II. Development of crispr/cas9 technology.

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II. DEVELOPMENT OF CRISPR/Cas9 TECHNOLOGY.

If the standard systems don’t contain the protein, called Cas9, a few proteins are collected in a complex that bind CRISPR RNA, and then this complex recognizes viral DNA-target (the portion of DNA that you want to edit) and attracts another protein, has been directly by cutting the viral DNA, in systems containing Cas9, the latest protein performs these functions: binds to CRISPR RNA, and recognizes the target, and cuts it. This tool is very convenient and easy to use, relatively standard systems.

In nature, CRISPR RNA is first encoded in CRISPR-cassette, and then associated proteins and only then recognizes the target. It turned out that can also be unnatural CRISPR RNA by chemical or enzymatic synthesis. With the spacer in such an RNA is the sequence chosen by the researcher. Thus, a protein called Cas9 that can recognize and connect with such a synthetic, non-natural СRISPR RNA becomes programmed to recognize and "cut" the appropriate place in the DNA.

III. THE MECHANISM OF GENOMIC EDITING USING CRISPR/Cas9.

Most human cells, except gametes, are diploid, that is, we have a double set of chromosomes — one from each parent. If one of the parent chromosomes are broken, the modified DNA sequence in some important gene, in this case, it may be as carriers of genetic diseases. If both copies are infringements, deviations from the norm — there will be genetic disease. A classic example hemophilia. In order to cure a genetic disease, you need to fix genetic information, as modified by the mutation. Hemophilia, like most genetic diseases, caused by changing only one letter of DNA, but only in our genome is about 6 billion letters. And to cope with the problem we need to find the a single mistake and try to fix it in the specified location without changing anything else, so as not to provoke other diseases. This is one of the main goals of genomic medicine. And to correct altered by the mutation of a gene, we need a very accurate molecular knife that will find a mutated sequence of nucleotides and can, cut out, delete it from the DNA. This "knife" is Cas9. With the help of a guide RNA (viral CRISPR RNA), the sequence of which coincides with the desired position, you need to delete, change, it can make the gap in the genome. Recognition of the target occurs at the segment with a length of 20 to 30 nucleotides. Cell under intervention of viral RNA, and not die from any break in the DNA chain, as it will be fixed by healthy copies of the paired chromosomes by the natural process of reparation. If no paired chromosomes, as, for example, in the case of hemophilia, then it is possible to make the cage part of the right specified by researcher gene simultaneously with the Cas9 and the RNA guide and use it as a matrix to fill the gap made.

Also using the CRISPR/Cas9 enable simultaneous editing of several mutated genes. You may enter the different Cas9 and multiple RNA guides. Each of the guides will direct Cas9 to its target, that will help holistically to eliminate disease from the genome.

The described mechanism operates by the principle of complementarity. Chains of the DNA double helix recognize each other according to the rules of this principle. CRISPR RNA recognize their target the same way. (A-T, G-C).

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