Cas12a domain flexibility guides R-loop formation and forces RuvC resetting.

The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables target recognition and nuclease activation, we used cryo-electron microscopy to capture wild-type Acidaminococcussp. Cas12a R-loop intermediates and DNA delivery into the RuvC active site. Stages of Cas12a R-loop formation-starting from a 5-bp seed-are marked by distinct REC domain arrangements. Dramatic domain flexibility limits contacts until nearly complete R-loop formation, when the non-target strand is pulled across the RuvC nuclease and coordinated domain docking promotes efficient cleavage. Next, substantial domain movements enable target strand repositioning into the RuvC active site. Between cleavage events, the RuvC lid conformationally resets to occlude the active site, requiring re-activation. These snapshots build a structural model depicting Cas12a DNA targeting that rationalizes observed specificity and highlights mechanistic comparisons to other class 2 effectors.

Mechanism of darunavir binding to monomeric HIV-1 protease: a step forward in the rational design of dimerization inhibitors.

HIV protease (HIVPR) is a key target in AIDS therapeutics. All ten FDA-approved drugs that compete with substrates in binding to this dimeric enzyme's active site have become ineffective due to the emergence of drug resistant mutants. Blocking the dimerization interface of HIVPR is thus being explored as an alternate strategy. The latest drug, darunavir (DRV), which exhibited a high genetic barrier to viral resistance, is said to have a dual mode of action - (i) binding to the dimeric active site, and (ii) preventing the dimerization by binding to the HIVPR monomer. Despite several reports on DRV complexation with dimeric HIVPR, the mode and mechanism of the binding of DRV to the HIVPR monomer are poorly understood. In this study, we utilized all-atomic MD simulations and umbrella sampling techniques to identify the best possible binding mode of DRV to the monomeric HIVPR and its mechanism of association. The results suggest that DRV binds between the active site and the flap of the monomer, and the flap plays a crucial role in directing the drug to bind and driving the other protein domains to undergo induced fit changes for stronger complexation. The obtained binding mode of DRV was validated by comparing with various mutational data from clinical isolates to reported in vitro mutations. The identified binding pose was also able to successfully reproduce the experimental Ki value in the picomolar range. The residue-level information extracted from this study could accelerate the structure-based drug designing approaches targeting HIVPR dimerization.

Ameliorative effects of biochar on persistency, dissipation, and toxicity of atrazine in three contrasting soils.

The presence of atrazine a persistent herbicide in soil poses a serious threat to the ecosystem. The biochar amendment in soil altered the fate of this herbicide by modifying the soil properties. The present study examines the dissipation and toxicity of atrazine in three contrasting soils (silty clay, sandy loam, and sandy clay) without and with biochar amendment (4%). The experiment was performed for 150 days with three application rates of atrazine (4, 8, and 10 mg kg-1). The speciation and degradation of atrazine, metabolite content, microbial biomass, and enzymatic activities were evaluated in all treatments. Three kinetic models and soil enzyme index were calculated to scrutinize the degradation of atrazine and its toxicity on soil biota, respectively. The goodness of fit statistical indices suggested that the first-order double exponential decay (FODE) model best described the degradation of atrazine in silty clay soil. However, a single first order with plateau (SFOP) was best fitted for atrazine degradation in sandy loam and sandy clay soils. The half-life of atrazine was higher in sandy clay soil (27-106 day-1) than silty clay (28-77 day-1) and sandy loam soil (27-83 day-1). The variations in the dissipation kinetics and half-life of the atrazine in three soil were associated with atrazine partitioning, availability of mineral content (silica, aluminum, and iron), and soil microbial biomass carbon. Biochar amendment significantly reduced the plateau in the kinetic curve and also reduced the atrazine toxicity on soil microbiota. Overall, biochar was more effective in sandy clay soil for the restoration of soil microbial activities under atrazine stress due to modulation in the pH and more improved soil quality.

Biochar amendment reduced the risk associated with metal uptake and improved metabolite content in medicinal herbs.

