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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.

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.

CRISPR-Cas9 is a cutting-edge genome editing technology, which uses the endonuclease Cas9 to introduce mutations at desired sites of the genome. This revolutionary tool is promising to treat a myriad of human genetic diseases. Nevertheless, the molecular basis of DNA cleavage, which is a fundamental step for genome editing, has not been established. Here, quantum-classical molecular dynamics (MD) and free energy methods are used to disclose the two-metal-dependent mechanism of phosphodiester bond cleavage in CRISPR-Cas9. Ab initio MD reveals a conformational rearrangement of the Mg2+-bound RuvC active site, which entails the relocation of H983 to act as a general base. Then, the DNA cleavage proceeds through a concerted associative pathway fundamentally assisted by the joint dynamics of the two Mg2+ ions. This clarifies previous controversial experimental evidence, which could not fully establish the catalytic role of the conserved H983 and the metal cluster conformation. The comparison with other two-metal-dependent enzymes supports the identified mechanism and suggests a common catalytic strategy for genome editing and recombination. Overall, the non-target DNA cleavage catalysis described here resolves a fundamental open question in the CRISPR-Cas9 biology and provides valuable insights for improving the catalytic efficiency and the metal-dependent function of the Cas9 enzyme, which are at the basis of the development of genome editing tools.

CRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, all-atom enhanced molecular dynamics (MD) simulations – using Gaussian accelerated MD (GaMD) – are used to decipher the mechanism of off-target binding at the molecular level. GaMD reveals that base pair mismatches in the target DNA at specific distal sites with respect to the Protospacer Adjacent Motif (PAM) induce an extended opening of the RNA:DNA heteroduplex, which leads to newly discovered interactions between the unwound nucleic acids and the protein counterpart. The conserved interactions between the target DNA strand and the L2 loop of the catalytic HNH domain constitute a “lock” effectively decreasing the conformational freedom of the HNH domain and its activation for cleavage. Remarkably, depending on their position at PAM distal sites, DNA mismatches leading to off-target cleavages are unable to “lock” the HNH domain, thereby identifying the ability to “lock” HNH as a key determinant. Consistently, off-target sequences hampering the catalysis have been shown to “trap” somehow the HNH domain in an inactive “conformational checkpoint” state (Dagdas et al. Sci Adv, 2017). As such, this mechanism identifies the molecular basis underlying off-target cleavages and contributes in clarifying a long-lasting open issue of the CRISPR-Cas9 function. It also poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the “locking” interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.

Abstract CRISPR-Cas9 is the state-of-the-art technology for editing and manipulating nucleic acids. However, the occurrence of off-target mutations can limit its applicability. Here, all-atom enhanced molecular dynamics (MD) simulations – using Gaussian accelerated MD (GaMD) – are used to decipher the mechanism of off-target binding at the molecular level. GaMD reveals that base pair mismatches in the target DNA at specific distal sites with respect to the Protospacer Adjacent Motif (PAM) induce an extended opening of the RNA:DNA heteroduplex, which leads to newly discovered interactions between the unwound nucleic acids and the protein counterpart. The conserved interactions between the target DNA strand and the L2 loop of the catalytic HNH domain constitute a “lock” effectively decreasing the conformational freedom of the HNH domain and its activation for cleavage. Remarkably, depending on their position at PAM distal sites, DNA mismatches leading to off-target cleavages are unable to “lock” the HNH domain, thereby identifying the ability to “lock” HNH as a key determinant. Consistently, off-target sequences hampering the catalysis have been shown to “trap” somehow the HNH domain in an inactive “conformational checkpoint” state (Dagdas et al. Sci Adv, 2017). As such, this mechanism identifies the molecular basis underlying off-target cleavages and contributes in clarifying a long-lasting open issue of the CRISPR-Cas9 function. It also poses the foundation for designing novel and more specific Cas9 variants, which could be obtained by magnifying the “locking” interactions between HNH and the target DNA in the presence of any incorrect off-target sequence, thus preventing undesired cleavages.

Understanding the conformational dynamics of CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 is of the utmost importance for improving its genome editing capability. Here, molecular dynamics simulations performed using Anton-2 - a specialized supercomputer capturing micro-to-millisecond biophysical events in real time and at atomic-level resolution - reveal the activation process of the endonuclease Cas9 toward DNA cleavage. Over the unbiased simulation, we observe that the spontaneous approach of the catalytic domain HNH to the DNA cleavage site is accompanied by a remarkable structural remodeling of the recognition (REC) lobe, which exerts a key role for DNA cleavage. Specifically, the significant conformational changes and the collective conformational dynamics of the REC lobe indicate a mechanism by which the REC1-3 regions 'sense' nucleic acids, 'regulate' the HNH conformational transition, and ultimately 'lock' the HNH domain at the cleavage site, contributing to its catalytic competence. By integrating additional independent simulations and existing experimental data, we provide a solid validation of the activated HNH conformation, which had been so far poorly characterized, and we deliver a comprehensive understanding of the role of REC1-3 in the activation process. Considering the importance of the REC lobe in the specificity of Cas9, this study poses the basis for fully understanding how the REC components control the cleavage of off-target sequences, laying the foundation for future engineering efforts toward improved genome editing.

