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

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.

GPU-Enhanced DFTB Metadynamics for Efficiently Predicting Free Energies of Biochemical Systems.

Metadynamics calculations of large chemical systems with ab initio methods are computationally prohibitive due to the extensive sampling required to simulate the large degrees of freedom in these systems. To address this computational bottleneck, we utilized a GPU-enhanced density functional tight binding (DFTB) approach on a massively parallelized cloud computing platform to efficiently calculate the thermodynamics and metadynamics of biochemical systems. To first validate our approach, we calculated the free-energy surfaces of alanine dipeptide and showed that our GPU-enhanced DFTB calculations qualitatively agree with computationally-intensive hybrid DFT benchmarks, whereas classical force fields give significant errors. Most importantly, we show that our GPU-accelerated DFTB calculations are significantly faster than previous approaches by up to two orders of magnitude. To further extend our GPU-enhanced DFTB approach, we also carried out a 10 ns metadynamics simulation of remdesivir, which is prohibitively out of reach for routine DFT-based metadynamics calculations. We find that the free-energy surfaces of remdesivir obtained from DFTB and classical force fields differ significantly, where the latter overestimates the internal energy contribution of high free-energy states. Taken together, our benchmark tests, analyses, and extensions to large biochemical systems highlight the use of GPU-enhanced DFTB simulations for efficiently predicting the free-energy surfaces/thermodynamics of large biochemical systems.

Structural basis for Cas9 off-target activity.

The target DNA specificity of the CRISPR-associated genome editor nuclease Cas9 is determined by complementarity to a 20-nucleotide segment in its guide RNA. However, Cas9 can bind and cleave partially complementary off-target sequences, which raises safety concerns for its use in clinical applications. Here, we report crystallographic structures of Cas9 bound to bona fide off-target substrates, revealing that off-target binding is enabled by a range of noncanonical base-pairing interactions within the guide:off-target heteroduplex. Off-target substrates containing single-nucleotide deletions relative to the guide RNA are accommodated by base skipping or multiple noncanonical base pairs rather than RNA bulge formation. Finally, PAM-distal mismatches result in duplex unpairing and induce a conformational change in the Cas9 REC lobe that perturbs its conformational activation. Together, these insights provide a structural rationale for the off-target activity of Cas9 and contribute to the improved rational design of guide RNAs and off-target prediction algorithms.

Enhancement of CRISPR/Cas12a trans-cleavage activity using hairpin DNA reporters.

The RNA programmed non-specific (trans) nuclease activity of CRISPR-Cas Type V and VI systems has opened a new era in the field of nucleic acid-based detection. Here, we report on the enhancement of trans-cleavage activity of Cas12a enzymes using hairpin DNA sequences as FRET-based reporters. We discover faster rate of trans-cleavage activity of Cas12a due to its improved affinity (Km) for hairpin DNA structures, and provide mechanistic insights of our findings through Molecular Dynamics simulations. Using hairpin DNA probes we significantly enhance FRET-based signal transduction compared to the widely used linear single stranded DNA reporters. Our signal transduction enables faster detection of clinically relevant double stranded DNA targets with improved sensitivity and specificity either in the presence or in the absence of an upstream pre-amplification step.

Dynamics and mechanisms of CRISPR-Cas9 through the lens of computational methods.

The clustered regularly interspaced short palindromic repeat (CRISPR) genome-editing revolution established the beginning of a new era in life sciences. Here, we review the role of state-of-the-art computations in the CRISPR-Cas9 revolution, from the early refinement of cryo-EM data to enhanced simulations of large-scale conformational transitions. Molecular simulations reported a mechanism for RNA binding and the formation of a catalytically competent Cas9 enzyme, in agreement with subsequent structural studies. Inspired by single-molecule experiments, molecular dynamics offered a rationale for the onset of off-target effects, while graph theory unveiled the allosteric regulation. Finally, the use of a mixed quantum-classical approach established the catalytic mechanism of DNA cleavage. Overall, molecular simulations have been instrumental in understanding the dynamics and mechanism of CRISPR-Cas9, contributing to understanding function, catalysis, allostery, and specificity.

Establishing the allosteric mechanism in CRISPR-Cas9.

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.

Frontiers of metal-coordinating drug design.

Introduction: The occurrence of metal ions in biomolecules is required to exert vital cellular functions. Metal-containing biomolecules can be modulated by small-molecule inhibitors targeting their metal-moiety. As well, the discovery of cisplatin ushered the rational discovery of metal-containing-drugs. The use of both drug types exploiting metal-ligand interactions is well established to treat distinct pathologies. Therefore, characterizing and leveraging metal-coordinating drugs is a pivotal, yet challenging, part of medicinal chemistry.Area covered: Atomic-level simulations are increasingly employed to overcome the challenges met by traditional drug-discovery approaches and to complement wet-lab experiments in elucidating the mechanisms of drugs' action. Multiscale simulations, allow deciphering the mechanism of metal-binding inhibitors and metallo-containing-drugs, enabling a reliable description of metal-complexes in their biological environment. In this compendium, the authors review selected applications exploiting the metal-ligand interactions by focusing on understanding the mechanism and design of (i) inhibitors targeting iron and zinc-enzymes, and (ii) ruthenium and gold-based anticancer agents targeting the nucleosome and aquaporin protein, respectively.Expert opinion: The showcased applications exemplify the current role and the potential of atomic-level simulations and reveal how their synergic use with experiments can contribute to uncover fundamental mechanistic facets and exploit metal-ligand interactions in medicinal chemistry.

Molecular Dynamics Reveals a DNA-Induced Dynamic Switch Triggering Activation of CRISPR-Cas12a.

CRISPR-Cas12a is a genome-editing system, recently also harnessed for nucleic acid detection, which is promising for the diagnosis of the SARS-CoV-2 coronavirus through the DETECTR technology. Here, a collective ensemble of multimicrosecond molecular dynamics characterizes the key dynamic determinants allowing nucleic acid processing in CRISPR-Cas12a. We show that DNA binding induces a switch in the conformational dynamics of Cas12a, which results in the activation of the peripheral REC2 and Nuc domains to enable cleavage of nucleic acids. The simulations reveal that large-amplitude motions of the Nuc domain could favor the conformational activation of the system toward DNA cleavages. In this process, the REC lobe plays a critical role. Accordingly, the joint dynamics of REC and Nuc shows the tendency to prime the conformational transition of the DNA target strand toward the catalytic site. Most notably, the highly coupled dynamics of the REC2 region and Nuc domain suggests that REC2 could act as a regulator of the Nuc function, similar to what was observed previously for the HNH domain in the CRISPR-associated nuclease Cas9. These mutual domain dynamics could be critical for the nonspecific binding of DNA and thereby for the underlying mechanistic functioning of the DETECTR technology. Considering that REC is a key determinant in the system's specificity, our findings provide a rational basis for future biophysical studies aimed at characterizing its function in CRISPR-Cas12a. Overall, our outcomes advance our mechanistic understanding of CRISPR-Cas12a and provide grounds for novel engineering efforts to improve genome editing and viral detection.