Contaminations of heavy metals such as lead (Pb) and cadmium (Cd) in medicinal plants (MPs) not only restrict their safe consumption due to health hazards but also lower their productivity. Biochar amendments in the soil are supposed to immobilize the toxic metals, improve the soil quality and agricultural productivity. However, the impact of biochar on growth attributes, metal accumulation, pharmacologically active compounds of MPs, and health risk is less explored. An experiment was performed on three medicinal plants (Bacopa monnieri (L.), Andrographis paniculata (Burmf.) Nees, and Withaniasomnifera (L.)) grown in a greenhouse in soil co-contaminated with Pb and Cd (at two concentrations) without and with biochar amendments (2 and 4% application rates). The fractionation of Pb and Cd, plant growth parameters, stress enzymes, photosynthetic capacity, pharmacologically active compounds, nutrient content, uptake and translocation of metals, antioxidant activities, and metabolite content were examined in the three MPs. The accumulation of Pb and Cd varied from 3.25-228 mg kg1 and 1.29-20.2 mg kg-1 , respectively, in the three MPs, while it was reduced to 0.08-18 mg kg-1 and 0.03-6.05 mg kg-1 upon biochar treatments. Plants grown in Pb and Cd co-contaminated soil had reduced plant biomass (5-50% depending on the species) compared to control and a deleterious effect on photosynthetic attributes and protein content. However, biochar amendments significantly improved plant biomass (21-175%), as well as photosynthesis attributes, chlorophyll, and protein contents. Biochar amendments in Pb and Cd co-contaminated soil significantly reduced the health hazard quotient (HQ) estimated for the consumption of these medicinal herbs grown on metal-rich soil. An enhancement in secondary metabolite content and antioxidant properties was also observed upon biochar treatments. These multiple beneficial effects of biochar supplementation in Pb and Cd co-contaminated soil suggested that a biochar amendment is a sustainable approach for the safe cultivation of MPs. This article is protected by copyright. All rights reserved.

Experimental Solubility, Thermodynamic/Computational Validations, and GastroPlus-Based In Silico Prediction for Subcutaneous Delivery of Rifampicin.

We focused to explore a suitable solvent for rifampicin (RIF) recommended for subcutaneous (sub-Q) delivery [ethylene glycol (EG), propylene glycol (PG), tween 20, polyethylene glycol-400 (PEG400), oleic acid (OA), N-methyl-2-pyrrolidone (NMP), cremophor-EL (CEL), ethyl oleate (EO), methanol, and glycerol] followed by computational validations and in-silico prediction using GastroPlus. The experimental solubility was conducted over temperature ranges T = 298.2-318.2 K) and fixed pressure (p = 0.1 MPa) followed by validation employing computational models (Apelblat, and van't Hoff). Moreover, the HSPiP solubility software provided the Hansen solubility parameters. At T = 318.2K, the estimated maximum solubility (in term of mole fraction) values of the drug were in order of NMP (11.9 × 10-2) ˃ methanol (6.8 × 10-2) ˃ PEG400 (4.8 × 10-2) ˃ tween 20 (3.4 × 10-2). The drug dissolution was endothermic process and entropy driven as evident from "apparent thermodynamic analysis". The activity coefficients confirmed facilitated RIF-NMP interactions for increased solubility among them. Eventually, GastroPlus predicted the impact of critical input parameters on major pharmacokinetics responses after sub-Q delivery as compared to oral delivery. Thus, NMP may be the best solvent for sub-Q delivery of RIF to treat skin tuberculosis (local and systemic) and cutaneous related disease at explored concentration.

Electrostatics Plays a Crucial Role in HIV-1 Protease Substrate Binding, Drugs Fail to Take Advantage.

