Study and visualize DNA editing mechanisms on the nanoscale

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

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)

Bo Sun and his group at Shanghai Tech utilize Dynamic Single-Molecule microscopy with the C-Trap to take a closer look at the interactions of Cas9 and its substrate. They were able to identify and characterize stable interactions between different Cas9 variants and DNA beyond the 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
• Unzipping DNA with bound Cas9 variants reveals that the stable interaction is mediated by electrostatic interactions between specific amino acids and the DNA backbone
• AsCas12f1, utilized as a versatile gene delivery platform, combines out-of-protospacer DNA unwinding with exonuclease activity in the sequential target cleavage

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