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1. Principles of Target DNA Cleavage and Role of Mg2+ in the Catalysis of CRISPR-Cas9.

   Ł. Nierzwicki, K. W. (October 2022, Nature catalysis)

   Jan Steven Voeller (Ed.)

   At the core of the CRISPR-Cas9 genome-editing technology, the endonuclease Cas9 introduces site-specific breaks in DNA. Here, multi-microsecond molecular dynamics, free-energy and multiscale simulations are combined with solution NMR and DNA cleavage experiments to resolve the catalytic mechanism of target DNA cleavage. We show that the conformation of an active HNH nuclease is tightly dependent on the catalytic Mg2+, unveiling its cardinal structural role. Solution NMR, DNA cleavage assays and molecular simulations of the Mg2+-bound HNH convey on the formation of the active state and show that the protonation state of catalytic H840 is strongly affected by active site mutations. Finally, ab-initio QM(DFT)/MM simulations and metadynamics establish that DNA cleavage occurs through the identified active state, showing that the catalysis is activated by H840 and aided by K866, in line with DNA cleavage experiments. This information is critical to ameliorating Cas9 function, and helping the development of genome-editing tools. 

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2. Two-metal ion mechanism of DNA cleavage in CRISPR-Cas9

   https://doi.org/10.26434/chemrxiv.9784082  

   Casalino Lorenzo, Jinek Martin (January 2019, ChemRxiv)

   CRISPR-Cas9 is a cutting-edge genome-editing technology, which employs the endonuclease Cas9 to cleave DNA sequences of interest. However, the catalytic mechanism of DNA cleavage and the critical role of the Mg2+ ions have remained elusive. Here, quantum–classical QM(Car-Parrinello)/MM simulations are used to disclose the two-Mg2+ aided mechanism of phosphodiester bond cleavage in the RuvC domain. We reveal that the catalysis proceeds through an associative pathway activated by H983 and fundamentally assisted by the joint dynamics of the two Mg2+ ions, which cooperatively act to properly orient the reactants and lead the chemical step to completion. Cross-validation of this mechanism is achieved by evaluating alternative reaction pathways and in light of experimental data, delivering fundamental insights on how CRISPR-Cas9 cleaves nucleic acids. This knowledge is critical for improving the Cas9 catalytic efficiency and its metal-dependent function, helping also the development of novel Cas9-based genome-editing tools. 

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3. A CRISPR-Cas9–integrase complex generates precise DNA fragments for genome integration

   https://doi.org/10.1093/nar/gkab123  

   Jakhanwal, Shrutee; Cress, Brady F; Maguin, Pascal; Lobba, Marco J; Marraffini, Luciano A; Doudna, Jennifer A (March 2021, Nucleic acids research)

   null (Ed.)

   CRISPR-Cas9 is an RNA-guided DNA endonuclease involved in bacterial adaptive immunity and widely repurposed for genome editing in human cells, animals and plants. In bacteria, RNA molecules that guide Cas9′s activity derive from foreign DNA fragments that are captured and integrated into the host CRISPR genomic locus by the Cas1-Cas2 CRISPR integrase. How cells generate the specific lengths of DNA required for integrase capture is a central unanswered question of type II-A CRISPR-based adaptive immunity. Here, we show that an integrase supercomplex comprising guide RNA and the proteins Cas1, Cas2, Csn2 and Cas9 generates precisely trimmed 30-base pair DNA molecules required for genome integration. The HNH active site of Cas9 catalyzes exonucleolytic DNA trimming by a mechanism that is independent of the guide RNA sequence. These results show that Cas9 possesses a distinct catalytic capacity for generating immunological memory in prokaryotes. 

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4. An alpha-helical lid guides the target DNA toward catalysis in CRISPR-Cas12a

   https://doi.org/10.1038/s41467-024-45762-6  

   Saha, Aakash; Ahsan, Mohd; Arantes, Pablo R; Schmitz, Michael; Chanez, Christelle; Jinek, Martin; Palermo, Giulia (December 2024, Nature Communications)

   Abstract CRISPR-Cas12a is a powerful RNA-guided genome-editing system that generates double-strand DNA breaks using its single RuvC nuclease domain by a sequential mechanism in which initial cleavage of the non-target strand is followed by target strand cleavage. How the spatially distant DNA target strand traverses toward the RuvC catalytic core is presently not understood. Here, continuous tens of microsecond-long molecular dynamics and free-energy simulations reveal that an α-helical lid, located within the RuvC domain, plays a pivotal role in the traversal of the DNA target strand by anchoring the crRNA:target strand duplex and guiding the target strand toward the RuvC core, as also corroborated by DNA cleavage experiments. In this mechanism, the REC2 domain pushes the crRNA:target strand duplex toward the core of the enzyme, while the Nuc domain aids the bending and accommodation of the target strand within the RuvC core by bending inward. Understanding of this critical process underlying Cas12a activity will enrich fundamental knowledge and facilitate further engineering strategies for genome editing. 

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5. Design Rules for Expanding PAM Compatibility in CRISPR-Cas9 from the VQR, VRER and EQR variants

   https://doi.org/10.1101/2025.09.03.673821  

   Vieya, Francisco H; Pindi, Chinmai; Lisi, George P; Morzan, Uriel; Palermo, Giulia (September 2025, bioRxiv)

   Expanding the range of Protospacer Adjacent Motifs (PAMs) recognized by CRISPR-Cas9 is essential for broadening genome-editing applications. Here, we combine molecular dynamics simulations with graph-theory and centrality analyses to dissect the principles of PAM recognition in three Cas9 variants - VQR, VRER, and EQR - that target non-canonical PAMs. We show that efficient recognition is not dictated solely by direct contacts between PAM-interacting residues and DNA, but also by a distal network that stabilizes the PAM-binding domain and preserves long-range communication with REC3, a hub that relays signals to the HNH nuclease. A key role emerges for the D1135V/E substitution, which enables stable DNA binding by K1107 and preserves key DNA phosphate locking interactions via S1109, securing stable PAM engagement. In contrast, variants carrying only R-to-Q substitutions at PAM-contacting residues, though predicted to enhance adenine recognition, destabilize the PAM-binding cleft, perturb REC3 dynamics, and disrupt allosteric coupling to HNH. Together, these findings establish that PAM recognition requires local stabilization, distal coupling, and entropic tuning, rather than a simple consequence of base-specific contacts. This framework provides guiding principles for engineering Cas9 variants with expanded PAM compatibility and improved editing efficiency. 

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