Live-cell imaging reveals the trade-off between target search flexibility and efficiency for Cas9 and Cas12a.

CRISPR-Cas systems have widely been adopted as genome editing tools, with two frequently employed Cas nucleases being SpyCas9 and LbCas12a. Although both nucleases use RNA guides to find and cleave target DNA sites, the two enzymes differ in terms of protospacer-adjacent motif (PAM) requirements, guide architecture and cleavage mechanism. In the last years, rational engineering led to the creation of PAM-relaxed variants SpRYCas9 and impLbCas12a to broaden the targetable DNA space. By employing their catalytically inactive variants (dCas9/dCas12a), we quantified how the protein-specific characteristics impact the target search process. To allow quantification, we fused these nucleases to the photoactivatable fluorescent protein PAmCherry2.1 and performed single-particle tracking in cells of Escherichia coli. From our tracking analysis, we derived kinetic parameters for each nuclease with a non-targeting RNA guide, strongly suggesting that interrogation of DNA by LbdCas12a variants proceeds faster than that of SpydCas9. In the presence of a targeting RNA guide, both simulations and imaging of cells confirmed that LbdCas12a variants are faster and more efficient in finding a specific target site. Our work demonstrates the trade-off of relaxing PAM requirements in SpydCas9 and LbdCas12a using a powerful framework, which can be applied to other nucleases to quantify their DNA target search.

Cas3-Derived Target DNA Degradation Fragments Fuel Primed CRISPR Adaptation.

Prokaryotes use a mechanism called priming to update their CRISPR immunological memory to rapidly counter revisiting, mutated viruses, and plasmids. Here we have determined how new spacers are produced and selected for integration into the CRISPR array during priming. We show that Cas3 couples CRISPR interference to adaptation by producing DNA breakdown products that fuel the spacer integration process in a two-step, PAM-associated manner. The helicase-nuclease Cas3 pre-processes target DNA into fragments of about 30-100 nt enriched for thymine-stretches in their 3' ends. The Cas1-2 complex further processes these fragments and integrates them sequence-specifically into CRISPR repeats by coupling of a 3' cytosine of the fragment. Our results highlight that the selection of PAM-compliant spacers during priming is enhanced by the combined sequence specificities of Cas3 and the Cas1-2 complex, leading to an increased propensity of integrating functional CTT-containing spacers.

Quantitative parameters of bacterial RNA polymerase open-complex formation, stabilization and disruption on a consensus promoter.

Transcription initiation is the first step in gene expression, and is therefore strongly regulated in all domains of life. The RNA polymerase (RNAP) first associates with the initiation factor $\sigma$ to form a holoenzyme, which binds, bends and opens the promoter in a succession of reversible states. These states are critical for transcription regulation, but remain poorly understood. Here, we addressed the mechanism of open complex formation by monitoring its assembly/disassembly kinetics on individual consensus lacUV5 promoters using high-throughput single-molecule magnetic tweezers. We probed the key protein-DNA interactions governing the open-complex formation and dissociation pathway by modulating the dynamics at different concentrations of monovalent salts and varying temperatures. Consistent with ensemble studies, we observed that RNAP-promoter open (RPO) complex is a stable, slowly reversible state that is preceded by a kinetically significant open intermediate (RPI), from which the holoenzyme dissociates. A strong anion concentration and type dependence indicates that the RPO stabilization may involve sequence-independent interactions between the DNA and the holoenzyme, driven by a non-Coulombic effect consistent with the non-template DNA strand interacting with $\sigma$ and the RNAP $\beta$ subunit. The temperature dependence provides the energy scale of open-complex formation and further supports the existence of additional intermediates.

Temperature controlled high-throughput magnetic tweezers show striking difference in activation energies of replicating viral RNA-dependent RNA polymerases.

