A new type of CRISPR systems

CRISPR systems using reverse transcription have been found

Yulia Kondratenko, "Biomolecule"

Some bacteria are able to preserve fragments of genomes of infectious agents, using not only DNA, but also RNA as a source material. Such bacteria can develop immunity to viruses with RNA genomes. In addition, thanks to the CRISPR system using RNA, bacterial immunity learns to respond to the most active genes of pathogens.

In 1987, Japanese scientists discovered areas with a regular structure in the genomes of bacteria – short identical sequences alternated with unique fragments that had nothing in common with different bacteria. Such sites were called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). 10 years later, it was proved that these sites are responsible for the adaptive immunity of bacteria, and in unique sequences – spacers – information about the genomes of viruses is stored, against which the bacterium is able to fight. The RNA that is synthesized from such sites, in combination with Cas proteins, destroys the corresponding DNA of viruses if they attack the cell. And the most remarkable thing is that a bacterium can replenish its library of viral DNA when it encounters unfamiliar pathogens [1]. That is why CRISPR's immunity is adaptive – it improves and learns to resist new types of infection. CRISPR is an interesting example of Lamarckian evolution: the events of an organism's life directly affect its DNA, changing it so that the organism becomes more adapted. On the other hand, CRISPR is interesting for applied science – it turned out that by providing Cas proteins with arbitrary RNA, they can be forced to split the DNA of cells in the appropriate place*.

* – Earlier, biomolecula repeatedly wrote about the achievements in the use of CRISPR/Cas systems for genome editing: "CRISPR systems: immunization of prokaryotes", "Mutagenic chain reaction: genome editing on the verge of fiction" and "Cure Duchenne myodystrophy: competition of groups, unity of techniques" [2-4]. – Ed.

With the help of such systems, you can simply and accurately edit the genomes of a wide variety of cells (it turned out that bacterial CRISPR works well in eukaryotic cells). The capabilities of CRISPR are so remarkable that a rare issue of an authoritative journal today dispenses with an article explaining new details of the operation of these systems or telling about their new application.

Although no one doubts that CRISPR is capable of much, on February 26, 2016, an article was published in Science telling about the type of these systems with new, even more incredible capabilities [5]. It turned out that some of the CRISPR systems are able to write information into memory not only from DNA, but also from the RNA of pathogens.

The idea that bacteria should have such an opportunity was suggested by some observations made earlier. Firstly, it was known that CRISPR helps bacteria fight against viruses with the RNA genome, in the life cycle of which DNA is not used at all. So, there must be a way to rewrite the information recorded in RNA molecules, not just in DNA, into the "memory" of the immune system. In addition, in some bacteria, among the CRISPR-associated genes that are located near the immune memory cassettes, sites encoding reverse transcriptase domains were found. Sometimes the reverse transcriptase was encoded as a separate protein, and sometimes as a chimera with the Cas1 protein, which usually inserts the virus DNA into CRISPR cassettes. Therefore, it suggested that bacteria could use reverse transcriptase to rewrite information from the RNA of the virus in the form of DNA and store it in this form.

To test their guesses, the scientists chose the CRISPR system of easily cultured Martelella mediterranea bacteria. A chimeric protein with the functions of Cas1 and reverse transcriptase was found in its composition. To make it easier to observe the work of the protein, the researchers obtained transgenic strains of bacteria with an additional copy of the chimeric protein gene placed under a strong promoter. At the same time, the protein with the functions of Cas1 and reverse transcriptase was developed by cells in huge quantities. Under such conditions, new fragments coinciding with the bacterium's own genetic material began to be embedded in CRISPR cassettes (Fig. 1). At the same time, fragments of those DNA sections that were most actively transcribed were most often embedded in cassettes. These data indirectly confirmed the participation of RNA in storing information in CRISPR memory, but could not be considered direct evidence of the role of RNA in the formation of new spacers. For example, the CRISPR system could preferably use the DNA of actively transcribed genes, because such DNA is cleared of proteins, and it could be more convenient for CRISPR components to approach it.

