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Cutting the DNA - Discovery of Cas9 Enzyme
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Expensive, complex, and very rarely performed procedure of cutting the DNA plays a role in replacing damaged genes to cure genetic diseases and AIDS. But scientists have made to the development of simple, accurate and inexpensive method of cutting the DNA, which could lead to a significant shift in genetic medicine.

The new technique was first discovered last year, but when cutting the DNA of bacteria. This discovery, published as a research paper in June 2012, is already declared as an outstanding creative achievement in the review of scientific contributions in 2012 of the journal Nature Biotechnology.

After that, the inventors of new method, Jennifer Doudna from the Howard Hughes Medical Institute, Jinek Martin from the University of Berkeley and Emmanuelle Charpentier from the Laboratory for Molecular Infection in Sweden, published two new papers in the journal Science Express, which have proven successful innovative methods for cutting DNA of human cells.

New Technology To Cross The Barriers

Major obstacle in the research and development of gene therapy in humans are modifications of gene technology, which are also the main precondition for the overall understanding of biology and human health. However, by using the new technology of DNA cutting, these barriers should not represent a problem any more, so almost everyone will be able to use this genetic reprogramming in mammals, and quite probably in other eukaryotic organisms, said Doudna, a professor of molecular and cell biology and chemistry and a researcher at the Howard Hughes Medical Institute and Lawrence Berkeley National Laboratory.

The scientists explain how, on the basis of feedback received, can be concluded that this method will lead to a revolution in genetic engineering of plants and animals. Not only makes programming easier, but it is probably just as strong as the polymerase chain reaction (PCR). George Church, professor of genetics at Harvard Medical School and lead author of an article in Science Express, adds that the ease of use and compactness of these techniques will allow the reproduction of small segments of DNA into millions of copies that will cause major changes in biological and medical research genetics.

The Principles of the New Method of DNA Cutting

Previous techniques that use cutting DNA synthesized zinc-ring nuclease (ZFN) and protein TALEN(Transcription Activator-Like Effector nucleases) Science magazine named as one of the top 10 scientific discoveries in year 2012. They named them "cruise missiles" as they allow researchers to "land" in a certain part of the genome and precisely cut the DNA double helix at this specific place. Using two of these cuts it is possible to remove the part of DNA, and to replace it with a new part. In this way, the damaged or mutated genes are replaced by healthy ones. Clinical studies conducted by bio-pharmaceutical company Sangamo Biosciences, have shown that the replacement of a specific gene in individuals infected with HIV, may make them resistant to AIDS.

By comparing the new technique, which uses an enzyme Cas9 with TALEN method, Church proved that the technique is five times more efficient in inserting genes into mammalian cells. This difference is attributed to the size of Cas9 that is smaller than TALEN, proteins and is therefore possible to use it to cut a small pieces of DNA. Since the smaller part of the DNA consists of a short series of nucleotides, that means the transfer of the nucleotide sequence will require RNA with less nucleotids which significantly facilitates the modification and synthesis of new DNA. Not only is it easier, but it is also possible to use Cas9 to reprogram hundreds of DNA sequences simultaneously. It is also shown that this technique is less toxic to cells than other methods. Church says it is too early to declare complete victory over the ZFN proteins and TALEN, but promising new technique works.

Discovery of Cas9 Enzyme

Doudna came to the discovery of the enzyme Cas9 while studying the immune system of bacteria that have developed an enzyme to cut the DNA as a way to defend form viruses. Using this enzyme, bacteria cut the viral DNA, then insert DNA parts into its own DNA, and uses it to produce RNAs which bind to the virus and deactivate it.

For this unusual antiviral immune system Doudna was intrigued by Jill Banfield, a professor of Earth and planetary systems at the University of Berkeley. But, in her research she focused on the study of the ways in which cells use RNA that are essentially copies of genes from DNA and are essential for its synthesis and replication.

Doudna, together with her associates Jinek East and Alexander and Aaron Cheng and Ma Enbom from the Department of Molecular and Cellular Biology at the University of Berkeley, continued to investigate how the DNA is being cut using an enzyme-RNA complex. First, the protein Cas9 binds to both the ends of the RNA, and then this complex binds to one particular piece of DNA which is determined by the RNA sequence. Researchers have simplified this whole system to use only one part of the RNA, rather than the whole RNA chain, to cut and target specific parts of the bacterial DNA, as presented in the previous article journal Science.

The main advantage of this system is the use of only one enzyme for genetic modification. There is no need to change the enzyme for every part of DNA we want to cut, because it just reprograms itself using different RNA transcripts. Comparing to the method used in the past few decades, this method is much easier to design and implement, says Doudna. Scientists claim this is the best solution so far that would allow to interfere with human genome.

Last three articles pay attention to the effectiveness of the studied bacterial DNA cutting system in human cells. Out of partly unknown bacterial immune system, the new technology is being developed, that completely changes the way of studying the cells of different animals and plants, and the way we manipulate with them. This opens the way for major scientific discoveries that would have a significant impact on human health, says Doudna.
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