HIV-1 protease (HIVPR) is an important drug target for combating AIDS. This enzyme is an aspartyl protease that is functionally active in its dimeric form. Nuclear magnetic resonance reports have convincingly shown that a pseudosymmetry exists at the HIVPR active site, where only one of the two aspartates remains protonated over the pH range of 2.5-7.0. To date, all HIVPR-targeted drug design strategies focused on maximizing the size-shape complementarity and van der Waals interactions of the small molecule drugs with the deprotonated, symmetric active site envelope of crystallized HIVPR. However, these strategies were ineffective with the emergence of drug resistant protease variants, primarily due to the steric clashes at the active site. In this study, we traced a specificity in the substrate binding motif that emerges primarily from the asymmetrical electrostatic potential present in the protease active site due to the uneven protonation. Our detailed results from atomistic molecular dynamics simulations show that while such a specific mode of substrate binding involves significant electrostatic interactions, none of the existing drugs or inhibitors could utilize this electrostatic hot spot. As the electrostatic is long-range interaction, it can provide sufficient binding strength without the necessity of increasing the bulkiness of the inhibitors. We propose that introducing the electrostatic component along with optimal fitting at the binding pocket could pave the way for promising designs that might be more effective against both wild type and HIVPR resistant variants.

Steering the Lipid Transfer To Unravel the Mechanism of Cholesteryl Ester Transfer Protein Inhibition.

Human plasma cholesteryl ester transfer protein (CETP) mediates the transfer of neutral lipids from antiatherogenic high-density lipoproteins (HDLs) to proatherogenic low-density lipoproteins (LDLs). Recent cryo-electron microscopy studies have suggested that CETP penetrates its N- and C-terminal domains in HDL and LDL to form a ternary complex, which facilitates the lipid transfer between different lipoproteins. Inhibition of CETP lipid transfer activity has been shown to increase the plasma HDL-C levels and, therefore, became an effective strategy for combating cardiovascular diseases. Thus, understanding the molecular mechanism of inhibition of lipid transfer through CETP is of paramount importance. Recently reported inhibitors, torcetrapib and anacetrapib, exhibited low potency in addition to severe side effects, which essentially demanded a thorough knowledge of the inhibition mechanism. Here, we employ steered molecular dynamics simulations to understand how inhibitors interfere with the neutral lipid transfer mechanism of CETP. Our study revealed that inhibitors physically occlude the tunnel posing a high energy barrier for lipid transfer. In addition, inhibitors bring about the conformational changes in CETP that hamper CE passage and expose protein residues that disrupt the optimal hydrophobicity of the CE transfer path. The atomic level details presented here could accelerate the designing of safe and efficacious CETP inhibitors.

Enhanced stability and permeation potential of nanoemulsion containing sefsol-218 oil for topical delivery of amphotericin B.

AIM: To characterize the enhanced stability and permeation potential of amphotericin B nanoemulsion comprising sefsol-218 oil at varying pH and temperature of aqueous continuous phase. METHODOLOGY: Several batches of amphotericin B loaded nanoemulsion were prepared and evaluated for their physical and chemical stability at different pH and temperature. Also, a comparative study of ex vivo drug permeation across the albino rat skin was investigated with commercial Fungisome® and drug solution at 37 °C for 24 h. The extent of drug penetrated through the rat skin was thereby evaluated using the confocal laser scanning microscopy (CLSM). RESULTS AND CONCLUSIONS: The optimized nanoemulsion demonstrated the highest flux rate 17.85 ± 0.5 µg/cm(2)/h than drug solution (5.37 ± 0.01 µg/cm(2)/h) and Fungisome® (7.97 ± 0.01 µg/cm(2)/h). Ex vivo drug penetration mechanism from the developed formulations at pH 6.8 and pH 7.4 of aqueous phase pH using the CLSM revealed enhanced penetration. Ex vivo drug penetration studies of developed formulation comprising of CLSM revealed enhanced penetration in aqueous phase at pH 6.8 and 7.4. The aggregation behavior of nanoemulsion at both the pH was found to be minimum and non-nephrotoxic. The stability of amphotericin B was obtained in terms of pH, optical density, globular size, polydispersity index and zeta potential value at different temperature for 90 days. The slowest drug degradation was observed in aqueous phase at pH 7.4 with shelf life 20.03-folds higher when stored at 4 °C (3.8 years) and 5-fold higher at 25 °C (0.951 years) than at 40 °C. The combined results suggested that nanoemulsion may hold an alternative for enhanced and sustained topical delivery system for amphotericin B.

