The CRISPR/Cas gene scissors revolutionized genetics and gene medicine and created completely new possibilities for gene editing. Researchers have now discovered a completely new variant of this gene scissors in bacteria. Unlike previous versions, CRISPR/Cas12a2 recognizes specific RNA sections instead of DNA sections - for example the RNA of viruses. This triggers a response from the gene scissors that turns them into genome destroyers: they grab the nearest strand of DNA or RNA, bend it to expose the molecular "backbone" of the strand, and cut it. This leads to the destruction of virus-infected or otherwise defective cells, but can also be used as a highly sensitive virus detection.
Hardly any other molecular biological process has caused as much furore in recent years as the CRISPR/Cas gene scissors. The molecular complex, which was originally used by bacteria to recognize and defend against viruses, has become a versatile and important tool in genetic engineering, medicine and genetics. It enables specific DNA bases or genes to be excised and replaced easily, inexpensively and with pinpoint accuracy. This is possible because the CRISPR/Cas complex contains various modules that are used to find and specifically cut DNA. CRISPR – short for Clustered Regularly Interspaced Short Palindromic Repeats – includes the part of the molecule that is used for identification. Among other things, it contains a short piece of RNA, the guide RNA, which is complementary to the DNA sequence of the DNA section being searched for. If the molecule finds a suitable piece of gene, it attaches itself there and the scissor part of the molecule is used. In the most common version, CRISPR/Cas9, the Cas9 enzyme cuts out the “marked” part of the DNA. But there are also versions with other enzymes that then, for example, shut down the gene you are looking for only by attaching a methyl group or react to the RNA of viruses such as Sars-CoV-2.
Cas12a2 enzyme breaks down and destroys DNA and RNA
Another variant of the CRISPR gene scissors has now been discovered by Oleg Dmytrenko from the Helmholtz Center for Infection Research in Würzburg and his colleagues. As part of their study, they examined CRISPR/Cas enzymes from the so-called Cas12a family, which support the immune defense of bacteria in a different way than the gene scissors that have been used up to now. If these enzymes become active, the entire affected cell is shut down or killed - and the viral infection is thus contained. "This strategy, called abortive infection, is used by bacteria and archaea to limit the spread of viruses and other pathogens," explains co-author Thomson Hallmark of Utah State University. However, while exploring this strategy, the team came across a new variant of the Cas12a enzyme, which they dubbed Cas12a2. "This nuclease does something completely different from Cas12a, but also from all CRISPR nucleases that we know of," says Dmytrenko.
To analyze this enzyme and its function in more detail, the researchers introduced the genes for a CRISPR/Cas12a2 construct into the bacterium Escherichia coli and observed it in action. It turned out that unlike Cas9, these gene scissors do not bind to a DNA sequence as a target, but to RNA sections that match its guide RNA. Once this attachment has happened, the entire bilobed molecule changes its conformation. It can now close like a kind of pliers and grab any strand of RNA or DNA. The “captured” strands are bent 90 degrees until the long framework of the double helix is exposed and fits into a notch in the Cas12a2 enzyme, analysis using cryo-electron microscopy has shown. "This is an extraordinary process, the mere observation of which caused audible surprise among my colleagues," reports Ryan Jackson of Utah State University.
Targeted elimination of defective or infected cells
Unlike Cas9, the new gene scissors not only cut specific sections of DNA, but randomly dismember all types of single and double-stranded DNA and RNA in the target cell as soon as Cas12a2 has been activated there by contact with suitable RNA. The newly discovered gene scissors CRSIPR/Cas12a2 thus work differently than the previously known variants. "A CRISPR-based defense mechanism that uses just a single nuclease to recognize an intruder and degrade the DNA and RNA of the infected cell has never been observed before," says Dmytrenko. While the indiscriminate destruction of genetic material initially appears like a dangerous killing spree, it is nevertheless useful - for the bacteria, but also for medicine. Because by specifically destroying only those cells that contain the target RNA that matches the guide RNA, these gene scissors can selectively eradicate cells that are infected with viruses, cells that have been degenerated by mutations, or cells that are otherwise affected by genetic defects.
"If Cas12a2 could be used to target, attack and destroy cells at the genetic level, it would have significant therapeutic implications," says Jackson. The gene scissors could be used, for example, in cancer therapy to destroy tumor cells, in gene therapy or in combating viruses. Another, less destructive use is to detect a viral infection: In a first test, Dmytrenko and his team equipped DNA in a solution with a fluorescent marker that lights up as soon as the DNA strand is cut by the Cas12a2 enzyme. If this is combined with a guide RNA that, for example, only responds to the presence of viral RNA in the cell, the gene scissors could be used as a highly sensitive virus test.
Source: Oleg Dmytrenko (Helmholtz Center for Infection Research, Würzburg) et al., Nature, doi: 10.1038/s41586-022-05559-3; Jack Bravo (University of Texas, Austin) et al., Nature, doi: 10.1038/s41586-022-05560-w