RNA virus survival depends on efficient viral genome replication, which is performed by the viral RNA dependent RNA polymerase (RdRp). The recent development of high throughput magnetic tweezers has enabled the simultaneous observation of dozens of viral RdRp elongation traces on kilobases long templates, and this has shown that RdRp nucleotide addition kinetics is stochastically interrupted by rare pauses of 1-1000 s duration, of which the short-lived ones (1-10 s) are the temporal signature of a low fidelity catalytic pathway. We present a simple and precise temperature controlled system for magnetic tweezers to characterize the replication kinetics temperature dependence between 25°C and 45°C of RdRps from three RNA viruses, i.e. the double-stranded RNA bacteriophage Φ6, and the positive-sense single-stranded RNA poliovirus (PV) and human rhinovirus C (HRV-C). We found that Φ6 RdRp is largely temperature insensitive, while PV and HRV-C RdRps replication kinetics are activated by temperature. Furthermore, the activation energies we measured for PV RdRp catalytic state corroborate previous estimations from ensemble pre-steady state kinetic studies, further confirming the catalytic origin of the short pauses and their link to temperature independent RdRp fidelity. This work will enable future temperature controlled study of biomolecular complex at the single molecule level.

What is all this fuss about Tus? Comparison of recent findings from biophysical and biochemical experiments.

Synchronizing the convergence of the two-oppositely moving DNA replication machineries at specific termination sites is a tightly coordinated process in bacteria. In Escherichia coli, a "replication fork trap" - found within a chromosomal region where forks are allowed to enter but not leave - is set by the protein-DNA roadblock Tus-Ter. The exact sequence of events by which Tus-Ter blocks replisomes approaching from one direction but not the other has been the subject of controversy for many decades. Specific protein-protein interactions between the nonpermissive face of Tus and the approaching helicase were challenged by biochemical and structural studies. These studies show that it is the helicase-induced strand separation that triggers the formation of new Tus-Ter interactions at the nonpermissive face - interactions that result in a highly stable "locked" complex. This controversy recently gained renewed attention as three single-molecule-based studies scrutinized this elusive Tus-Ter mechanism - leading to new findings and refinement of existing models, but also generating new questions. Here, we discuss and compare the findings of each of the single-molecule studies to find their common ground, pinpoint the crucial differences that remain, and push the understanding of this bipartite DNA-protein system further.

Why Argonaute is needed to make microRNA target search fast and reliable.

MicroRNA (miRNA) interferes with the translation of cognate messenger RNA (mRNA) by finding, preferentially binding, and marking it for degradation. To facilitate the search process, Argonaute (Ago) proteins come together with miRNA, forming a dynamic search complex. In this review we use the language of free-energy landscapes to discuss recent single-molecule and high-resolution structural data in the light of theoretical work appropriated from the study of transcription-factor search. We suggest that experimentally observed internal states of the Ago-miRNA search complex may have the explicit biological function of speeding up search while maintaining specificity.

Crowding-induced transcriptional bursts dictate polymerase and nucleosome density profiles along genes.

During eukaryotic transcription, RNA polymerase (RNAP) translocates along DNA molecules covered with nucleosomes and other DNA binding proteins. Though the interactions between a single nucleosome and RNAP are by now fairly well understood, this understanding has not been synthesized into a description of transcription on crowded genes, where multiple RNAP transcribe through nucleosomes while preserving the nucleosome coverage. We here take a deductive modeling approach to establish the consequences of RNAP-nucleosome interactions for transcription in crowded environments. We show that under physiologically crowded conditions, the interactions of RNAP with nucleosomes induce a strong kinetic attraction between RNAP molecules, causing them to self-organize into stable and moving pelotons. The peloton formation quantitatively explains the observed nucleosome and RNAP depletion close to the initiation site on heavily transcribed genes. Pelotons further translate into short-timescale transcriptional bursts at termination, resulting in burst characteristics consistent with instances of bursty transcription observed in vivo. To facilitate experimental testing of our proposed mechanism, we present several analytic relations that make testable quantitative predictions.

Switching between Exonucleolysis and Replication by T7 DNA Polymerase Ensures High Fidelity.

DNA polymerase catalyzes the accurate transfer of genetic information from one generation to the next, and thus it is vitally important for replication to be faithful. DNA polymerase fulfills the strict requirements for fidelity by a combination of mechanisms: 1) high selectivity for correct nucleotide incorporation, 2) a slowing down of the replication rate after misincorporation, and 3) proofreading by excision of misincorporated bases. To elucidate the kinetic interplay between replication and proofreading, we used high-resolution optical tweezers to probe how DNA-duplex stability affects replication by bacteriophage T7 DNA polymerase. Our data show highly irregular replication dynamics, with frequent pauses and direction reversals as the polymerase cycles through the states that govern the mechanochemistry behind high-fidelity T7 DNA replication. We constructed a kinetic model that incorporates both existing biochemical data and the, to our knowledge, novel states we observed. We fit the model directly to the acquired pause-time and run-time distributions. Our findings indicate that the main pathway for error correction is DNA polymerase dissociation-mediated DNA transfer, followed by biased binding into the exonuclease active site. The number of bases removed by this proofreading mechanism is much larger than the number of erroneous bases that would be expected to be incorporated, ensuring a high-fidelity replication of the bacteriophage T7 genome.