Formulation by design of felodipine loaded liquid and solid self nanoemulsifying drug delivery systems using Box-Behnken design.

OBJECTIVE: To design and develop liquid and solid self-nanoemulsifying drug delivery systems (SNEDDS and S-SNEDDS) of felodipine (FLD) using Box-Behnken design (BBD). METHODS: Solubility study was carried out in various vehicles. Ternary phase diagram was constructed to delineate the boundaries of the nanoemulsion domain. The content of formulation variables, X1 (Acconon E), X2 (Cremophor EL) and X3 (Lutrol E300) were optimized by assessment of 15 formulations (as per BBD) for mean globule sizes in Millipore water (Y1), 0.1 N HCl (Y2), phosphate buffer (pH 6.4) (Y3); emulsification time (Y4) and T85% (Y5). The responses (Y1-Y5) were evaluated statistically by analysis of variance and response surface plots to obtain optimum points. The optimized formulations were solidified by adsorption to solid carrier technique using Aerosil 200 (AER). RESULTS AND DISCUSSION: Transmission electron microscopy images confirmed the spherical shape of globules with the size range concordant with the globule size analysis by dynamic light scattering technique (<60 nm). The surface morphology of S-SNEDDS (before release) by scanning electron microscopy and atomic force microscopy indicated that SNEDDS are adsorbed uniformly on the surface of AER. The dried residue of S-SNEDDS (after release) revealed the presence of nanometric pores vacated by the previously adsorbed SNEDDS onto AER. Differential scanning calorimetry and X-ray powder diffraction studies illustrated the change of FLD from crystalline to amorphous state. CONCLUSION: This study indicates that owing to nanosize, SNEDDS and S-SNEDDS of FLD have potential to enhance its absorption and may serve an efficient oral delivery.

Cardioprotective effect of green tea extract on doxorubicin-induced cardiotoxicity in rats.

The in vivo antioxidant properties of green tea extract (GTE) were investigated against doxorubicin (DOX) induced cardiotoxicity in rats. In this experiment, 48 Wistar albino rats (200-250 g) were divided into eight groups (n = 6). Control group received normal saline for 30 days. Cardiotoxicity was induced by DOX (20 mg/kg ip.), once on 29th day of study and were treated with GTE (100, 200 and 400 mg/kg, p.o.) for 30 days. Aspartate aminotransferase (AST), creatinine kinase (CK), lactate dehydrogenase (LDH), lipid peroxidation (LPO), cytochrome P450 (CYP), blood glutathione, tissue glutathione, enzymatic and non-enzymatic antioxidants were evaluated along with histopathological studies. DOX treated rats showed a significant increased levels of AST, CK, LDH, LPO and CYP, which were restored by oral administration of GTE at doses 100, 200 and 400 mg/kg for 30 days. Moreover, GTE administration significantly increased the activities of glutathione peroxidase (GPX), glutathione reductase (GR), glutathione s-transferase (GST), superoxide dismutase (SOD) and catalase (CAT), in heart, which were reduced by DOX treatment. In this study, we have found that oral administration of GTE prevented DOX-induced cardiotoxicity by accelerating heart antioxidant defense mechanisms and down regulating the LPO levels to the normal levels.

High-fidelity, hyper-accurate, and evolved mutants rewire atomic-level communication in CRISPR-Cas9.

