Genetic engineering mechanism visualized



One of the techniques used in genetic engineering -- the process of artificially modifying the genome of a living organism -- involves the so-called CRISPR-Cas9 nuclease system.

Using this system, a cell's DNA can be cut at a desired site, where genes can be deleted or added. 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.

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.

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 (Figure 2).

The work of Shibata advances our understanding of the CRISPR-Cas9 genome-editing mechanism. In the words of the researchers: "... 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."

Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy Mikihiro Shibata, Hiroshi Nishimasu, Noriyuki Kodera, Seiichi Hirano, Toshio Ando, Takayuki Uchihashi & Osamu Nureki Nature Communications 8, Article number: 1430 (2017) doi:10.1038/s41467-017-01466-8

Kanazawa University

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