The mechanism of recording new information in CRISPR is described

Anna Kaznadzei, N+1

Scientists from the University of California at Berkeley have found out how new sections of DNA are embedded in bacterial CRISPR systems, which are necessary for protection against infections and are widely used in modern genetic engineering methods. It turned out that for correct and accurate embedding, the cooperation of the Cas1-Cas2 integrase system and the IHF factor is necessary, and the DNA bends like a horseshoe, and the key factor here is the correct geometry of the mechanism, and not the exact nucleotide sequences inside the CRISPR cassette. The study is published in Science (Wright et al., Structures of the CRISPR genome integration complex). 

The CRISPR cassette is arranged as follows: it is a section of the genome containing a series of repeats of 20-50 nucleotides long, separated by "spacers" – DNA sections, for example, viral, which the system uses as a kind of reference in the fight against infections. If the DNA that has entered the cell is similar to what is in the reference book, special proteins recognize it and cut it – this is called the immune response of the bacterial cell.

The new spacers are inserted into CRISPR using an integrase complex consisting of 4 Cas1 proteins and two Cas2 proteins. The complex inserts a new spacer at the beginning of the cassette, before its first repeat, which follows an AT-rich leader sequence. The place where the new spacer is inserted must be chosen correctly, because when DNA is embedded in an arbitrary part of the genome, among other things, bacterial genes may malfunction.

It is known that in CRISPR systems, the leader sequence, the first repeat and, in particular, the inverted repeated GC-rich region inside it participate in the integration process of spacers. The IHF (Integration Host Factor) binding factor, similar to eukaryotic histones, binds to the leader sequence and connects the Cas1-Cas2 complex to the process. Scientists decided to investigate this mechanism, the details of which were still unclear.

Using the methods of electron microscopy and X-ray crystallography, they were able to obtain the structures of integrase complexes and substrate (infectious) and genomic DNA at the intermediate and final stages of the integration process.

It turned out that due to the structure of the cassette (in particular, the presence of repetitions in it), IHF can bend it like a horseshoe, while opening the integrase access to the areas it needs. IHF contacts two sites for this. At the same time, there is almost no direct chemical contact (formation of hydrogen bonds) between integrase and genomic DNA, with the exception of only a few hydrogen bonds at the place where the seventh α-helix Cas1 enters the small groove of the DNA helix near the leader sequence. The main factor in the integration process, however, is not hydrogen bonds, but its correct geometry, which is achieved due to a certain arrangement of protein binding sites and the DNA structure of the cassette itself. GC-rich inverted repeat section allows DNA to be bent, the section in the middle of the first repeat acts as a door hinge, and IHF holds the whole structure. The integrase complex itself does not perform accurate recognition of sites by nucleotides, which once again emphasizes the non-selective nature of the Cas1 trasposase described in many studies.

IHF bends DNA into a horseshoe-shaped structure, after which it occurs
interaction of Cas1 with its sites (from an article in Science).

It also turned out that in the absence of IHF, the embedding of substrate DNA into arbitrary sections of the bacterial genome is much more common. When transferring the site of its binding by five nucleotides to the side, this does not happen, but the efficiency of integration decreases. Thus, the correct interaction of IHF and the Cas1-Cas2 integrase complex turns out to be key for the accurate and efficient embedding of new spacers into the CRISPR cassette.

At the same time, as mentioned above, the structure of the object is important to the integrase complex, where it will embed new sequences, but not specific nucleotide sequences inside it. Perhaps this feature is due to the huge variety of CRISPR cassettes in bacteria. Scientists believe that such a unique mechanism of the Cas1-Cas2 integrase complex can be used as a "molecular recording device" for barcoding genomes or for non-standard locus-specific embeddings.

And you can read about how tomatoes that do not need pollination were created using the CRISPR system here.

Portal "Eternal youth" http://vechnayamolodost.ru  24.07.2017