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A revolutionary genetic tool

In the past few years a new gene splicing method has fundamentally changed the game for genetics: Crispr/Cas9. These “gene scissors” allow DNA genome building blocks to be modified with previously unimaginable precision

At the end of November last year there was an international outcry: Chinese biophysicist Hè Jiànkuí from the University of Shēnzhèn reported the birth of twin girls whose genetic makeup had been genetically modified in the embryonic stage so that they were resistant to HIV. Shēnzhèn University was “profoundly shocked” by this transgression, and countless scientists and politicians were likewise outraged. Shortly afterwards the public authorities arrested Hè, and he is facing punishment. This manipulation of the genome was made possible by a new technique that is considered one of the greatest developments in molecular biology: the “gene scissors” Crispr/Cas9.

In the beginning: Bacteria fight off viruses
As is so often the case with groundbreaking discoveries, Crispr/Cas9 began with an observation: bacteria can effectively defend themselves against hostile viruses. This defense is based on the so-called Crispr/Cas system. When a virus binds to a bacterial cell and injects its genetic material, a short section of it is inserted between the Crispr sequences of the bacterial DNA. These sections are a kind of library of all pathogens the cell has confronted in the past. In the event of a new infection, Crispr/Cas provides a “memory” for the bacteria’s defense against infection, enabling it to cut up the virus and render it harmless. This library is preserved for generations, because it is passed on from the bacterium to its descendants. Thus, as with epigenetics an acquired property is inherited – a mechanism that violates Darwin’s concept of evolution. Today we know that about onehalf of all known bacteria have a Crispr/ Cas defense system. Depending on the type, two large Crispr/Cas classes are distinguished. Class-I systems comprise protein complexes consisting of many molecules, whereas class-II systems comprise only one cutting protein each.

Targeted mutations
In 2011 and 2012, Emmanuelle Charpentier and Jennifer Doudna of Berkeley University, California, published the basic research results on bacterias’ Crispr/ Cas9 defense in the leading profession al journals Nature and Science. One year later, Zhāng Fēng of the Broad Institute of Cambridge published how the method can be applied to higher organisms as well, for Crispr/Cas9 works not only in bacteria but also in cells with nuclei, i.e., in plants, animals and humans.

The Crispr/Cas system is based on three components: 1) A short RNA molecule serves as a genetic recognition sequence. Such a “probe” can be produced relatively easily and matches the nucleotide pattern of the respective DNA target sequence. 2) It is linked to the socalled tracrRNA. 3) This RNA complex in turn attaches itself as a “guide” to an enzymatic cutting tool, the Cas9 protein. This completes the molecular “gene scissors” consisting of RNA recognition sequence, tracrRNA and Cas9 scissors. Now the triple complex binds to a specific location on a target DNA and cuts it up with the Cas9 scissors. The American scientists realized the potential of this mechanism. Since the recognition RNA sequence can be varied easily, it is now possible to determine exactly where the molecular gene scissors bind and cut the target DNA. It is true that a cell is able to repair such a cut; however, this repair is usually incomplete, resulting in reading errors. In other words, by cutting up the target DNA, genes can be specifically “switched off.” In addition, individual DNA building blocks or larger functional DNA sections can also be inserted into the cut, and thus completely new properties implanted very precisely into the genome.

No chance for chance
Crispr/Cas9 and other methods of socalled “genome editing” promise a plethora of application possibilities. In plant and animal breeding, for example, geneticists are trying to create more productive or disease-resistant varieties and breeds. These include, for example, mildew-resistant wheat, starch-enriched corn or potatoes that can be stored at low temperatures. The basic mechanism – induction of a double-strand break and subsequent cellular repair – is the same mechanism that follows natural mutations. Mutation breeding in plants is likewise based on this process. Previously, however, such breaks were triggered in an uncontrolled manner, often through irradiation or chemicals. So it was a matter of chance at which point in the genome of a plant the new, additional gene might be integrated.

With genome editing and especially with Crispr/Cas9, results are no longer left to chance, because editing occurs at single, pre-determined points. However, even with Crispr/Cas9, unintentional mutations can occur, albeit rarely. Since such so-called “off-target” mutations might have serious consequences, especially in the medical field, scientists have cautiously continued the development of Crispr/Cas9 and other protein scissors to improve accuracy. For example, new Crispr/Cas9 variants cut only a single DNA strand, which significantly reduces the number of missing or additional base pairs (22). If the two single strands are cut at staggered positions, producing “sticky ends,” i.e. DNA ends with complementary over-hangs, the accuracy of the genetic modification is significantly improved.

Many things still remain unclear
For years scientists have been trying to address certain diseases by specifically altering the genetic makeup, but mostly unsuccessfully. Since the discovery of the Crispr/Cas9 system, hopes have risen. The first positive results have been reported: a treatment for Duchenne muscular dystrophy (DMD). This condition is based on the mutation of a gene that produces the protein dystrophin – an important component of muscle fibers. After a Crispr/Cas9 treatment, slightly elevated levels of the protein could be detected. In initial clinical studies, the new genome editing methods have also been tested in HIV and cancer patients. However, scientists are still struggling: so far gene repair works in comparatively few human cells, since the repair mechanism is active only in reproducing cells; but most cells in the body do not replicate. In addition there is a question of how to get the gene scissors to their site of action within the body’s cells. Both the stomach and the immunocytes in blood destroy such proteins. It is possible that vehicles such as nanoparticles (e.g., liposomes) might be able to transport Crispr/Cas9 molecules directly inside cells. Harmless or artificially inactivated viruses are also being tested as transport vehicles.

Whether defective genes already in the germ line should be repaired – i.e., in egg and sperm cells or in embryos, as seems to have happened to the Chinese twin girls – is highly controversial, for ethical reasons. Most scientists disagree with this approach, since it would pave the way to “designed humans.”

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