Strand separation establishes a sustained lock at the Tus-Ter replication fork barrier.

The bidirectional replication of a circular chromosome by many bacteria necessitates proper termination to avoid the head-on collision of the opposing replisomes. In Escherichia coli, replisome progression beyond the termination site is prevented by Tus proteins bound to asymmetric Ter sites. Structural evidence indicates that strand separation on the blocking (nonpermissive) side of Tus-Ter triggers roadblock formation, but biochemical evidence also suggests roles for protein-protein interactions. Here DNA unzipping experiments demonstrate that nonpermissively oriented Tus-Ter forms a tight lock in the absence of replicative proteins, whereas permissively oriented Tus-Ter allows nearly unhindered strand separation. Quantifying the lock strength reveals the existence of several intermediate lock states that are impacted by mutations in the lock domain but not by mutations in the DNA-binding domain. Lock formation is highly specific and exceeds reported in vivo efficiencies. We postulate that protein-protein interactions may actually hinder, rather than promote, proper lock formation.

Backtracking behavior in viral RNA-dependent RNA polymerase provides the basis for a second initiation site.

Transcription in RNA viruses is highly dynamic, with a variety of pauses interrupting nucleotide addition by RNA-dependent RNA polymerase (RdRp). For example, rare but lengthy pauses (>20 s) have been linked to backtracking for viral single-subunit RdRps. However, while such backtracking has been well characterized for multi-subunit RNA polymerases (RNAPs) from bacteria and yeast, little is known about the details of viral RdRp backtracking and its biological roles. Using high-throughput magnetic tweezers, we quantify the backtracking by RdRp from the double-stranded (ds) RNA bacteriophage Φ6, a model system for RdRps. We characterize the probability of entering long backtracks as a function of force and propose a model in which the bias toward backtracking is determined by the base paring at the dsRNA fork. We further discover that extensive backtracking provides access to a new 3'-end that allows for the de novo initiation of a second RdRp. This previously unidentified behavior provides a new mechanism for rapid RNA synthesis using coupled RdRps and hints at a possible regulatory pathway for gene expression during viral RNA transcription.

Visualization and quantification of nascent RAD51 filament formation at single-monomer resolution.

During recombinational repair of double-stranded DNA breaks, RAD51 recombinase assembles as a nucleoprotein filament around single-stranded DNA to form a catalytically proficient structure able to promote homology recognition and strand exchange. Mediators and accessory factors guide the action and control the dynamics of RAD51 filaments. Elucidation of these control mechanisms necessitates development of approaches to quantitatively probe transient aspects of RAD51 filament dynamics. Here, we combine fluorescence microscopy, optical tweezers, and microfluidics to visualize the assembly of RAD51 filaments on bare single-stranded DNA and quantify the process with single-monomer sensitivity. We show that filaments are seeded from RAD51 nuclei that are heterogeneous in size. This heterogeneity appears to arise from the energetic balance between RAD51 self-assembly in solution and the size-dependent interaction time of the nuclei with DNA. We show that nucleation intrinsically is substrate selective, strongly favoring filament formation on bare single-stranded DNA. Furthermore, we devised a single-molecule fluorescence recovery after photobleaching assay to independently observe filament nucleation and growth, permitting direct measurement of their contributions to filament formation. Our findings yield a comprehensive, quantitative understanding of RAD51 filament formation on bare single-stranded DNA that will serve as a basis to elucidate how mediators help RAD51 filament assembly and accessory factors control filament dynamics.

Anisotropies in cortical tension reveal the physical basis of polarizing cortical flows.