The high-fidelity (HF1), hyper-accurate (Hypa), and evolved (Evo) variants of the CRISPR-associated protein 9 (Cas9) endonuclease are critical tools to mitigate off-target effects in the application of CRISPR-Cas9 technology. The mechanisms by which mutations in recognition subdomain 3 (Rec3) mediate specificity in these variants are poorly understood. Here, solution nuclear magnetic resonance and molecular dynamics simulations establish the structural and dynamic effects of high-specificity mutations in Rec3, and how they propagate the allosteric signal of Cas9. We reveal conserved structural changes and dynamic differences at regions of Rec3 that interface with the RNA:DNA hybrid, transducing chemical signals from Rec3 to the catalytic His-Asn-His (HNH) domain. The variants remodel the communication sourcing from the Rec3 α helix 37, previously shown to sense target DNA complementarity, either directly or allosterically. This mechanism increases communication between the DNA mismatch recognition helix and the HNH active site, shedding light on the structure and dynamics underlying Cas9 specificity and providing insight for future engineering principles.

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.

RNA targeting and cleavage by the type III-Dv CRISPR effector complex.

CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction1-5. Target RNA cleavage at regular intervals is characteristic of type III effector complexes6-8. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors9,10, in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes.

The Electronic Structure of Genome Editors from the First Principles.

Genome editing based on the CRISPR-Cas9 system has paved new avenues for medicine, pharmaceutics, biotechnology, and beyond. This article reports the role of first-principles (ab-initio) molecular dynamics (MD) in the CRISPR-Cas9 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 characterize 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 of the Cas9 enzyme, providing valuable insights that can serve for the design of genome editing tools with improved catalytic efficiency or controllable activity.

Structural Insights into the Antiparallel G-Quadruplex in the Presence of K+ and Mg2+ Ions.

G-Quadruplex (GQ) is a secondary structural unit of DNA, formed at the telomere region of the chromosome with a high guanine content. It is reported that the GQs can hinder many biological processes. Thus, research thrives to explore the structural stability of GQs. Studies based on circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments established the vital role of cations such as K+ and Mg2+ in the stability of antiparallel G-quadruplexes (AGQs). However, there is a need to understand how stability in AGQ is attained in the presence of cations. Here, we employed molecular dynamics (MD) simulations, steered MD (SMD) simulations, and QM/MM calculations to understand the biophysical and electronic bases of the stability imparted to AGQs via cation binding. Our results showed that Mg2+ prefers to bind in the plane with the guanine tetrad, whereas K+ binds in between the AGQ tetrads. Thus, three Mg2+ cations or two K+ ions are needed to stabilize an AGQ molecule, where each and every tetrad binds to Mg2+ more robustly with a higher binding affinity. SMD revealed that the traversal of K+ through the AGQ central channel required less force than that of Mg2+, illustrating the presence of more strong interactions between Mg2+ and AGQ tetrads compared to K+. The stabilization in the AGQ tetrads due to cation binding is reassessed by employing ab initio simulations. Mixed QM/MM calculations confirmed that Mg2+ binds strongly to AGQ compared to K+, and it induces higher interactions between the guanine tetrads. However, K+ binding to AGQ induces a higher stabilization energy than Mg2+ binding to AGQ tetrads. Despite the higher binding energy, Mg2+ binding imparts lower stabilization to AGQ due to its unfavorable fermionic quantum energy.

Machines on Genes through the Computational Microscope.

Macromolecular machines acting on genes are at the core of life's fundamental processes, including DNA replication and repair, gene transcription and regulation, chromatin packaging, RNA splicing, and genome editing. Here, we report the increasing role of computational biophysics in characterizing the mechanisms of "machines on genes", focusing on innovative applications of computational methods and their integration with structural and biophysical experiments. We showcase how state-of-the-art computational methods, including classical and ab initio molecular dynamics to enhanced sampling techniques, and coarse-grained approaches are used for understanding and exploring gene machines for real-world applications. As this review unfolds, advanced computational methods describe the biophysical function that is unseen through experimental techniques, accomplishing the power of the "computational microscope", an expression coined by Klaus Schulten to highlight the extraordinary capability of computer simulations. Pushing the frontiers of computational biophysics toward a pragmatic representation of large multimegadalton biomolecular complexes is instrumental in bridging the gap between experimentally obtained macroscopic observables and the molecular principles playing at the microscopic level. This understanding will help harness molecular machines for medical, pharmaceutical, and biotechnological purposes.

