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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
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
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:
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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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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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.
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 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:
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)
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.