How Does CRISPR-Cas9 Edit DNA? Under the Computational Microscope

You know what was truly mind-blowing while working on my novel, Zagreb? My editor’s meticulous attention to detail. Obsessively poring over every comma, every period, every word, ensuring each element conveyed the intended message. Now, imagine applying that level of precision to DNA—pinpoint accuracy in replacing just the right genetic letters to potentially rewrite the very blueprint of life. Scary but fascinating, right? Well, that’s exactly what CRISPR-Cas9 allows us to do—the gene editing tool that’s reshaping our era. I’ve been mulling over this for quite a while: How does CRISPR-Cas9 work? And what’s the secret behind its ability to cleave DNA? These questions led me to the recent work of Lorenzo Casalino, who has been employing quantum-chemistry-based simulations to solve CRISPR-Cas9’s remaining puzzles. You might recall our previous story on Casalino’s majestic 305-million atom molecular dynamics simulations, the computational microscope of the SARS-CoV-2 virus. A fortuitous exchange on LinkedIn, followed by discovering our shared history at SISSA in Trieste, Italy, only fueled my curiosity further. What insights did Casalino and team gain from their simulations on a monstrous system like CRISPR-Cas9? Today, dear reader, we explore CRISPR-Cas9’s DNA cleavage through the lens of Car-Parrinello molecular dynamics simulations. Are you ready? Let’s go.

How Does CRISPR-Cas9 Edit DNA? Under the Computational Microscope | From Atoms To Words | Arturo Robertazzi

Introducing CRISPR-Cas9: A Genetic Revolution

What if we could tailor medical treatments to fit each person’s unique genetic makeup? Or wipe out inherited diseases like cystic fibrosis or sickle cell anemia before they even begin?

Clustered Regularly Interspaced Short Palindromic Repeats paired with associated protein 9, introducing CRISPR-Cas9: the groundbreaking marvel has brought us into a bold new era in our ability to edit the human genome.

Birthed from the mechanisms of bacterial defense and refined into an exquisitely precise tool for editing life’s very code, this game-changing tool opens the door to personalized medicine—therapies that are customized to be the most effective and significantly reduce or even eliminate side effects.

But its impact doesn’t stop there. Through germline editing, CRISPR-Cas9 holds the potential to free future generations from the burden of such conditions. And with the organ shortage crisis looming, CRISPR-Cas9 is making strides in editing pig genomes to create animal organs compatible with the human body.

Perhaps the most groundbreaking aspect of CRISPR-Cas9 is its accessibility. This isn’t a tool locked away in ivory towers; it’s becoming increasingly available to researchers worldwide, democratizing the potential for discovery and innovation.

Ethical regulations, anyone?

Now, CRISPR-Cas9’s power resides in its unique ability to make precise double-stranded breaks at specific DNA locations, setting the stage for gene editing that can correct mutations or disrupt harmful genes. Originating from Streptococcus pyogenes, the Cas9 enzyme stands out as an unparalleled device for targeted genomic modifications, enabling exact breaks at designated sites. Hailed as the Breakthrough of the Year in 2015 and earning its developers, Charpentier and Doudna, the Nobel Prize in Chemistry in 2020, CRISPR-Cas9 is the foundation for present and future genetic breakthroughs.

And now, with the historical approval of the first CRISPR-based drug for treating sickle-cell disease and β-thalassemia, we’re witnessing a monumental shift in gene therapies’ journey—from a promising research topic to a transformative medical solution.

Exciting and scary times we live in, wouldn’t you agree?

How Does CRISPR-Cas9 Edit DNA? Under the Computational Microscope | From Atoms To Words | Arturo Robertazzi
Catalytically active CRISPR-Cas9-RNA-DNA Complex Primed for DNA Cleavage | PDB: 5F9R

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CRISPR-CAS9: What Do We Know? What Don’t We?

We often toss around the word revolutionary (mea culpa!) when discussing technologies, but with CRISPR-Cas9, I think it’s safe to say that it’s warranted.

The catalytic machinery resides within Cas9, the enzyme that orchestrates the key biological function of cleaving the DNA phosphodiester bond—the bond that links the sugars of DNA nucleotides. Now, we’ve already learned a lot about it. For instance, we know it functions as a metal-dependent nuclease, requiring magnesium ions for precise DNA cutting, while calcium ions act as inhibitors. This specificity is crucial for Cas9’s unparalleled precision in gene editing.

So, how does it accomplish this feat? By aligning a guide RNA with the target DNA, positioning it within the RuvC domain—a process similar to that of RNA Ribonucleases, indicating a two-metal-ion mechanism at play.

What does this domain, the active site, look like?

