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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. Establishing the allosteric mechanism in CRISPR‐Cas9

   https://doi.org/10.1002/wcms.1503  

   Nierzwicki, Łukasz ; Arantes, Pablo Ricardo ; Saha, Aakash ; Palermo, Giulia ( October 2020 , WIREs Computational Molecular Science)

   Allostery is a fundamental property of proteins, which regulates biochemical information transfer between spatially distant sites. Here, we report on the critical role of molecular dynamics (MD) simulations in discovering the mechanism of allosteric communication within CRISPR‐Cas9, a leading genome editing machinery with enormous promises for medicine and biotechnology. MD revealed how allostery intervenes during at least three steps of the CRISPR‐Cas9 function: affecting DNA recognition, mediating the cleavage and interfering with the off‐target activity. An allosteric communication that activates concerted DNA cleavages was found to led through the L1/L2 loops, which connect the HNH and RuvC catalytic domains. The identification of these “allosteric transducers” inspired the development of novel variants of the Cas9 protein with improved specificity, opening a new avenue for controlling the CRISPR‐Cas9 activity. Discussed studies also highlight the critical role of the recognition lobe in the conformational activation of the catalytic HNH domain. Specifically, the REC3 region was found to modulate the dynamics of HNH by sensing the formation of the RNA:DNA hybrid. The role of REC3 was revealed to be particularly relevant in the presence of DNA mismatches. Indeed, interference of REC3 with the RNA:DNA hybrid containing mismatched pairs at specific positions resulted in locking HNH in an inactive “conformational checkpoint” conformation, thereby hampering off‐target cleavages. Overall, MD simulations established the fundamental mechanisms underlying the allosterism of CRISPR‐Cas9, aiding engineering strategies to develop new CRISPR‐Cas9 variants for improved genome editing.

   This article is categorized under:

   Structure and Mechanism > Computational Biochemistry and Biophysics

    

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3. The electronic structure of genome editors from the first principles

   https://doi.org/10.1088/2516-1075/acb410  

   Nierzwicki, Łukasz ; Ahsan, Mohd ; Palermo, Giulia ( February 2023 , Electronic Structure)

   Abstract Ab-initio molecular dynamics enables following the dynamics of biological systems from the first principles, describing the electronic structure and offering the opportunity to “watch” the evolution of biochemical processes with unique resolution, beyond the capabilities of state-of-the-art experimental techniques. This article reports the role of first-principles ( ab-initio ) molecular dynamics (MD) in the CRISPR-Cas9 genome editing revolution, achieving a profound understanding of the enzymatic function and offering valuable insights for enzyme engineering. We introduce the methodologies and explain the use of ab-initio MD simulations to establish the two-metal dependent mechanism of DNA cleavage in the RuvC domain of the Cas9 enzyme, and how a second catalytic domain, HNH, cleaves the target DNA with the aid of a single metal ion. A detailed description of how ab-initio MD is combined with free-energy methods—i.e., thermodynamic integration and metadynamics—to break and form chemical bonds is given, explaining the use of these methods to determine the chemical landscape and establish the catalytic mechanism in CRISPR-Cas9. The critical role of classical methods is also discussed, explaining theory and application of constant pH MD simulations, used to accurately predict the catalytic residues’ protonation states. Overall, first-principles methods are shown to unravel the electronic structure and reveal the catalytic mechanism of the Cas9 enzyme, providing valuable insights that can serve for the design of genome editing tools with improved catalytic efficiency or controllable activity. 

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4. Enhanced specificity mutations perturb allosteric signaling in CRISPR-Cas9

   https://doi.org/10.7554/eLife.73601  

   Nierzwicki, Lukasz ; East, Kyle W ; Morzan, Uriel N ; Arantes, Pablo R ; Batista, Victor S ; Lisi, George P ; Palermo, Giulia ( December 2021 , eLife)

   CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat and associated Cas9 protein) is a molecular tool with transformative genome editing capabilities. At the molecular level, an intricate allosteric signaling is critical for DNA cleavage, but its role in the specificity enhancement of the Cas9 endonuclease is poorly understood. Here, multi-microsecond molecular dynamics is combined with solution NMR and graph theory-derived models to probe the allosteric role of key specificity-enhancing mutations. We show that mutations responsible for increasing the specificity of Cas9 alter the allosteric structure of the catalytic HNH domain, impacting the signal transmission from the DNA recognition region to the catalytic sites for cleavage. Specifically, the K855A mutation strongly disrupts the allosteric connectivity of the HNH domain, exerting the highest perturbation on the signaling transfer, while K810A and K848A result in more moderate effects on the allosteric communication. This differential perturbation of the allosteric signal correlates to the order of specificity enhancement (K855A > K848A ~ K810A) observed in biochemical studies, with the mutation achieving the highest specificity most strongly perturbing the signaling transfer. These findings suggest that alterations of the allosteric communication from DNA recognition to cleavage are critical to increasing the specificity of Cas9 and that allosteric hotspots can be targeted through mutational studies for improving the system’s function. 

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5. 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 ( February 2024 , Nature Communications)

   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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