Asymmetric cell divisions are essential for the development of multicellular organisms. To proceed, they require an initially symmetric cell to polarize. In Caenorhabditis elegans zygotes, anteroposterior polarization is facilitated by a large-scale flow of the actomyosin cortex, which directs the asymmetry of the first mitotic division. Cortical flows appear in many contexts of development, but their underlying forces and physical principles remain poorly understood. How actomyosin contractility and cortical tension interact to generate large-scale flow is unclear. Here we report on the subcellular distribution of cortical tension in the polarizing C. elegans zygote, which we determined using position- and direction-sensitive laser ablation. We demonstrate that cortical flow is associated with anisotropies in cortical tension and is not driven by gradients in cortical tension, which contradicts previous proposals. These experiments, in conjunction with a theoretical description of active cortical mechanics, identify two prerequisites for large-scale cortical flow: a gradient in actomyosin contractility to drive flow and a sufficiently large viscosity of the cortex to allow flow to be long-ranged. We thus reveal the physical requirements of large-scale intracellular cortical flow that ensure the efficient polarization of the C. elegans zygote.

Active multistage coarsening of actin networks driven by myosin motors.

In cells, many vital processes involve myosin-driven motility that actively remodels the actin cytoskeleton and changes cell shape. Here we study how the collective action of myosin motors organizes actin filaments into contractile structures in a simplified model system devoid of biochemical regulation. We show that this self-organization occurs through an active multistage coarsening process. First, motors form dense foci by moving along the actin network structure followed by coalescence. Then the foci accumulate actin filaments in a shell around them. These actomyosin condensates eventually cluster due to motor-driven coalescence. We propose that the physical origin of this multistage aggregation is the highly asymmetric load response of actin filaments: they can support large tensions but buckle easily under piconewton compressive loads. Because the motor-generated forces well exceed this threshold, buckling is induced on the connected actin network that resists motor-driven filament sliding. We show how this buckling can give rise to the accumulation of actin shells around myosin foci and subsequent coalescence of foci into superaggregates. This new physical mechanism provides an explanation for the formation and contractile dynamics of disordered condensed actomyosin states observed in vivo.

Stress-enhanced gelation: a dynamic nonlinearity of elasticity.

A hallmark of biopolymer networks is their sensitivity to stress, reflected by pronounced nonlinear elastic stiffening. Here, we demonstrate a distinct dynamical nonlinearity in biopolymer networks consisting of filamentous actin cross-linked by α-actinin-4. Applied stress delays the onset of relaxation and flow, markedly enhancing gelation and extending the regime of solidlike behavior to much lower frequencies. We show that this macroscopic network response can be accounted for at the single molecule level by the increased binding affinity of the cross-linker under load, characteristic of catch-bond-like behavior.

High-throughput single-molecule experiments reveal heterogeneity, state switching, and three interconnected pause states in transcription.

Pausing by bacterial RNA polymerase (RNAp) is vital in the recruitment of regulatory factors, RNA folding, and coupled translation. While backtracking and intra-structural isomerization have been proposed to trigger pausing, our mechanistic understanding of backtrack-associated pauses and catalytic recovery remains incomplete. Using high-throughput magnetic tweezers, we examine the Escherichia coli RNAp transcription dynamics over a wide range of forces and NTP concentrations. Dwell-time analysis and stochastic modeling identify, in addition to a short-lived elemental pause, two distinct long-lived backtrack pause states differing in recovery rates. We identify two stochastic sources of transcription heterogeneity: alterations in short-pause frequency that underlies elongation-rate switching, and variations in RNA cleavage rates in long-lived backtrack states. Together with effects of force and Gre factors, we demonstrate that recovery from deep backtracks is governed by intrinsic RNA cleavage rather than diffusional Brownian dynamics. We introduce a consensus mechanistic model that unifies our findings with prior models.

A kinetic model predicts SpCas9 activity, improves off-target classification, and reveals the physical basis of targeting fidelity.

The S. pyogenes (Sp) Cas9 endonuclease is an important gene-editing tool. SpCas9 is directed to target sites based on complementarity to a complexed single-guide RNA (sgRNA). However, SpCas9-sgRNA also binds and cleaves genomic off-targets with only partial complementarity. To date, we lack the ability to predict cleavage and binding activity quantitatively, and rely on binary classification schemes to identify strong off-targets. We report a quantitative kinetic model that captures the SpCas9-mediated strand-replacement reaction in free-energy terms. The model predicts binding and cleavage activity as a function of time, target, and experimental conditions. Trained and validated on high-throughput bulk-biochemical data, our model predicts the intermediate R-loop state recently observed in single-molecule experiments, as well as the associated conversion rates. Finally, we show that our quantitative activity predictor can be reduced to a binary off-target classifier that outperforms the established state-of-the-art. Our approach is extensible, and can characterize any CRISPR-Cas nuclease - benchmarking natural and future high-fidelity variants against SpCas9; elucidating determinants of CRISPR fidelity; and revealing pathways to increased specificity and efficiency in engineered systems.

