RESUMO
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.
Assuntos
Sistemas CRISPR-Cas , RNA Guia de Cinetoplastídeos , RNA Guia de Cinetoplastídeos/metabolismo , Endonucleases/metabolismo , Pareamento de Bases , Nucleotídeos , Edição de GenesRESUMO
Cas13a is a recent addition to the CRISPR-Cas toolkit that exclusively targets RNA, which makes it a promising tool for RNA detection. It utilizes a CRISPR RNA (crRNA) to target RNA sequences and trigger a composite active site formed by two 'Higher Eukaryotes and Prokaryotes Nucleotide' (HEPN) domains, cleaving any solvent-exposed RNA. In this system, an intriguing form of allosteric communication controls the RNA cleavage activity, yet its molecular details are unknown. Here, multiple-microsecond molecular dynamics simulations are combined with graph theory to decipher this intricate activation mechanism. We show that the binding of a target RNA acts as an allosteric effector, by amplifying the communication signals over the dynamical noise through interactions of the crRNA at the buried HEPN1-2 interface. By introducing a novel Signal-to-Noise Ratio (SNR) of communication efficiency, we reveal critical allosteric residues-R377, N378, and R973-that rearrange their interactions upon target RNA binding. Alanine mutation of these residues is shown to select target RNA over an extended complementary sequence beyond guide-target duplex for RNA cleavage, establishing the functional significance of these hotspots. Collectively our findings offer a fundamental understanding of the Cas13a mechanism of action and pave new avenues for the development of highly selective RNA-based cleavage and detection tools.
Assuntos
Proteínas Associadas a CRISPR , RNA Guia de Sistemas CRISPR-Cas , Regulação Alostérica , Sistemas CRISPR-Cas , Mutação , RNA/genética , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismoRESUMO
An increasingly pressing need for clinical diagnostics has required the development of novel nucleic acid-based detection technologies that are sensitive, fast, and inexpensive, and that can be deployed at point-of-care. Recently, the RNA-guided ribonuclease CRISPR-Cas13 has been successfully harnessed for such purposes. However, developing assays for detection of genetic variability, for example single-nucleotide polymorphisms, is still challenging and previously described design strategies are not always generalizable. Here, we expanded our characterization of LbuCas13a RNA-detection specificity by performing a combination of experimental RNA mismatch tolerance profiling, molecular dynamics simulations, protein, and crRNA engineering. We found certain positions in the crRNA-target-RNA duplex that are particularly sensitive to mismatches and establish the effect of RNA concentration in mismatch tolerance. Additionally, we determined that shortening the crRNA spacer or modifying the direct repeat of the crRNA leads to stricter specificities. Furthermore, we harnessed our understanding of LbuCas13a allosteric activation pathways through molecular dynamics and structure-guided engineering to develop novel Cas13a variants that display increased sensitivities to single-nucleotide mismatches. We deployed these Cas13a variants and crRNA design strategies to achieve superior discrimination of SARS-CoV-2 strains compared to wild-type LbuCas13a. Together, our work provides new design criteria and Cas13a variants to use in future easier-to-implement Cas13-based RNA detection applications.
Assuntos
RNA Guia de Sistemas CRISPR-Cas , RNA , RNA/genética , Sistemas CRISPR-CasRESUMO
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.
Assuntos
Proteínas Associadas a CRISPR , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , DNA/genética , Clivagem do DNA , DNA de Cadeia Simples/genéticaRESUMO
A hallmark of tightly regulated high-fidelity enzymes is that they become activated only after encountering cognate substrates, often by an induced-fit mechanism rather than conformational selection. Upon analysis of molecular dynamics trajectories, we recently discovered that the Cas9 HNH domain exists in three conformations: 1) Y836 (which is two residues away from the catalytic D839 and H840 residues) is hydrogen bonded to the D829 backbone amide, 2) Y836 is hydrogen bonded to the backbone amide of D861 (which is one residue away from the third catalytic residue N863), and 3) Y836 is not hydrogen bonded to either residue. Each of the three conformers differs from the active state of HNH. The conversion between the inactive and active states involves a local unfolding-refolding process that displaces the Cα and side chain of the catalytic N863 residue by â¼5 Å and â¼10 Å, respectively. In this study, we report the two largest principal components of coordinate variance of the HNH domain throughout molecular dynamics trajectories to establish the interconversion pathways of these conformations. We show that conformation 2 is an obligate step between conformations 1 and 3, which are not directly interconvertible without conformation 2. The loss of hydrogen bonding of the Y836 side chain in conformation 3 likely plays an essential role in activation during local unfolding-refolding of an α-helix containing the catalytic N863. Three single Lys-to-Ala mutants appear to eliminate this substrate-independent activation pathway of the wild-type HNH nuclease, thereby enhancing the fidelity of HNH cleavage.
Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Simulação de Dinâmica Molecular , Hidrogênio/metabolismo , AmidasRESUMO
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.
RESUMO
Many bacteria possess type-II immunity against invading phages or plasmids known as the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated 9 (Cas9) system to detect and degrade the foreign DNA sequences. The Cas9 protein has two endonucleases responsible for double-strand breaks (the HNH domain for cleaving the target strand of DNA duplexes and RuvC domain for the nontarget strand, respectively) and a single-guide RNA-binding domain where the RNA and target DNA strands are base-paired. Three engineered single Lys-to-Ala HNH mutants (K810A, K848A, and K855A) exhibit an enhanced substrate specificity for cleavage of the target DNA strand. We report in this study that in the wild-type (wt) enzyme, D835, Y836, and D837 within the Y836-containing loop (comprising E827-D837) adjacent to the catalytic site have uncharacterizable broadened 1H15N nuclear magnetic resonance (NMR) features, whereas remaining residues in the loop have different extents of broadened NMR spectra. We find that this loop in the wt enzyme exhibits three distinct conformations over the duration of the molecular dynamics simulations, whereas the three Lys-to-Ala mutants retain only one conformation. The versatility of multiple alternate conformations of this loop in the wt enzyme could help to recruit noncognate DNA substrates into the HNH active site for cleavage, thereby reducing its substrate specificity relative to the three mutants. Our study provides further experimental and computational evidence that Lys-to-Ala substitutions reduce dynamics of proteins and thus increase their stability.
Assuntos
Sistemas CRISPR-Cas , Endonucleases , Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA/química , DNA/genética , Endonucleases/químicaRESUMO
CRISPR-Cas9 is a widely used biochemical tool with applications in molecular biology and precision medicine. The RNA-guided Cas9 protein uses its HNH endonuclease domain to cleave the DNA strand complementary to its endogenous guide RNA. In this study, novel constructs of HNH from two divergent organisms, G. stearothermophilus (GeoHNH) and S. pyogenes (SpHNH) were engineered from their respective full-length Cas9 proteins. Despite low sequence similarity, the X-ray crystal structures of these constructs reveal that the core of HNH surrounding the active site is conserved. Structure prediction of the full-length GeoCas9 protein using Phyre2 and AlphaFold2 also showed that the crystallographic construct of GeoHNH represents the structure of the domain within the full-length GeoCas9 protein. However, significant differences are observed in the solution dynamics of structurally conserved regions of GeoHNH and SpHNH, the latter of which was shown to use such molecular motions to propagate the DNA cleavage signal. Indeed, molecular simulations show that the intradomain signaling pathways, which drive SpHNH function, are non-specific and poorly formed in GeoHNH. Taken together, these outcomes suggest mechanistic differences between mesophilic and thermophilic Cas9 species.
Assuntos
Sistemas CRISPR-Cas , Simulação de Dinâmica Molecular , Proteína 9 Associada à CRISPR/química , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Streptococcus pyogenes/metabolismoRESUMO
We present a user-friendly front-end for running molecular dynamics (MD) simulations using the OpenMM toolkit on the Google Colab framework. Our goals are (1) to highlight the usage of a cloud-computing scheme for educational purposes for a hands-on approach when learning MD simulations and (2) to exemplify how low-income research groups can perform MD simulations in the microsecond time scale. We hope this work facilitates teaching and learning of molecular simulation throughout the community.
Assuntos
Computação em Nuvem , Simulação de Dinâmica MolecularRESUMO
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.