Figure 1. Scheme of embedding new spacers into CRISPR cassettes by an enzyme combining the functions of the Cas1 protein and reverse transcriptase. As a starting material, the enzyme can use both RNA molecules (upper part of the figure) and DNA molecules (lower part). In the first case, reverse transcriptase completes a complementary DNA chain to the RNA inserted into the cassette. Figure from [1].

To clarify the question of the role of RNA in the formation of new inserts, scientists synthesized a special DNA sequence encoding self-splicing RNA. Such RNA is capable of cutting out a certain fragment from itself. Therefore, the variant of the molecule recorded in DNA was different from the RNA variant, and it was possible to distinguish in CRISPR cassettes information recorded on the basis of DNA information or the corresponding RNA. Synthetic DNA was fed to cells in large quantities to make it an attractive target for the formation of new inserts into CRISPR cassettes. After analyzing the sequences of cassettes, the scientists found spacers in them, which could only be formed if the bacterium recorded information from RNA molecules into the memory of the immune system.

Interestingly, when using a gene with a broken reverse transcriptase domain, bacteria did not stop inserting new sequences into CRISPR cassettes, but their tendency to insert fragments corresponding to the most actively transcribed genes disappeared. This led to the assumption that chimeric Cas1 can rewrite both information written in RNA and information written in DNA into the memory of the immune system. This would be beneficial for bacteria, because it would allow them to attack all types of nucleic acids of the virus at once. In order to finally understand the mechanism of the protein, scientists conducted clean experiments in a test tube. At the same time, they used certain types of matrices – only DNA or only RNA. Experiments have confirmed that both types of molecules are suitable as a source of information for recording in the memory of the bacterial immune system. The mechanism of recording information from RNA into cassettes turned out to be particularly interesting – it turned out that RNA itself is ligated in the DNA of cassettes, and a working domain of reverse transcriptase is required for ligation. The RNA can orient itself in any direction, and, apparently, there are no mechanisms that would help to position it in the correct orientation. Then, if DNA nucleotides are added to the test tube, a complementary DNA chain is completed on the basis of RNA. Probably, in the cell, the RNA in the new CRISPR fragment is destroyed by RNase H, which recognizes RNA and DNA hybrids, and then replaced by a second DNA chain, so that no RNA component remains in the cassettes.

The new type of CRISPR systems can be considered the first discovered example of adaptive use of reverse transcription by cellular life forms. It will be interesting to set up experiments that will allow us to quantify the effect of this type of immunity on the survival of bacteria that resist infection. Although the system can help bacteria target precisely those genes that work most actively in the pathogen, it also has a drawback – the already mentioned lack of directional orientation when embedding RNA fragments in CRISPR cassettes. At the same time, it turns out that about half of the spacers obtained on the basis of RNA will be located backwards and will direct the immune system of bacteria to look for non-existent targets.

Anyway, the new data supplemented our knowledge about the mechanisms of CRISPR systems. The potential practical applications of the discovery are also interesting: now bioengineers will have a way to edit only those genetic variants that are actively working in a certain cell.

Literature

1. Sontheimer E.J. and Marraffini L.A. (2016). CRISPR goes retro. Science. 351, 920–921;
2. Biomolecule: "CRISPR systems: immunization of prokaryotes";
3. Biomolecule: "Mutagenic chain reaction: genome editing on the verge of fiction";
4. Biomolecule: "Cure Duchenne myodystrophy: competition of groups, unity of techniques";
5. Silas S., Mohr G., Sidote D.J., Markham L.M., Sanchez-Amat A., Bhaya D. et al. (2016). Direct CRISPR spacer acquisition from RNA by a natural reverse transcriptase-Cas1 fusion protein. Science. 351, aad4234.

Portal "Eternal youth" http://vechnayamolodost.ru  10.03.2015