The RuvC domain is known to host three carboxylates (D10, D986, and E762), which are part of the highly conserved DDE motif, a hallmark of two-metal-dependent nucleases. The X-ray structures, which feature manganese ions instead of the catalytically active magnesium, demonstrate how this motif coordinates the two metals. Additionally, the H983 residue plays a crucial role, coordinating with the active site metal through a nitrogen ligand. Mutating H983 confirms its importance as it results in the loss of RuvC activity.

See, it’s quite amazing what we have managed to learn already. So, what’s there more to discover? Plenty! Enough to ensure student grants for years to come.

How do the catalytically active magnesium ions, as opposed to the manganese ions observed in crystallography, arrange themselves to catalyze the reaction? Where does the nucleophile that initiates the chemical cleavage of the phosphodiester bond come from? And how does the critical histidine amino acid, H983 contribute to the catalytic mechanism?

While experimental work has provided valuable insights, at From Atoms To Words, as you know by now, we steer our exploration towards simulations. And that’s where Lorenzo Casalino’s quantum-chemistry-based simulations come into the picture.

Casalino’s computational microscope has previously examined CRISPR-Cas9’s conformational dynamics, selectivity, and allosteric functions, giving us a glimpse into the enzyme‘s inner workings. Today, we explore their most recent work that delves into a detailed analysis of the chemical pathway of DNA cleavage.

Why is this important? Because rationalizing this mechanism, as is the case with many chemical systems, is essential for fine-tuning, optimizing, and fully leveraging the technology’s potential.

So, what exactly did Casalino find out? And how did they do it?

How Does CRISPR-Cas9 Edit DNA? Under the Computational Microscope | From Atoms To Words | Arturo Robertazzi
Active site configuration with water nucleophile near H983 and scissile phosphate. Catalytically active magnesium ions are shown (A, B) | Zoomed-in view of the quantum chemistry layer in Casalino’s CRISPR-Cas9 models

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Setting Up the Computational Microscope

Tackling the lingering questions surrounding CRISPR-Cas9’s molecular complexities is no small feat; it demands substantial scientific effort. And this is precisely what Casalino brought to the table with their computational microscope. Remember their mission? To shed light on the precise mechanisms behind Cas9’s DNA cleavage, a pivotal phase in genome editing.

But before we dive into Casalino’s findings, let’s unpack the computational framework they deployed. Ready for a peek behind the curtain?

  • Step 1: Model Building – Casalino’s computational journey begins with crafting a structural model, anchored in the most comprehensive X-ray structure of the Streptococcus pyogenes CRISPR-Cas9 complex available. Placed within a realistic water environment and balanced with counterions, this model set the stage for all simulations that follow, marking the first step in investigating Cas9’s DNA cleavage activity. Overall, the model features 340,000 atoms.
  • Step 2: Classical Molecular Dynamics – With the model in place, Casalino and team were ready to perform molecular dynamics simulations, offering a computational microscope view into the CRISPR-Cas9 complex. The molecular dynamics simulations, spanning approximately 10 nanoseconds per run, unveiled the behavior of atoms and molecules in an environment that closely mimics its natural cellular surroundings. They also provided initial insights into the dynamics at play and set the stage for the next step: quantum-chemistry-based simulations.
  • Step 3: Car-Parrinello Hybrid Molecular Dynamics Simulations – Now, it’s time to bring out the big guns and delve deeper into the model by deploying dynamical QM/MM simulations. Here, Casalino examined the RuvC active site at a quantum level, focusing on 105 atoms out of the total 340,000 contained in the model. Each simulation ran for approximately 40 picoseconds. As we have seen in previous stories, QM/MM simulations serve as a bridge, connecting the broad strokes of classical mechanics with the electronic details of the quantum methods. They offer a detailed glimpse into the catalytic processes of magnesium ions and protein residues, crucial for DNA cleavage.
  • Step 4: Free Energy Simulations – To map the energetic landscapes of DNA cleavage, Casalino turned to free energy simulations. These simulations are key in calculating energy changes throughout the reaction, quantifying the chemical path from reactants to products and unveiling both the energetics and the structural details of the DNA cleavage mechanism. To accomplish this, Casalino performed QM/MM molecular dynamics simulations, totaling approximately 640 picoseconds. Given the intensive computational demand of these simulations, this sampling represents the pinnacle of what current advanced supercomputing facilities can achieve.

Impressive, right? But beyond the wow factor, what did this powerful computational microscope actually show us about Cas9’s DNA cleavage? What did Casalino find out? Let’s dive deeper.

Structural model of the CRISPR-Cas9 complex | Casalino et al. 2020

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CRISPR-Cas9: A Peep Through The Computational Microscope

Now that we’ve seen what Casalino’s computational microscope looks like, let’s explore what it unveiled about the inner workings of CRISPR-Cas9. Their sophisticated blend of quantum-classical molecular dynamics simulations and free energy calculations has mapped out the phosphodiester bond cleavage mechanism within the RuvC active site.

