Catalytic Mechanism of Non-Target DNA Cleavage in CRISPR-Cas9 Revealed by Ab Initio Molecular Dynamics

Abstract

CRISPR-Cas9 is a cutting-edge genome editing technology, which uses the endonuclease Cas9 to introduce mutations at desired sites of the genome. This revolutionary tool is promising to treat a myriad of human genetic diseases. Nevertheless, the molecular basis of DNA cleavage, which is a fundamental step for genome editing, has not been established. Here, quantum-classical molecular dynamics (MD) and free energy methods are used to disclose the two-metal-dependent mechanism of phosphodiester bond cleavage in CRISPR-Cas9. Ab initio MD reveals a conformational rearrangement of the Mg²⁺-bound RuvC active site, which entails the relocation of H983 to act as a general base. Then, the DNA cleavage proceeds through a concerted associative pathway fundamentally assisted by the joint dynamics of the two Mg²⁺ ions. This clarifies previous controversial experimental evidence, which could not fully establish the catalytic role of the conserved H983 and the metal cluster conformation. The comparison with other two-metal-dependent enzymes supports the identified mechanism and suggests a common catalytic strategy for genome editing and recombination. Overall, the non-target DNA cleavage catalysis described here resolves a fundamental open question in the CRISPR-Cas9 biology and provides valuable insights for improving the catalytic efficiency and the metal-dependent function of the Cas9 enzyme, which are at the basis of the development of genome editing tools.

Keywords: CRISPR-Cas9; QM/MM; free energy simulations; genome editing; magnesium-aided catalysis; non-coding RNA; phosphodiester bond cleavage; protein/nucleic acid interactions.

Figure 1.

Overview of the CRISPR-Cas9 complex bound to a guide RNA and to a target DNA. (A) Cas9 protein is shown in the molecular surface, highlighting the HNH (green) and RuvC (blue) domains. The RNA (violet) and the DNA (black) strands are shown as ribbons. (B) Inset of the RuvC active site, displaying the catalytic metals (A and B, orange), the surrounding protein residues, and water molecules (left panel). The configuration of the active site resulting from ab initio molecular dynamics (MD) is shown in the right panel. The water nucleophile locates in between H983 and the scissile phosphate, positioning for the chemical reaction. (C) Time evolution along ~40 ps of ab initio MD of the interaction network established by H983 and by the water nucleophile. The complete set of interactions established by the metal cluster is reported in Figures S3 and S4.

Figure 2.

Structural and energetic properties of the two Mg²⁺-aided catalysis in CRISPR-Cas9. (A) Representative snapshots of the reactant (R), (B) proton transfer (PT), (C) transition state (TS^‡), and (D) product (P) states along phosphodiester bond cleavage. (E) Free energy profile (ΔF#, in kcal mol⁻¹) has been computed by using the difference in distance between the breaking (O3′_DNA–P_DNA, d1) and forming (O_WAT–P_DNA, d2) P–O bonds as a reaction coordinate (RC = d1 – d2, highlighted in panel A. The RC windows corresponding to the R, PT, TS^‡, and P states are highlighted using colored bars.

Figure 3.

Interaction distances along phosphodiester bond cleavage. (A) Variation of the significant interaction distances (i.e., the Mg–Mg and the d1–d7 distances, shown on the right panel), computed at each step of the reaction coordinate (RC) in the associative reaction pathway activated by H983 (described in Figure 2). (B) Variation of the critical interaction distances along the alternative dissociative reaction pathway (described in Figure 4). The RC windows corresponding to the reactant (R), proton transfer (PT), transition state (TS^‡), and product (P) states are highlighted using colored bars. The RC (i.e., difference in distance between the breaking and forming P–O bonds) and the interaction distances are described on the R in the right panels.

Figure 4.

Alternative phosphate-mediated reaction pathway. (A) Free energy profile (ΔF#, in kcal mol⁻¹) for the phosphate-mediated dissociative mechanism (blue line), highlighting regions corresponding to the reactant (R′), transition state (TS^‡′), proton transfer (PT′), and products (P′). The free energy profile for the associative pathway activated by H983 is also shown (red line). (B) Snapshots of the R′, TS^‡′, PT′, and P′ states, as from the dissociative pathway.