Assuntos
COVID-19/diagnóstico , DNA Viral/análise , DNA Viral/genética , SARS-CoV-2/genética , Sistemas CRISPR-Cas , Domínio Catalítico , Clivagem do DNA , Edição de Genes , Humanos , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Transição de Fase , Especificidade por SubstratoRESUMO
Lipid-linked oligosaccharides (LLOs) play an important role in the N-glycosylation pathway as the donor substrate of oligosaccharyltransferases (OSTs), which are responsible for the en bloc transfer of glycan chains onto a nascent polypeptide. The lipid component of LLO in both eukarya and archaea consists of a dolichol, and an undecaprenol in prokarya, whereas the number of isoprene units may change between species. Given the potential relevance of LLOs and their related enzymes to diverse biotechnological applications, obtaining reliable LLO models from distinct domains of life could support further studies on complex formation and their processing by OSTs, as well as protein engineering on such systems. In this work, molecular modeling techniques, such as quantum mechanics calculations, molecular dynamics simulations, and metadynamics were employed to study eukaryotic (Glc3-Man9-GlcNAc2-PP-Dolichol), bacterial (Glc1-GalNAc5-Bac1-PP-Undecaprenol), and archaeal (Glc1-Man1-Gal1-Man1-Glc1-Gal1-Glc1-P-Dolichol) LLOs in membrane bilayers. Microsecond molecular dynamics simulations and metadynamics calculations of LLOs revealed that glycan chains are more prone to interact with the membrane lipid head groups, while the PP linkages are positioned at the lipid phosphate head groups level. The dynamics of isoprenoid chains embedded within the bilayer are described, and membrane dynamics and related properties are also investigated. Overall, there are similarities regarding the structure and dynamics of the eukaryotic, the bacterial, and the archaeal LLOs in bilayers, which can support the comprehension of their association with OSTs. These data may support future studies on the transferring mechanism of the oligosaccharide chain to an acceptor protein.
Assuntos
Metabolismo dos Lipídeos , Modelos Moleculares , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Configuração de Carboidratos , Membrana Celular/metabolismo , GlicosilaçãoRESUMO
The aerial parts of Ipomoea batatas are described herein to produce four new resin glycosides, designated as ipomotaosides A, B, C, and D. Ipomotaoside A was found to present inhibitory activity on both cyclooxygenases. However, the conformational elucidation of these molecules may be difficult due to their high flexibility. In this context, the current work presents a conformational characterization of ipomotaosides A-D in aqueous and nonaqueous solvents. The employed protocol includes metadynamics evaluation and unrestrained molecular dynamics simulations (MD). The obtained data provided structural models for the ipomotaosides in good agreement with previous ROESY distances measured in pyridine. Accordingly, the most abundant conformation of ipomotaoside A in solution was employed in flexible docking studies, providing a structural basis for the compound's inhibition of COX enzymes. The so-obtained complex supports resin glycosides' role as original scaffolds for future studies, aiming at structural optimization and development of potential new anti-inflammatory agents.
Assuntos
Ciclo-Oxigenase 1/química , Ciclo-Oxigenase 2/química , Inibidores de Ciclo-Oxigenase/química , Glicosídeos/química , Ipomoea batatas/química , Configuração de Carboidratos , Sequência de Carboidratos , Espectroscopia de Ressonância Magnética , Simulação de Dinâmica Molecular , Extratos Vegetais/química , Ligação Proteica , Resinas Vegetais/químicaRESUMO
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.
Assuntos
Sistemas CRISPR-Cas , RNA Guia de Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , DNA/genética , Edição de Genes , CatáliseRESUMO
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.
Assuntos
Nucleossomos , Humanos , Nucleossomos/química , Simulação de Dinâmica Molecular , Replicação do DNA , Reparo do DNA , Splicing de RNA , Spliceossomos , Transcrição Gênica , Edição de GenesRESUMO
Cas13a is a recent addition to the CRISPR-Cas toolkit that exclusively targets RNA, which makes it a promising tool for RNA detection. The protein uses a CRISPR RNA (crRNA) to target RNA sequences, which are cleaved by a composite active site formed by two 'Higher Eukaryotes and Prokaryotes Nucleotide' (HEPN) catalytic domains. In this system, an intriguing form of allosteric communication controls RNA cleavage activity, yet its molecular details are unknown. Here, multiple-microsecond molecular dynamics simulations are combined with graph theory and RNA cleavage assays to decipher this activation mechanism. We show that the binding of a target RNA acts as an allosteric effector of the spatially distant HEPN catalytic cleft, by amplifying the allosteric signals over the dynamical noise, that passes through the buried HEPN interface. Critical residues in this region - N378, R973, and R377 - rearrange their interactions upon target RNA binding, and alter allosteric signalling. Alanine mutation of these residues is experimentally shown to select target RNA over an extended complementary sequence beyond guide-target duplex, for RNA cleavage. Altogether, our findings offer a fundamental understanding of the Cas13a mechanism of action and pave new avenues for the development of more selective RNA-based cleavage and detection tools.
