14 January 2013

Correction of the genome up to the nucleotide

Researchers at the Massachusetts Institute of Technology, the Broad Institute and Rockefeller University have developed a new technique that allows you to change the genomes of living cells with a high degree of accuracy by adding or removing genes. They claim that the new technology provides a new, easy-to-use and relatively cheap method of modifying organisms for the production of biofuels, creating animal models for studying human diseases, developing new therapies and many other purposes.

The first genetically modified mice were created in the 1980s by inserting small fragments of DNA into the nuclei of mouse embryonic cells. Currently, this method is widely used to create transgenic animals that act as models for studying human diseases. However, it has one serious drawback – DNA fragments are inserted into the genome randomly.

In recent years, experts have developed new more accurate methods of genome modification. One of them, known as homologous recombination, involves the use of a DNA fragment, in addition to the target gene containing sequences corresponding to the region of the genome in which the gene of interest to scientists needs to be embedded. However, the probability of success of this procedure is very low, since DNA recombination occurs very rarely in normal cells.

More recently, biologists have learned to increase the efficiency of this process with the help of DNA–cutting enzymes - nucleases. Protein complexes known as "zinc fingers" are usually used to deliver nucleases to the desired regions of the genome, but they cannot affect all possible DNA sequences, which limits the scope of their application. Moreover, the synthesis of these proteins is very laborious and requires large financial costs.

To cut DNA in certain regions, effector nucleases similar to transcription activators (transcription activator-like effector nucleases, TALENs) can also be used, but the synthesis of these complexes is also complex and costly.

The system developed by the authors is much more acceptable from a practical point of view. It is based on the use of natural bacterial complexes consisting of proteins and RNA. These complexes protect bacterial cells from invading viruses by recognizing and cutting viral RNA. Using these structures as a basis, the researchers created DNA-modifying complexes representing the Cas9 nuclease associated with short RNA strands. These sequences are connected by complementary DNA sequences, and Cas9 cuts them at the specified location.

This approach can be used both to block the work of a particular gene, and to replace it with the necessary genetic variant. To replace the gene, the necessary DNA sequence is introduced into the complex, which is copied to the incision site.

The accuracy of the new method is exceptionally high. Nuclease activation does not occur even when the DNA sequences of the genome and the guide RNA differ by one pair of nucleotides. The efficiency and cost of development also favorably distinguish it from previously used approaches.

The developers have placed all the genetic components necessary to create the system in the non-profit plasmid foundation Addgene, giving all colleagues the opportunity to use the new method. They also created a website that provides recommendations and tips on how to use the technique.

Among other things, the proposed system can be used to develop new methods of treating diseases caused by a defect in one gene, such as Huntington's disease. Currently, clinical studies of therapeutic approaches are already underway, consisting in blocking the activity of certain genes with the help of "zinc fingers". The new technology is potentially a more efficient alternative.

It may also be useful in the treatment of HIV by isolating the patient's lymphocytes and changing their gene encoding the CCR5 receptor, which is the "entrance gate" for the virus. After being injected back into the patient, such modified cells will prevent the development of the disease.

Another area of application of the new technology is the study of human diseases by inducing certain mutations in the genome of stem cells. The subsequent transformation of such cells into cardiomyocytes or neurons will allow us to study the effect of mutations on the biology of specialized cells.

Articles by Mali et al., RNA-Guided Human Genome Engineering via Cas9 (full text – on the Church Lab page on the Harvard University website) and Cong et al., Multiplex Genome Engineering Using CRISPR/Cas Systems (full text – on the Zhang Lab @MIT page) are published in the journal Science.

Evgeniya Ryabtseva
Portal "Eternal youth" http://vechnayamolodost.ru based on MIT materials: Editing the genome with high precision.

Portal "Eternal youth" http://vechnayamolodost.ru14.01.2013

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