CRISPR instead of RNA interference

The new CRISPR system will be able to "jam" genes at the RNA level

Alexander Ershov, N+1

An international group of scientists from Russia and the USA has discovered an enzyme that is able to specifically destroy the necessary RNA using an RNA guide. Nuclease is part of one of the varieties of the CRISPR system, however, unlike the well-known CRISPR/Cas9, it acts at the level of RNA, not DNA, which eliminates the entire method from the risk of destabilization of the genome due to the introduction of "incorrect" breaks. The work has not yet been reviewed and is available as a preprint in the database bioarXiv.org (Abudayyeh et al., C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector).

Structure of the Cas9 nuclease complex with Guide RNA

The CRISPR/Cas9 genome editing system created in 2012 is based on the bacterial antiviral immunity system. Such immunity allows bacteria to find fragments of virus DNA in their CRISPR "card file" (it is located in a certain part of the bacterial genome), and destroy viral DNA with the help of a special nuclease.

There are several types of CRISPR system and several nucleases in different bacteria and archaea, but for the purposes of genome editing in bioengineering, Cas9 nuclease is almost always used (this is reflected in the name of the method). The main advantage of this particular nuclease is that Cas9 works independently (it is a single protein), while most of the other CRISPR nucleases work in a complex of several enzymes, and using multi-subunit complexes for genome editing is inconvenient and often inefficient.

The CRISPR/Cas9 system has three key drawbacks. Firstly, Cas9 nuclease is a rather large protein, the gene of which often does not "fit" into those carriers (vectors) that are used to introduce genetic constructs in cells.

Secondly, Cas9 acts only on DNA, not RNA. This cannot be called a disadvantage if we really want to edit the genome of a cell, that is, change its DNA. However, often the desired result can be achieved simply by "turning off" the activity of the desired gene at the RNA level – by simply destroying all copies made from this gene. Such an intervention, known by the example of RNA interference, is potentially safer, since it does not introduce instability into the genome. For example, even if the nuclease makes a mistake in choosing a target, this does not lead to the appearance of mutations in the genome. Such a method could potentially be closer to therapeutic use than real genome editing using CRISPR/Cas9.

Thirdly, now the CRISPR/Cas9 genome editing method is the subject of a patent dispute, which may significantly delay its entry into clinical practice. All these reasons require the search for new nucleases that could be used to regulate gene activity.

Earlier, the same group of researchers managed to create a bioinformatic system for searching for new types of CRISPR immunity in bacterial genomic data and found a new class of CRISPR systems, including the hypothetical C2c2 nuclease. This is a small protein that, as shown by sequence analysis, most likely acts not on DNA, but on RNA.

In the new work, scientists were able to confirm these assumptions and test the new CRISPR/C2c2 system in action. To do this, the authors transferred the sequences of CRISPR, C2c2 and other components of the system from Leptotrichia (Leptotrichia shahii), in whose genome it was found, to the genome of E. coli. The scientists then infected the bacteria with the MS2 RNA virus and looked at the surviving cells. Thus, it was possible to detect fragments of the virus that are most vulnerable to the action of CRISPR/C2c2, and to determine the substrate preferences of the C2c2 enzyme.

As a test of the bioengineering properties of the CRISPR/C2c2 system, the authors learned to turn off the red glow of bacteria by destroying the RNA of the red fluorescent protein gene previously introduced into cells. According to the scientists, the shutdown efficiency ranged from 20 to 92 percent, depending on the selected target on the fluorescent protein RNA.

This efficiency is comparable to the efficiency of RNA interference, which works in a similar way, but at the expense of other molecular biological mechanisms. At the same time, RNA interference has its own disadvantages. For example, short RNAs that are injected into cells to turn off genes are very quickly destroyed by non-specific RNases and often cannot penetrate into the cell, which is why the therapeutic effectiveness is significantly reduced.

Portal "Eternal youth" http://vechnayamolodost.ru  24.05.2016