Researchers at Kanazawa University and the University of Tokyo report in Nature Communications the visualization of the dynamics of ‘molecular scissors’ — the main mechanism of the CRISPR-Cas9 genetic-engineering technique. 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.
CRISPR-Cas9 is a genome editing tool that is creating a buzz in the science world. It is faster, cheaper and more accurate than previous techniques of editing DNA and has a wide range of potential applications.Selection of the site to be cut is done by a ‘guide RNA’ molecule bound to the Cas9 protein. Now, a team of researchers led by Mikihiro Shibata from Kanazawa University and Osamu Nureki from the University of Tokyo has visualized the dynamics of the CRISPR-Cas9 complex, in particular how it cuts DNA, providing valuable insights into the CRISPR-Cas9-mediated DNA cleavage mechanism.
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From left to right: Cas9 alone (apo-Cas9), Cas9 bound to RNA (Cas9–RNA), Cas9–RNA bound to its single-stranded DNA target (Cas9–RNA–DNA), Cas9–RNA bound to a partial DNA duplex (Cas9–RNA–DNA) and Cas9–RNA bound to its double-stranded DNA target (Cas9–RNA–DNA).[/caption]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 of Shibata and colleagues enabled extremely fast, repeated scans — convertible into movies — of the biomolecules taking part in the molecular scissoring action.
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Fluctuations of the nuclease domain are indicated by magenta arrows. The cleavage products released from Cas9–RNA are indicated by blue arrows.[/caption]First, the scientists compared Cas9 without and with RNA attached (Cas9–RNA). They found that the former was able to flexibly adopt various conformations, while the latter has 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. They confirmed 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.
Article
Title: Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy
Journal: Nature Communications
Authors: Mikihiro Shibata, Hiroshi Nishimasu, Noriyuki Kodera, Seiichi Hirano, Toshio Ando, Takayuki Uchihashi, and Osamu Nureki
Doi: 10.1038/s41467-017-01466-8
Funders
The Kao Foundation for Arts and Science, The Brain Science Foundation, JST/PRESTO, JST/CREST, The Basic Science and Platform Technology Program for Innovative Biological Medicine from AMED, JSPS KAKENHI
regulator CDC25B, and tumor suppressor p53, which suggest the roles of this kinase in stress related transcription and cell cycle regulation, as well as in genotoxic stress response. Four alternatively spliced transcript variants of this gene encoding distinct isoforms have been reported.
constitutively expressed in specific neurons of the brain and inducible nitric oxide synthase (iNOS), whose expression is typically induced in inflammatory diseases. eNOS is primarily responsible for the generation of NO in the vascular endothelium, a monolayer of flat cells lining the interior surface of blood vessels, at the interface between circulating blood in the lumen and the remainder of the vessel wall. NO produced by eNOS in the vascular endothelium plays crucial roles in regulating vascular tone, cellular proliferation, leukocyte adhesion, and platelet aggregation. Therefore, a functional eNOS is essential for a healthy cardiovascular system.
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