Visualization of CRISPR-Cas9 genetic-engineering technique

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.

[caption id="attachment_1065" align="aligncenter" width="728"] 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.

[caption id="attachment_1066" align="aligncenter" width="376"] 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