Nanotechnology

The researchers visualized the activity of the CRISPR genetic scissors

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July 12, 2023

(Nanowerk News) Scientists at the University of Leipzig, in collaboration with colleagues at the University of Vilnius in Lithuania, have developed a new method for measuring the twists and torques of the smallest molecules in milliseconds. This method makes it possible to trace the gene recognition of the CRISPR-Cas protein complex, also known as the “genetic scissors”, in real time and with the highest resolution. With the data obtained, the recognition process can be accurately characterized and modeled to increase the precision of genetic shears.

The results obtained by the team led by Professors Ralf Seidel and Dominik Kauert from the Faculty of Physics and Earth Sciences have now been published in the journal Natural Structural and Molecular Biology (“Energy landscape for R-loop formation by the CRISPR-Cas Cascade complex”). researchers fabricated DNA rotor arms and attached gold nanoparticles to the ends to observe the movement during DNA unwinding The researchers fabricated DNA rotor arms and attached gold nanoparticles to the ends to observe the movement during DNA unwinding. (Image: Dominik Kauert)

When bacteria are attacked by viruses, they can defend themselves by mechanisms that fend off genetic material introduced by intruders. The key is the CRISPR-Cas protein complex. Only in the last decades has its function for adaptive immunity in microorganisms been discovered and described. With the help of the embedded RNA, the CRISPR complex recognizes short sequences in the attacker’s DNA.

The mechanism of sequence recognition by RNA has since been used to selectively switch off and modify genes in any organism. This discovery revolutionized genetic engineering and has been honored in 2020 with the Nobel Prize in Chemistry awarded to Emmanuelle Charpentier and Jennifer A. Doudna.

However, sometimes the CRISPR complex also reacts to gene segments that differ slightly from the sequence specified by the RNA. This causes unwanted side effects in medical applications. “The causes are not well understood, as the process was not directly observable until now,” said Dominik Kauert, who worked on the project as a PhD student.

The nanoscale process is traced in detail

To better understand the recognition process, the team led by Professors Ralf Seidel and Dominik Kauert took advantage of the fact that the DNA double helix of the target sequence unwinds during recognition to allow base pairing with RNA. “The main question of this project is whether the shedding of a piece of DNA that is only 10 nanometers (nm) long can be tracked in real time,” said Kauert.

To observe the discharge process in detail, scientists must make it visible with a microscope. To achieve this goal, the team leveraged the achievements of DNA nanotechnology, which can be used to create three-dimensional DNA nanostructures. Using this so-called DNA origami technique, the researchers fabricated 75 nm long DNA rotor arms with gold nanoparticles attached to the ends. In the experiment, loose strands of DNA 2 nm thin and 10 nm long were transferred to the rotation of the gold nanoparticles along a circle with a diameter of 160 nm – this motion can be magnified and tracked using a special microscope setup.

With this new method, researchers can observe sequence recognition by the CRISPR Cascade complex almost base pair by base pair. Surprisingly, base pairing with RNA is energetically disadvantageous, meaning that the complex only binds unstable during sequence recognition. Only when the entire sequence is recognized does stable binding occur and the DNA is then destroyed. If it is an “incorrect” target sequence, the process is aborted.

The findings will assist in selecting suitable RNA sequences

The fact that the recognition process sometimes produces erroneous results is due to its stochastic nature, that is, the random movement of molecules, as researchers can now show. “Sequence recognition is driven by thermal fluctuations in base pairs,” says Kauert. With the data obtained, it is possible to construct a sequence recognition thermodynamic model that describes the recognition of aberrant sequence segments. In the future, this will enable better selection of RNA sequences that recognize only the target sequence of interest, thereby optimizing the precision of genetic manipulation.

Because the designed nanorotor is universal in its suitability for measuring spin and torque in single molecules, they can also be used for other CRISPR-Cas complexes or biomolecules.



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