CRISPR-Cas9 is a genome editing technology with major impact in life sciences. In this system, the endonuclease Cas9 generates double strand breaks in DNA upon RNA-guided recognition of a complementary DNA sequence, which strictly requires the presence of a protospacer adjacent motif (PAM) next to the target site. Although PAM recognition is essential for cleavage, it is unknown whether and how PAM binding activates Cas9 for DNA cleavage at spatially distant sites. Here, we find evidence of a PAM-induced allosteric mechanism revealed by microsecond molecular dynamics simulations. PAM acts as an allosteric effector and triggers the interdependent conformational dynamics of the Cas9 catalytic domains (HNH and RuvC), responsible for concerted cleavage of the two DNA strands. Targeting such an allosteric mechanism should enable control of CRISPR-Cas9 functionality.

CRISPR-Cas9 has become a facile genome editing technology, yet the structural and mechanistic features underlying its function are unclear. Here, we perform extensive molecular simulations in an enhanced sampling regime, using a Gaussian-accelerated molecular dynamics (GaMD) methodology, which probes displacements over hundreds of microseconds to milliseconds, to reveal the conformational dynamics of the endonuclease Cas9 during its activation toward catalysis. We disclose the conformational transition of Cas9 from its apo form to the RNA-bound form, suggesting a mechanism for RNA recruitment in which the domain relocations cause the formation of a positively charged cavity for nucleic acid binding. GaMD also reveals the conformation of a catalytically competent Cas9, which is prone for catalysis and whose experimental characterization is still limited. We show that, upon DNA binding, the conformational dynamics of the HNH domain triggers the formation of the active state, explaining how the HNH domain exerts a conformational control domain over DNA cleavage [Sternberg SH et al. (2015) Nature, 527, 110-113]. These results provide atomic-level information on the molecular mechanism of CRISPR-Cas9 that will inspire future experimental investigations aimed at fully clarifying the biophysics of this unique genome editing machinery and at developing new tools for nucleic acid manipulation based on CRISPR-Cas9.

The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system recently emerged as a transformative genome-editing technology that is innovating basic bioscience and applied medicine and biotechnology. The endonuclease Cas9 associates with a guide RNA to match and cleave complementary sequences in double stranded DNA, forming an RNA:DNA hybrid and a displaced non-target DNA strand. Although extensive structural studies are ongoing, the conformational dynamics of Cas9 and its interplay with the nucleic acids during association and DNA cleavage are largely unclear. Here, by employing multi-microsecond time scale molecular dynamics, we reveal the conformational plasticity of Cas9 and identify key determinants that allow its large-scale conformational changes during nucleic acid binding and processing. We show how the "closure" of the protein, which accompanies nucleic acid binding, fundamentally relies on highly coupled and specific motions of the protein domains, collectively initiating the prominent conformational changes needed for nucleic acid association. We further reveal a key role of the non-target DNA during the process of activation of the nuclease HNH domain, showing how the nontarget DNA positioning triggers local conformational changes that favor the formation of a catalytically competent Cas9. Finally, a remarkable conformational plasticity is identified as an intrinsic property of the HNH domain, constituting a necessary element that allows for the HNH repositioning. These novel findings constitute a reference for future experimental studies aimed at a full characterization of the dynamic features of the CRISPR-Cas9 system, and-more importantly-call for novel structure engineering efforts that are of fundamental importance for the rational design of new genome-engineering applications.

A framework for open discourse on the use of CRISPR-Cas9 technology to manipulate the human genome is urgently needed

Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.

In many bacteria and archaea, small RNAs derived from clustered regularly interspaced short palindromic repeats (CRISPRs) associate with CRISPR-associated (Cas) proteins to target foreign DNA for destruction. In Type I and III CRISPR/Cas systems, the Cas6 family of endoribonucleases generates functional CRISPR-derived RNAs by site-specific cleavage of repeat sequences in precursor transcripts. CRISPR repeats differ widely in both sequence and structure, with varying propensity to form hairpin folds immediately preceding the cleavage site. To investigate the evolution of distinct mechanisms for the recognition of diverse CRISPR repeats by Cas6 enzymes, we determined crystal structures of two Thermus thermophilus Cas6 enzymes both alone and bound to substrate and product RNAs. These structures show how the scaffold common to all Cas6 endonucleases has evolved two binding sites with distinct modes of RNA recognition: one specific for a hairpin fold and the other for a single-stranded 5'-terminal segment preceding the hairpin. These findings explain how divergent Cas6 enzymes have emerged to mediate highly selective pre-CRISPR-derived RNA processing across diverse CRISPR systems.

