CRISPR Caught In The Act

Using high-speed atomic-force microscopy, scientists in Japan have observed how the CRISPR/Cas9 complex cuts DNA in real-time.

AsianScientist (Nov. 20, 2017) – A team of researchers from Kanazawa University have captured live footage of how the CRISPR-Cas9 complex cuts DNA. These findings, published in Nature Communications, provide valuable insight into how the increasingly popular gene editing technique works.

The CRISPR-Cas9 nuclease system allows scientists to cut DNA at a specific site determined by guide RNA molecules. In recent years, CRISPR-based gene editing has been successfully used for editing genes in rice, pigs and even humans.

In the present study, a team of researchers led by Associate Professor Mikihiro Shibata from Kanazawa University and Professor Osamu Nureki from the University of Tokyo has visualized the dynamics of the CRISPR-Cas9 complex in real-time.

For their visualization studies, the scientists used high-speed atomic-force microscopy (HS-AFM), a method for imaging surfaces. A surface is probed by moving a tiny cantilever over it; the force experienced by the probe can be converted into a height measure. A scan of the whole surface then results in a height map of the sample. The high-speed experimental set-up enabled extremely fast, repeated scans of the biomolecules taking part in the molecular scissoring action.

First, the scientists compared Cas9 without and with the guide RNA attached. They found that without guide RNA, Cas9 was able to flexibly adopt various conformations, while the presence of RNA gave the protein a fixed, two-lobe structure, highlighting the conformational-stabilization ability of the guide RNA.

Then, Shibata and colleagues looked at how the stabilized Cas9-RNA complex targets DNA, confirming that it binds to a pre-selected protospacer adjacent motif (PAM) site in the DNA. A PAM is a short nucleotide sequence located next to the DNA’s target site, which is complementary to the guide RNA.

The research team’s high-speed movies further revealed that targeting (‘DNA interrogation’) is achieved through 3D diffusion of the Cas9-RNA complex. Finally, the researchers managed to visualize the dynamics of the cleavage process itself: they observed how the region of ‘molecular scissors’ undergoes conformational fluctuations after Cas9-RNA locally unwinds the double-stranded DNA.

“This study provides unprecedented details about the functional dynamics of CRISPR-Cas9, and highlights the potential of HS-AFM to elucidate the action mechanisms of RNA-guided effector nucleases from distinct CRISPR-Cas systems,” the authors said.



The article can be found at: Shibata et al. (2017) Real-space and Real-time Dynamics of CRISPR-Cas9 Visualized by High-speed Atomic Force Microscopy.

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Source: Kanazawa University.
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