Study and visualize DNA editing mechanisms on the nanoscale

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Dynamic Single-Molecule

Revealing biomolecular insights never before available

Dynamic Single-Molecule page
Why Dynamic Single-Molecule?

The precision and dynamics behind genome engineering

Genome editing holds the promise of transforming medicine, but the molecular details behind these powerful tools often remain hidden. Conventional assays average out complex interactions and miss rare events that can shape editing outcomes. Without the ability to visualize and control these processes at the single-molecule level, key questions about efficiency, specificity, and mechanism remain unanswered.
Overcome these challenges with Dynamic Single-Molecule technology through:
  • Directly observe the cleavage, binding, and mobility of gene-editing proteins on DNA at the single-molecule level
  • Measure the efficiency and specificity of genome-editing tools like CRISPR-Cas systems in real time
  • Uncover the mechanisms driving off-target effects, repair pathway choices, and editing outcomes
  • Understand off-target activities of Cas for safer gene editing

    CRISPR/Cas9 is a highly popular genome-editing tool and a trending research topic. Current research is focused on fully understanding Cas9 function under different circumstances to exclude off-target binding events and ensure its precision during DNA editing applications. However, there are still many challenges in performing this research, including missing control of when and where exactly off-target events occur, together with the inability to directly visualize these binding events. For a deep understanding of the CRISPR/Cas9 mechanism and its off-target binding to DNA, it is crucial to visualize the binding location and dynamics at the base-pair level and in real-time, as well as have the tool to manipulate the DNA structure to uncover how different DNA states influence Cas9 interactions.

    Case study

    Mechanical stress on DNA – the missing link to understand off-target activity?

    CRISPR, CAS9
    David Rueda, PhD
    Professor and Chair

    Guillermo Montoya, PhD
    Professor and Head of Research

    DNA experiences mechanical stress in a multitude of processes including chromatin formation and replication. But how does the mechanical state of DNA influence Cas binding and what does that mean for the safety and efficacy of gene editing tools?

    Current CRISPR/Cas research is focused on fully understandingCas function under different circumstances to exclude off-target binding events and ensure its precision during DNA editing applications. With experiment and data analysis automation, the C-Trap enables a fast workflow for precise localization of DNA-binding proteins. Critical parameters like the location and probability of off-target binding and activity under different conditions become readily accessible.

    Dynamic single-molecule research by the groups of David Rueda(MRC-LMS) and Guillermo Montoya (University of Copenhagen) investigates thisrelationship and, enabled by direct real-time tracking of Cas-DNA interactions,revealed that

    • Moderate tension on DNA induces Cas9 and Cas12aoff-target binding
    • Off-target binding sites relate to the localstability of the DNA double-strand and thus its sequence
    • Off-target dsDNA-ssDNA intermediates rather thanstable ssDNA attract Cas
    • Supercoiling has a similar effect as tension andincreases unspecific interactions

    Taken together, these findings improve our understanding of CRISPR/Casgene editing mechanisms and open up new approaches of increasing gene editingreliability and safety.

    "We can see the different proteins coming in and working on the DNA, and that direct visualisation is a great advance" - Simon Boulton, PhD(Principal Group Leader - Assistant Research Director, The Francis Crick Institute)

    Further reading:

    • Newton et al. Nat Struct Mol Biol (2019)
    • Losito et al Phys. Chem. Chem. Phys. (2021)
    • Paul et al. Nucleic Acids Research (2021)
    • Newton et al. Molecular Cell (2023)

    Fluorescence images showing dCas9 on-target binding to DNA at contour length (top) and additional off-target binding (bottom) to stretched DNA that has formed bubbles after applying 20 pN force. Left and right fluorescent spheres are optically trapped polystyrene beads that are holding the DNA molecule (unlabelled) in between.

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    Unlocking the power of Cas12a: Novel insights into engineered endonucleases for safe and precise next generation genome editing

    Webinar

    The advent of powerful and precise gene-editing tools is transforming the way we approach health, medicine, and biological research, opening up possibilities that were once considered science fiction.

    Join us today in this webinar to unravel the secret of Cas12a endonuclease, a component of the revolutionary CRISPR-Cas gene-editing technology. Prof. Guillermo Montoya will guide us through his groundbreaking research that explores how Cas12a, an RNA-guided enzyme, interacts with bacteriophage λ-DNA. This journey into the world of genome editing will not only deepen our understanding of this complex tool but also inspire us to imagine the potential advancements in biomedicine and biotechnology.

    As we embark on this journey together, we are excited to share and explore the novel insights and possibilities that are becoming accessible through these advancements in gene-editing technologies. Let’s dive in and explore the extraordinary world of Cas12a and its implications in gene editing.

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    Unlock the true potential of CRISPR Cas9 technology through real-time direct visualization of Cas9 gene-editing

    App Notes

    CRISPR Cas9 is a gene editing tool that has increased in popularity due to its simplicity to use. It allows researchers to seamlessly edit DNA sequences by combining a sequence identifying guide RNA (gRNA) with the Cas9 endonuclease enzyme. However, its applicability as a gene-editing and therapeutic tool is impeded due to undesired off-target binding of Cas9.