Systematic decomposition of sequence determinants governing CRISPR/Cas9 specificity.

The specificity of CRISPR/Cas9 genome editing is largely determined by the sequences of guide RNA (gRNA) and the targeted DNA, yet the sequence-dependent rules underlying off-target effects are not fully understood. To systematically explore the sequence determinants governing CRISPR/Cas9 specificity, here we describe a dual-target system to measure the relative cleavage rate between off- and on-target sequences (off-on ratios) of 1902 gRNAs on 13,314 synthetic target sequences, and reveal a set of sequence rules involving 2 factors in off-targeting: 1) a guide-intrinsic mismatch tolerance (GMT) independent of the mismatch context; 2) an "epistasis-like" combinatorial effect of multiple mismatches, which are associated with the free-energy landscape in R-loop formation and are explainable by a multi-state kinetic model. These sequence rules lead to the development of MOFF, a model-based predictor of Cas9-mediated off-target effects. Moreover, the "epistasis-like" combinatorial effect suggests a strategy of allele-specific genome editing using mismatched guides. With the aid of MOFF prediction, this strategy significantly improves the selectivity and expands the application domain of Cas9-based allele-specific editing, as tested in a high-throughput allele-editing screen on 18 cancer hotspot mutations.

The nucleotide addition cycle of the SARS-CoV-2 polymerase.

Coronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase subunit (nsp12) and its intimately associated cofactors (nsp7 and nsp8). We use a high-throughput magnetic-tweezers approach to develop a mechanochemical description of this core polymerase. The core polymerase exists in at least three catalytically distinct conformations, one being kinetically consistent with incorporation of incorrect nucleotides. We provide evidence that the RNA-dependent RNA polymerase (RdRp) uses a thermal ratchet instead of a power stroke to transition from the pre- to post-translocated state. Ultra-stable magnetic tweezers enable the direct observation of coronavirus polymerase deep and long-lived backtracking that is strongly stimulated by secondary structures in the template. The framework we present here elucidates one of the most important structure-dynamics-function relationships in human health today and will form the grounds for understanding the regulation of this complex.

The nucleotide addition cycle of the SARS-CoV-2 polymerase.

Coronaviruses have evolved elaborate multisubunit machines to replicate and transcribe their genomes. Central to these machines are the RNA-dependent RNA polymerase subunit (nsp12) and its intimately associated cofactors (nsp7 and nsp8). We have used a high-throughput magnetic-tweezers approach to develop a mechanochemical description of this core polymerase. The core polymerase exists in at least three catalytically distinct conformations, one being kinetically consistent with incorporation of incorrect nucleotides. We provide the first evidence that an RdRp uses a thermal ratchet instead of a power stroke to transition from the pre- to post-translocated state. Ultra-stable magnetic tweezers enables the direct observation of coronavirus polymerase deep and long-lived backtrack that are strongly stimulated by secondary structure in the template. The framework presented here elucidates one of the most important structure-dynamics-function relationships in human health today, and will form the grounds for understanding the regulation of this complex.

Inhibition of SARS-CoV-2 polymerase by nucleotide analogs: a single molecule perspective.

The nucleotide analog Remdesivir (RDV) is the only FDA-approved antiviral therapy to treat infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The physical basis for efficient utilization of RDV by SARS-CoV-2 polymerase is unknown. Here, we characterize the impact of RDV and other nucleotide analogs on RNA synthesis by the polymerase using a high-throughput, single-molecule, magnetic-tweezers platform. The location of the modification in the ribose or in the base dictates the catalytic pathway(s) used for its incorporation. We reveal that RDV incorporation does not terminate viral RNA synthesis, but leads the polymerase into deep backtrack, which may appear as termination in traditional ensemble assays. SARS-CoV-2 is able to evade the endogenously synthesized product of the viperin antiviral protein, ddhCTP, though the polymerase incorporates this nucleotide analog well. This experimental paradigm is essential to the discovery and development of therapeutics targeting viral polymerases. TEASER: We revise Remdesivir's mechanism of action and reveal SARS-CoV-2 ability to evade interferon-induced antiviral ddhCTP.