RECQL5 is a member of the highly conserved RecQ family of DNA helicases involved in DNA repair. RECQL5 interacts with RNA polymerase II (Pol II) and inhibits transcription of protein-encoding genes by an unknown mechanism. We show that RECQL5 contacts the Rpb1 jaw domain of Pol II at a site that overlaps with the binding site for the transcription elongation factor TFIIS. Our cryo-EM structure of elongating Pol II arrested in complex with RECQL5 shows that the RECQL5 helicase domain is positioned to sterically block elongation. The crystal structure of the RECQL5 KIX domain reveals similarities with TFIIS, and binding of RECQL5 to Pol II interferes with the ability of TFIIS to promote transcriptional read-through in vitro. Together, our findings reveal a dual mode of transcriptional repression by RECQL5 that includes structural mimicry of the Pol II-TFIIS interaction.

Type II CRISPR immune systems in bacteria use a dual RNA-guided DNA endonuclease, Cas9, to cleave foreign DNA at specific sites. We show here that Cas9 assembles with hybrid guide RNAs in human cells and can induce the formation of double-strand DNA breaks (DSBs) at a site complementary to the guide RNA sequence in genomic DNA. This cleavage activity requires both Cas9 and the complementary binding of the guide RNA. Experiments using extracts from transfected cells show that RNA expression and/or assembly into Cas9 is the limiting factor for Cas9-mediated DNA cleavage. In addition, we find that extension of the RNA sequence at the 3' end enhances DNA targeting activity in vivo. These results show that RNA-programmed genome editing is a facile strategy for introducing site-specific genetic changes in human cells.DOI:http://dx.doi.org/10.7554/eLife.00471.001.

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.

Messenger RNA decay plays a central role in the regulation and surveillance of eukaryotic gene expression. The conserved multidomain exoribonuclease Xrn1 targets cytoplasmic RNA substrates marked by a 5' monophosphate for processive 5'-to-3' degradation by an unknown mechanism. Here, we report the crystal structure of an Xrn1-substrate complex. The single-stranded substrate is held in place by stacking of the 5'-terminal trinucleotide between aromatic side chains while a highly basic pocket specifically recognizes the 5' phosphate. Mutations of residues involved in binding the 5'-terminal nucleotide impair Xrn1 processivity. The substrate recognition mechanism allows Xrn1 to couple processive hydrolysis to duplex melting in RNA substrates with sufficiently long single-stranded 5' overhangs. The Xrn1-substrate complex structure thus rationalizes the exclusive specificity of Xrn1 for 5'-monophosphorylated substrates, ensuring fidelity of mRNA turnover, and posits a model for translocation-coupled unwinding of structured RNA substrates.

Many bacteria and archaea contain clustered regularly interspaced short palindromic repeats (CRISPRs) that confer resistance to invasive genetic elements. Central to this immune system is the production of CRISPR-derived RNAs (crRNAs) after transcription of the CRISPR locus. Here, we identify the endoribonuclease (Csy4) responsible for CRISPR transcript (pre-crRNA) processing in Pseudomonas aeruginosa. A 1.8 angstrom crystal structure of Csy4 bound to its cognate RNA reveals that Csy4 makes sequence-specific interactions in the major groove of the crRNA repeat stem-loop. Together with electrostatic contacts to the phosphate backbone, these enable Csy4 to bind selectively and cleave pre-crRNAs using phylogenetically conserved serine and histidine residues in the active site. The RNA recognition mechanism identified here explains sequence- and structure-specific processing by a large family of CRISPR-specific endoribonucleases.

GW182-family proteins are essential for microRNA-mediated translational repression and deadenylation in animal cells. Here we show that a conserved motif in the human GW182 paralog TNRC6C interacts with the C-terminal domain of polyadenylate binding protein 1 (PABC) and present the crystal structure of the complex. Mutations at the complex interface impair mRNA deadenylation in mammalian cell extracts, suggesting that the GW182-PABC interaction contributes to microRNA-mediated gene silencing.

MicroRNAs (miRNAs) inhibit mRNA expression in general by base pairing to the 3'UTR of target mRNAs and consequently inhibiting translation and/or initiating poly(A) tail deadenylation and mRNA destabilization. Here we examine the mechanism and kinetics of miRNA-mediated deadenylation in mouse Krebs-2 ascites extract. We demonstrate that miRNA-mediated mRNA deadenylation occurs subsequent to initial translational inhibition, indicating a two-step mechanism of miRNA action, which serves to consolidate repression. We show that a let-7 miRNA-loaded RNA-induced silencing complex (miRISC) interacts with the poly(A)-binding protein (PABP) and the CAF1 and CCR4 deadenylases. In addition, we demonstrate that miRNA-mediated deadenylation is dependent upon CAF1 activity and PABP, which serves as a bona fide miRNA coactivator. Importantly, we present evidence that GW182, a core component of the miRISC, directly interacts with PABP via its C-terminal region and that this interaction is required for miRNA-mediated deadenylation.