RESUMO
The pressing need for clinical diagnostics has required the development of novel nucleic acid-based detection technologies that are sensitive, fast, and inexpensive, and that can be deployed at point-of-care. Recently, the RNA-guided ribonuclease CRISPR-Cas13 has been successfully harnessed for such purposes. However, developing assays for detection of genetic variability, for example single-nucleotide polymorphisms, is still challenging and previously described design strategies are not always generalizable. Here, we expanded our characterization of LbuCas13a RNA-detection specificity by performing a combination of experimental RNA mismatch tolerance profiling, molecular dynamics simulations, protein, and crRNA engineering. We found certain positions in the crRNA-target-RNA duplex that are particularly sensitive to mismatches and establish the effect of RNA concentration in mismatch tolerance. Additionally, we determined that shortening the crRNA spacer or modifying the direct repeat of the crRNA leads to stricter specificities. Furthermore, we harnessed our understanding of LbuCas13a allosteric activation pathways through molecular dynamics and structure-guided engineering to develop novel Cas13a variants that display increased sensitivities to single-nucleotide mismatches. We deployed these Cas13a variants and crRNA design strategies to achieve superior discrimination of SARS-CoV-2 strains compared to wild-type LbuCas13a. Together, our work provides new design criteria and new Cas13a variants for easier-to-implement Cas13-based diagnostics. KEY POINTS: Certain positions in the Cas13a crRNA-target-RNA duplex are particularly sensitive to mismatches.Understanding Cas13a's allosteric activation pathway allowed us to develop novel high-fidelity Cas13a variants.These Cas13a variants and crRNA design strategies achieve superior discrimination of SARS-CoV-2 strains.
RESUMO
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.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Proteína 9 Associada à CRISPR , Clivagem do DNA , Edição de Genes/métodos , Simulação de Dinâmica MolecularRESUMO
Many large protein-nucleic acid complexes exhibit allosteric regulation. In these systems, the propagation of the allosteric signaling is strongly coupled to conformational dynamics and catalytic function, challenging state-of-the-art analytical methods. Here, we review established and innovative approaches used to elucidate allosteric mechanisms in these complexes. Specifically, we report network models derived from graph theory and centrality analyses in combination with molecular dynamics (MD) simulations, introducing novel schemes that implement the synergistic use of graph theory with enhanced simulations methods and ab-initio MD. Accelerated MD simulations are used to construct "enhanced network models", describing the allosteric response over long timescales and capturing the relation between allostery and conformational changes. "Ab-initio network models" combine graph theory with ab-initio MD and quantum mechanics/molecular mechanics (QM/MM) simulations to describe the allosteric regulation of catalysis by following the step-by-step dynamics of biochemical reactions. This approach characterizes how the allosteric regulation changes from reactants to products and how it affects the transition state, revealing a tense-to-relaxed allosteric regulation along the chemical step. Allosteric models and applications are showcased for three paradigmatic examples of allostery in protein-nucleic acid complexes: (i) the nucleosome core particle, (ii) the CRISPR-Cas9 genome editing system and (iii) the spliceosome. These methods and applications create innovative protocols to determine allosteric mechanisms in protein-nucleic acid complexes that show tremendous promise for medicine and bioengineering.
Assuntos
DNA , Proteínas , Regulação Alostérica , Sistemas CRISPR-Cas , DNA/química , Edição de Genes , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Nucleossomos/química , Conformação Proteica , Proteínas/química , Spliceossomos/químicaRESUMO
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.
RESUMO
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.