    Conventional bulk in vitro assays provide information on the DNA sequences but do not give insights into the changes in the local DNA structure. In this application note, dynamic single molecule (DSM) analysis is shown to be a novel and a more powerful investigative tool to directly visualize and manipulate CRISPR Cas9 – DNA interactions, and accelerate gene-editing researchand therapy development.

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    White Paper

    Real-time insights into gene editing mechanisms

    Understanding the molecular interactions that govern Cas-DNAbinding is crucial for the development of safe and reliable medicalapplications of the CRISPR/Cas gene editing system. Recent research indicatesthat sequence-matching interactions between the guide RNA and DNA template arenot the only drivers of Cas binding and catalytic activity.

    Case study

    Bo Sun, PhD
    Associate Professor

    Bo Sun and his group at Shanghai Tech utilize DynamicSingle-Molecule microscopy with the C-Trap to take a closer look at theinteractions of Cas9 and its substrate. They were able to identify andcharacterize stable interactions between different Cas9 variants and DNA beyondthe protospacer adjacent motif (PAM), leading to valuable insights:

    • Cas9 interacts with DNA sequences up to 14 basepairs downstream of the PAM
    • This interaction is critical for stable DNAbinding and cleavage activity of Cas9
    • Upon DNA cleavage, Cas9 releases the PAM-distalDNA, thus facilitating its dissociation
    • Use the force: Unzipping DNA with bound Cas9 variantsreveals that the stable interaction is mediated by electrostatic interactionsbetween specific amino acids and the DNA backbone
    • AsCas12f1, utilized as a versatile gene deliveryplatform, combines out-of-protospacer DNA unwinding with exonuclease activityin the sequential target cleavage

    Further reading: 

    Song et al. Nucleic Acids Research (2024)
    Zhang et al. Sci. Adv. (2019)
    Zhang et al. Nucleic Acids Research (2021)
    Zhang et al., Dynamics of Staphylococcus aureus Cas9 in DNA target Associationand Dissociation. EMBO Rep 21 (2020).

    A cartoon illustrating the single-molecule DNA unzipping experiment used to detect the interactions between AsCas12f1-gRNA and DNA. Image taken from Song et al. Nucleic Acids Research (2024)

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    Webinar
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    App Notes

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    White Paper
    Solutions

    C-Trap

    Biomolecular interactions re-imagined

    The C-Trap® provides the world’s first dynamic single-molecule microscope to allow simultaneous manipulation and visualization of single-molecule interactions in real time.

    Discover the C-Trap

    Publications

    Understand the key insights by reading up on our latest publications

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    Text Link

    DNA stretching induces Cas9 off-target activity

    Newton, M. et al.
    2019
    Nature Structural & Molecular Biology
    Author Empty
    DNA Editing
    Publication
    Text Link

    Dynamics of Staphylococcus aureus Cas9 in DNA target Association and Dissociation

    Zhang, S. et al.
    2020
    EMBO Rep
    Author Empty
    DNA Editing
    Publication
    Text Link

    Mechanics of CRISPR-Cas12a and engineered variants on λ-DNA

    Paul, B. et al.
    2021
    Nucleic Acids Research
    Author Empty
    DNA Editing
    Publication

    Relevant resources

    Learn as much as you can by reading up on our application notes or marathoning our webinars.

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    Unlocking the power of Cas12a: Novel insights into engineered endonucleases for safe and precise next generation genome editing

    Unlocking the power of Cas12a: Novel insights into engineered endonucleases for safe and precise next generation genome editing

    Webinar
    01-01-20
    01-01-20

    Unveiling Key Mechanisms in Kinetochore-Mediated Chromosome Segregation with the C-Trap

    Unveiling Key Mechanisms in Kinetochore-Mediated Chromosome Segregation with the C-Trap

    Scientific update
    Chenlu Yu, PhD

    DNA-Binding proteins

    DNA Editing

    DNA/RNA Structure

    DNA Organization

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    Real-time visualization of DNA structural transitions under mechanical stress

    Real-time visualization of DNA structural transitions under mechanical stress

    Application note
    01-01-20
    01-01-20

    Analyze Cas9 binding and cleavage properties in real-time while manipulating DNA structures

    Analyze Cas9 binding and cleavage properties in real-time while manipulating DNA structures

    Application note
    01-01-20
    01-01-20

    Unlock the true potential of CRISPR Cas9 technology through real-time direct visualization of Cas9 gene-editing

    Unlock the true potential of CRISPR Cas9 technology through real-time direct visualization of Cas9 gene-editing

    Application note
    01-01-20
    01-01-20

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    SITC 2025

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    April 22, 2025
    01-01-20

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    CAR-TCR Summit 2025

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    April 22, 2025
    01-01-20

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    CICON 2025

    Conference
    April 22, 2025
    01-01-20