High-Fidelity, Hyper-Accurate, and Evolved Mutants Rewire Atomic Level Communication in CRISPR-Cas9.

The Cas9-HF1, HypaCas9, and evoCas9 variants of the Cas9 endonuclease are critical tools to mitigate off-target effects in the application of CRISPR-Cas9 technology. The mechanisms by which mutations in the Rec3 domain mediate specificity in these variants are poorly understood. Here, solution NMR and molecular dynamics simulations establish the structural and dynamic effects of high-specificity mutations in Rec3, and how they propagate the allosteric signal of Cas9. We reveal conserved structural changes and peculiar dynamic differences at regions of Rec3 that interface with the RNA:DNA hybrid, transducing chemical signals from Rec3 to the catalytic HNH domain. The variants remodel the communication sourcing from the Rec3 α-helix 37, previously shown to sense target DNA complementarity, either directly or allosterically. This mechanism increases communication between the DNA mismatch recognition helix and the HNH active site, shedding light on the structure and dynamics underlying Cas9 specificity and providing insight for future engineering principles.

Type III CRISPR-Cas complexes act as protein-assisted ribozymes during target RNA cleavage.

CRISPR-Cas systems are an adaptive immune system in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction1-5. Target RNA cleavage at regular intervals is characteristic of type III effector complexes; however, the mechanism has remained enigmatic6,7. Here, we determine the structures of the Synechocystis type III-Dv complex, an evolutionary intermediate in type III effectors8,9, in pre- and post-cleavage states, which show metal ion coordination in the active sites. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we reveal the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Thus, type III CRISPR-Cas complexes function as protein-assisted ribozymes, and their programmable nature has important implications for how these complexes could be repurposed for applications.

Twisting and swiveling domain motions in Cas9 to recognize target DNA duplexes, make double-strand breaks, and release cleaved duplexes.

The CRISPR-associated protein 9 (Cas9) has been engineered as a precise gene editing tool to make double-strand breaks. CRISPR-associated protein 9 binds the folded guide RNA (gRNA) that serves as a binding scaffold to guide it to the target DNA duplex via a RecA-like strand-displacement mechanism but without ATP binding or hydrolysis. The target search begins with the protospacer adjacent motif or PAM-interacting domain, recognizing it at the major groove of the duplex and melting its downstream duplex where an RNA-DNA heteroduplex is formed at nanomolar affinity. The rate-limiting step is the formation of an R-loop structure where the HNH domain inserts between the target heteroduplex and the displaced non-target DNA strand. Once the R-loop structure is formed, the non-target strand is rapidly cleaved by RuvC and ejected from the active site. This event is immediately followed by cleavage of the target DNA strand by the HNH domain and product release. Within CRISPR-associated protein 9, the HNH domain is inserted into the RuvC domain near the RuvC active site via two linker loops that provide allosteric communication between the two active sites. Due to the high flexibility of these loops and active sites, biophysical techniques have been instrumental in characterizing the dynamics and mechanism of the CRISPR-associated protein 9 nucleases, aiding structural studies in the visualization of the complete active sites and relevant linker structures. Here, we review biochemical, structural, and biophysical studies on the underlying mechanism with emphasis on how CRISPR-associated protein 9 selects the target DNA duplex and rejects non-target sequences.

Principles of target DNA cleavage and the role of Mg2+ in the catalysis of CRISPR-Cas9.

At the core of the CRISPR-Cas9 genome-editing technology, the endonuclease Cas9 introduces site-specific breaks in DNA. However, precise mechanistic information to ameliorating Cas9 function is still missing. 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. This activated Mg2+-bound HNH is consistently described through molecular simulations, solution NMR and DNA cleavage assays, revealing also that the protonation state of the catalytic H840 is strongly affected by active site mutations. Finally, ab-initio QM(DFT)/MM simulations and metadynamics establish the catalytic mechanism, showing that the catalysis is activated by H840 and completed by K866, rationalising DNA cleavage experiments. This information is critical to enhance the enzymatic function of CRISPR-Cas9 toward improved genome-editing.