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Updated: Aug 8, 2025
Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage
Published on: May 6, 2020
Dynamics of Target DNA Binding and Cleavage by Staphylococcus aureus Cas9 as Revealed by High-Speed Atomic Force
Leonardo Puppulin1, Junichiro Ishikawa2, Ayumi Sumino1,3
1WPI Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, Kanazawa, Ishikawa 920-1192, Japan.
Abstract:
Programmable DNA binding and cleavage by CRISPR-Cas9 has revolutionized the life sciences. However, the off-target cleavage observed in DNA sequences with some homology to the target still represents a major limitation for a more widespread use of Cas9 in biology and medicine. For this reason, complete understanding of the dynamics of DNA binding, interrogation and cleavage by Cas9 is crucial to improve the efficiency of genome editing. Here, we use high-speed atomic force microscopy (HS-AFM) to investigate Staphylococcus aureus Cas9 (SaCas9) and its dynamics of DNA binding and cleavage. Upon binding to single-guide RNA (sgRNA), SaCas9 forms a close bilobed structure that transiently and flexibly adopts also an open configuration. The SaCas9-mediated DNA cleavage is characterized by release of cleaved DNA and immediate dissociation, confirming that SaCas9 operates as a multiple turnover endonuclease. According to present knowledge, the process of searching for target DNA is mainly governed by three-dimensional diffusion. Independent HS-AFM experiments show a potential long-range attractive interaction between SaCas9-sgRNA and its target DNA. The interaction precedes the formation of the stable ternary complex and is observed exclusively in the vicinity of the protospacer-adjacent motif (PAM), up to distances of several nanometers. The direct visualization of the process by sequential topographic images suggests that SaCas9-sgRNA binds to the target sequence first, while the following binding of the PAM is accompanied by local DNA bending and formation of the stable complex. Collectively, our HS-AFM data reveal a potential and unexpected behavior of SaCas9 during the search for DNA targets.
Insights
High-speed atomic force microscopy reveals Staphylococcus aureus Cas9 dynamics. SaCas9 binds DNA via a novel long-range interaction near the PAM, preceding stable complex formation and cleavage.
Area of Science:
- Molecular Biology
- Biophysics
- Genomics
Background:
- CRISPR-Cas9 technology has transformed life sciences but faces limitations due to off-target DNA cleavage.
- Understanding Cas9 dynamics is crucial for improving genome editing efficiency and safety.
Purpose of the Study:
- To investigate the DNA binding and cleavage dynamics of Staphylococcus aureus Cas9 (SaCas9) using high-speed atomic force microscopy (HS-AFM).
- To elucidate the mechanism of target DNA searching and complex formation by SaCas9.
Main Methods:
- High-speed atomic force microscopy (HS-AFM) was employed to visualize SaCas9-sgRNA interactions with target DNA in real-time.
- Analysis of structural changes and dynamic movements during DNA binding, interrogation, and cleavage.
Main Results:
- SaCas9 forms a bilobed structure upon sgRNA binding, transitioning between open and closed configurations.
- Cleavage results in rapid DNA release and enzyme dissociation, indicating multiple turnover activity.
- A novel, long-range attractive interaction between SaCas9-sgRNA and target DNA, localized near the PAM, was observed prior to stable complex formation.
Conclusions:
- SaCas9 exhibits unexpected target search behavior involving long-range attraction and PAM-proximal binding.
- The findings suggest a mechanism where initial binding to the target sequence is followed by PAM recognition and DNA bending.
- This study provides critical insights into SaCas9 dynamics, potentially guiding the development of more precise genome editing tools.
