISPR-Cas systems and are highly mobile within the Cas9 protein matrix and dynamically coordinated within the Cas catalytic sites.…”

“…In general, the Mg 2+ ions are administered commonly in concentrations ∼10 mM along the CRISPR-Cas systems and are highly mobile within the Cas9 protein matrix and dynamically coordinated within the Cas catalytic sites. Thus, Mg 2+ ions are known to stabilize cleavage-activated conformations, like the hybrid sgRNA–DNA intermediate at the PAM-distal site, in an allosteric but also concentration-dependent manner. , Herein, we have probed a low Mg 2+ concentration (3 mM) where all of the Mg 2+ ions are placed at key sites proposed in the literature or resolved in the crystal structure . These Mg 2+ ions exert very low mobility throughout the trajectories and simulate the physiological state of metal coordination within Cas9 (∼10 mM) .…”

“…These mutations determine the sensitivity to mismatches along the sgRNA–DNA hybrid duplex and are probed herein by computational approaches like classical molecular dynamics (MD), Markov state modeling (MSM), , an enhanced sampling technique, , and machine (deep) learning algorithms. − We focus on the dynamics of the catalytic His840 (HNH) and Arg976 (RuvC) residues, along with the Gln768 dynamics belonging to the linker domain between HNH and RuvC for the Cas9 wild type and mutants. The Arg976/Gln768 dynamics are also determined in relation to the presence of Mg 2+ ions that are indispensable for the action of Cas proteins . The long-time-scale behavior described hereafter refers only to a small part of Cas9, either to the 718–1001 Cas9 region (MSM) or to the 767–984 Cas9 region (machine learning analysis).…”

“…The Arg976/Gln768 dynamics are also determined in relation to the presence of Mg 2+ ions that are indispensable for the action of Cas proteins. 32 The long-time-scale behavior described hereafter refers only to a small part of Cas9, either to the 718–1001 Cas9 region (MSM) or to the 767–984 Cas9 region (machine learning analysis).…”