We've updated our Privacy Policy to make it clearer how we use your personal data.

We use cookies to provide you with a better experience. You can read our Cookie Policy here.


CRISPR Gene Editing Visualized at the Nano Level

A strand of DNA composed of small red and blue spheres.
Credit: Alexander Antropov/ Pixabay
Listen with
Register for free to listen to this article
Thank you. Listen to this article using the player above.

Want to listen to this article for FREE?

Complete the form below to unlock access to ALL audio articles.

Read time: 2 minutes

When bacteria are attacked by a virus, they can defend themselves with a mechanism that repels the introduced genetic material of the invader. The key to this is CRISPR-Cas protein complexes. Their function as adaptive immune systems in microorganisms was only discovered and elucidated in the last decade. With the help of an integrated RNA, CRISPR complexes recognize the attackers by a short sequence in their DNA and then destroy them. The mechanism of sequence recognition with the help of an RNA was then used to switch off and change specific genes in any organism. This discovery revolutionized genetic engineering and was honored with the 2020 Nobel Prize in Chemistry for Emmanuelle Charpentier and Jennifer A. Doudna. 

From time to time, however, CRISPR complexes also react to gene segments that deviate slightly from the sequence specified by the RNA. This leads to undesirable side effects in medical applications. "The reasons for this are not yet well understood, as the process has not yet been observed directly," says Dominik Kauert, who worked on the project as a doctoral student.

Want more breaking news?

Subscribe to Technology Networks’ daily newsletter, delivering breaking science news straight to your inbox every day.

Subscribe for FREE

Processes at the nano level followed in detail 

In order to better understand the recognition process, the team led by Prof. Dr. Ralf Seidel and Dominik Kauert take advantage of the fact that the DNA double helix of the target sequence is unwound during recognition to enable base pairing with the RNA. "The central question of the project was therefore whether the unwinding of a piece of DNA that is only 10 nanometers (nm) long can be tracked in real time at all," says Kauert. 

In order to be able to observe the unwinding process in detail, the scientists had to make it detectable for the microscope. To do this, the team resorted to the toolbox of DNA nanotechnology, which can be used to create any three-dimensional DNA nanostructures. Using this so-called DNA origami technique, the researchers constructed a 75 nm long DNA rotor blade and attached a gold nanoparticle to its end. In the experiment, the unwinding of the 2 nm thin and 10 nm long DNA sequence was transferred to the rotation of the gold nanoparticle along a circle with a diameter of 160 nm - this enlarged movement could be followed in a special microscope setup.

With this new method, the scientists were now able to observe sequence recognition by the CRISPR complex Cascade almost base pair by base pair. It was surprising that the base pairing with the RNA is energetically hardly advantageous, so that the complex is only labilely bound during sequence recognition. Only when the entire sequence has been recognized in full is there a stable bond and, as a result, the DNA is destroyed. If it is the "wrong" target sequence, the process is aborted.

Results can help in the future in the selection of suitable RNA sequences

The fact that the recognition process sometimes delivers incorrect results is due to its stochastic nature, namely random molecular movements, as the researchers have now been able to demonstrate. "Sequence recognition is driven by thermal fluctuations in base pairing," says Kauert. With the data obtained, a thermodynamic model of sequence recognition could be created, which describes the recognition of deviating sequence sections. In the future, this should enable a better selection of RNA sequences that only recognize the desired target sequence in order to optimize the precision of genetic manipulations. 

Since the designed nanorotors are universally suitable for measuring twists and torques in single molecules, they can also be used for other CRISPR-Cas complexes or biomolecules.

The work was funded by the European Research Council and the German Research Foundation and carried out in collaboration with the working group of Prof. Dr. Virginijus Siksnys from Vilnius University (Lithuania), who isolated and provided the CRISPR complexes used. 

Reference: Kauert DJ, Madariaga-Marcos J, Rutkauskas M, et al. The energy landscape for R-loop formation by the CRISPR–Cas Cascade complex. Nat Struct Mol Biol. 2023:1-8. doi: 10.1038/s41594-023-01019-2

This article has been republished from the following materials. Note: material may have been edited for length and content. For further information, please contact the cited source.