Crave some juice? Here’s what they found:

  • Key Conformational Change Identified: A crucial finding is the conformational change triggered by replacing crystallographic manganese ions with catalytically active magnesium ions. The investigation confirmed the reactant state’s stability and verified that the chemical prerequisites for a two-metal-ion catalysis were met. As a result, there was a conformational rearrangement of the catalytic site and the interaction network formed by H983 and the water nucleophile. The final configuration, with the DDE motif coordinating the magnesium ions and the scissile phosphate positioned between the two metal ions, is a key condition for catalysis, as it enables the phosphate to adopt a configuration suitable for a nucleophilic attack. This fundamental conformational shift is vital for the activation of the enzyme. Now, isn’t this amazing? A slight chemical change, from manganese to magnesium, and the whole site rearranges.
  • Two-Metal-Dependent Catalytic Mechanism: At the heart of Casalino’s findings lies the identification of a concerted SN2-like associative pathway for DNA cleavage. This pathway is activated by the H983 residue and is dynamically supported by the two catalytic magnesium ions. The reaction  proceeds with a calculated barrier of ∼16 kcal/mol, in virtually perfect agreement with the experimental catalytic rate of 3.5 s-1.
  • Alternative Cleavage Pathway: Casalino’s computational microscope also reveals an alternative phosphate-mediated cleavage pathway, characterized by a dissociative transition state and slightly less energy efficiency compared to the H983-activated mechanism—by a margin of just 2 kcal/mol. Given that this difference falls within statistical error, Casalino’s simulations suggest that the alternative pathway is also likely to occur. This finding highlights CRISPR-Cas9’s versatility and potential for different modes of action under various conditions. Such diversity could be harnessed to develop new CRISPR systems tailored for specific applications, increasing the toolkit available for genome editing.
  • Comparison with Other Enzymes: Finally, Casalino and team compared their outcomes with the mechanisms of other two-metal-dependent enzymes, finding parallels of the associative mechanism of DNA cleavage in studies of HIV-1 RNase H and DNA polymerase III. This demonstrates a universal strategy across genome editing and recombination processes, suggesting a shared evolutionary and functional framework among enzymes that utilize a two-metal ion approach. Such insights indicate potential cross-applicability in biotechnology and medicine.

Yup, there’s a treasure trove of structural, mechanistic, and energetic data in Casalino’s publication. And what awesome animations! If you’re keen to further dive into the nitty-gritty details and the quantitative findings, I highly recommend taking a closer look at their work. Here at From Atoms To Words, I tread carefully—go too deep, and we risk getting tangled in a thicket of science-speak. And losing you in that jungle is the last thing I want, dear reader.

What I can emphasize here is that Casalino’s work lays the groundwork for future computational studies on the CRISPR-Cas9 system. This is key to deepening our understanding and thus, setting the stage for the next surge of breakthroughs in genome editing.

How Does CRISPR-Cas9 Edit DNA? Under the Computational Microscope | From Atoms To Words | Arturo Robertazzi
Casalino’s simulation of the phosphodiester bond cleavage mechanism within the RuvC active site (Animation accelerated 3x) | Credit

A Final Personal Touch

And there we go, wrapping up another story. Wasn’t that a ride? Packed with insights, right down to the last drop of computational juice. Absolutely worth the trip, if you ask me.

So, what did we learn?

In our previous stories on From Atoms To Words, the significance of the computational microscope has been a recurring theme. Remember Krenn’s three dimensions? The computational microscope is an excellent tool that helps us move from discovery to scientific understanding. And haven’t we just witnessed that today?

We’ve caught a glimpse into the inner workings of CRISPR-Cas9 and how this remarkable ancient machinery performs its biological tricks. Just like an editor meticulously fine-tunes the words of a novel, CRISPR-Cas9 edits life’s genetic script.

Standing at the edge of this new frontier, it’s impossible not to get a rush. But Casalino’s study isn’t just a win for the science nerds (no offense). It’s signaling a future where diseases might be edited out of our lives, where treatments are as personalized as your DNA, and where the mysteries of biology are there for the unraveling.

Armed with such profound knowledge, the possibilities stretch as far as the imagination can roam. It’s exhilarating and, yes, a tad daunting.

And that’s good news. More work for scientists. And even more work for philosophers and ethicists—to ensure that this technology not only refrains from harming us but actively keeps us safe. But hey, let’s save that conversation for another day.

If you enjoyed this dive into CRISPR-CAS9 and the mechanism of DNA cleavage, I’d love to hear your thoughts. Agree, disagree, or have a totally wild theory of your own? Let’s connect! Subscribe to my LinkedIn newsletter and let’s keep the conversation rolling.


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