Nav: Home

Genetic engineering mechanism visualized

November 13, 2017

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."

[Background]

CRISPR-Cas9


CRISPR, short for "clustered regularly interspaced short palindromic repeats", refers to a set of bacterial DNA sequences containing fragments of the DNA of viruses having earlier attacked the bacteria. These fragments are used by the bacteria to prevent further attacks by the same viruses. "Cas" refers to CRISPR-associated genes; "Cas9" is a CRISPR-associated protein with two nuclease domains (A nuclease is an enzyme capable of cleaving nucleic acids, organic molecules present in DNA and RNA).

In recent years, a genetic-engineering technique where a CRISPR-Cas9 complex acts as 'molecular scissors' has been developed; the Cas9 nuclease binds to a guide RNA molecule that contains information about the DNA site to target. Using high-speed atomic force microscopy, Mikihiro Shibata from Kanazawa University and colleagues have now studied the dynamics of the CRISPR-Cas9 complex in great detail.

Atomic force microscopy

Atomic force microscopy (AFM) is an imaging technique in which the image is formed by scanning a surface with a very small tip. Horizontal scanning motion of the tip is controlled via piezoelectric elements, while vertical motion is converted into a height profile, resulting in a height distribution of the sample's surface. As the technique does not involve lenses, its resolution is not restricted by the so-called diffraction limit. In a high-speed setup, AFM can be used to produce movies of a sample's evolution in real time. High-speed AFM has been used successfully to study protein dynamics, for example myosin V walking on an actin filament, the photo-induced conformational change of bacteriorhodopsin, and the degradation of cellulose. Shibata and colleagues have now applied the high-speed AFM technique for visualizing the dynamics of DNA cleavage by CRISPR-Cas9.
-end-


Kanazawa University

Related Dna Articles:

Penn State DNA ladders: Inexpensive molecular rulers for DNA research
New license-free tools will allow researchers to estimate the size of DNA fragments for a fraction of the cost of currently available methods.
It is easier for a DNA knot...
How can long DNA filaments, which have convoluted and highly knotted structure, manage to pass through the tiny pores of biological systems?
How do metals interact with DNA?
Since a couple of decades, metal-containing drugs have been successfully used to fight against certain types of cancer.
Electrons use DNA like a wire for signaling DNA replication
A Caltech-led study has shown that the electrical wire-like behavior of DNA is involved in the molecule's replication.
Switched-on DNA
DNA, the stuff of life, may very well also pack quite the jolt for engineers trying to advance the development of tiny, low-cost electronic devices.
Researchers are first to see DNA 'blink'
Northwestern University biomedical engineers have developed imaging technology that is the first to see DNA 'blink,' or fluoresce.
Finding our way around DNA
A Salk team developed a tool that maps functional areas of the genome to better understand disease.
A 'strand' of DNA as never before
In a carefully designed polymer, researchers at the Institute of Physical Chemistry of the Polish Academy of Sciences have imprinted a sequence of a single strand of DNA.
Doubling down on DNA
The African clawed frog X. laevis genome contains two full sets of chromosomes from two extinct ancestors.
'Poring over' DNA
Church's team at Harvard's Wyss Institute for Biologically Inspired Engineering and the Harvard Medical School developed a new electronic DNA sequencing platform based on biologically engineered nanopores that could help overcome present limitations.

Related Dna Reading:

Blueprint: How DNA Makes Us Who We Are (The MIT Press)
by Robert Plomin (Author)

DNA: The Story of the Genetic Revolution
by James D. Watson (Author), Andrew Berry (Author), Kevin Davies (Author)

Dinosaur DNA: A Nonfiction Companion to the Films (Jurassic World)
by Marilyn Easton (Author)

The Family Tree Guide to DNA Testing and Genetic Genealogy
by Blaine T. Bettinger (Author)

Move Your DNA: Restore Your Health Through Natural Movement Expanded Edition
by Katy Bowman (Author)

The Innovator's DNA: Mastering the Five Skills of Disruptive Innovators
by Jeff Dyer (Author), Hal Gregersen (Author), Clayton M. Christensen (Author)

DNA (Science Readers: Content and Literacy)
by Teacher Created Materials (Author)

DNA: The Secret of Life
by James D. Watson (Author), Andrew Berry (Author)

DNA: A Graphic Guide to the Molecule that Shook the World
by Israel Rosenfield (Author), Edward Ziff (Author), Borin Van Loon (Author)

DNA Science: A First Course, Second Edition
by David Micklos (Author), Greg Freyer (Author)

Best Science Podcasts 2018

We have hand picked the best science podcasts for 2018. Sit back and enjoy new science podcasts updated daily from your favorite science news services and scientists.
Now Playing: TED Radio Hour

Hacking The Law
We have a vision of justice as blind, impartial, and fair — but in reality, the law often fails those who need it most. This hour, TED speakers explore radical ways to change the legal system. Guests include lawyer and social justice advocate Robin Steinberg, animal rights lawyer Steven Wise, political activist Brett Hennig, and lawyer and social entrepreneur Vivek Maru.
Now Playing: Science for the People

#495 Earth Science in Space
Some worlds are made of sand. Some are made of water. Some are even made of salt. In science fiction and fantasy, planet can be made of whatever you want. But what does that mean for how the planets themselves work? When in doubt, throw an asteroid at it. This is a live show recorded at the 2018 Dragon Con in Atlanta Georgia. Featuring Travor Valle, Mika McKinnon, David Moscato, Scott Harris, and moderated by our own Bethany Brookshire. Note: The sound isn't as good as we'd hoped but we love the guests and the conversation and we wanted to...