Researchers Visualize Activity of CRISPR Genetic Scissors
Published:18 Sep.2023 Source:Universität Leipzig
When bacteria are attacked by a virus, they can defend themselves with a mechanism that fends off the genetic material introduced by the intruder. The key is CRISPR-Cas protein complexes. With the help of an embedded RNA, the CRISPR complexes recognise a short sequence 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 revolutionised genetic engineering.
Occasionally, however, CRISPR complexes also react to gene segments that differ slightly from the sequence specified by the RNA. This leads to undesirable side effects in medical applications. "The causes of this are not yet well understood, as the process could not be observed directly until now," says Dominik Kauert, who worked on the project as a PhD student. To better understand the recognition process, the team led by Professor Ralf Seidel and Dominik Kauert took advantage of the fact that the DNA double helix of the target sequence is unwound during recognition to enable base pairing with the RNA. To achieve this goal, the team drew on the achievements of DNA nanotechnology, which can be used to create any three-dimensional DNA nanostructure. Surprisingly, base pairing with the RNA is not energetically advantageous, meaning that the complex is only unstably bound during sequence recognition.
The fact that the recognition process sometimes produces incorrect results is due to its stochastic nature, i.e. to 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, it was possible to create a thermodynamic model of sequence recognition that describes the recognition of deviating sequence segments. In the future, this should allow better selection of RNA sequences that recognise only the desired target sequence, thus optimising the precision of genetic manipulation. As the designed nanorotors are universal in their suitability for measuring twists and torques in single molecules, they can also be used for other CRISPR-Cas complexes or biomolecules.