For a long time, changing or editing genes in the genome has been a tedious and expensive affair. Because previous methods were not very accurate and enormously complex. But then, a few years ago, researchers discovered a mechanism by which bacteria fight back against virus attacks: so-called "Clustered Regularly Interspaced Short Palindromic Repeats" in their genome - in short, CRISPR. These gene segments, together with the enzyme Cas9, have the ability to selectively cut out DNA sequences or incorporate them into a specific site of the genome. Since then, gene clippers CRISPR / Cas9 have opened up new possibilities for genetic research to introduce genes into the genome and correct even pathological point mutations. Thus, researchers have already been able to repair the responsible for the sickle cell anemia gene change in blood cells in mice succeeded in the treatment of Duchenne muscular dystrophy. In China, scientists have already introduced the first gene segments into human embryos - a highly controversial intervention in the human germline.
However: Also CRISPR / Cas9 is not infallible. Sometimes off-target effects also occur in the new gene scissors by incorporating gene parts in unwanted organs or in wrong places of the DNA. "If you want to introduce a new gene into the mammalian genome, the difficulty may be finding the cheapest site, " explains study leader Yong Zhang of Northwest A & F University in Yangling. "You have to search the genome and look for a region where the change has the least impact on the neighboring genes." At the same time, the cell's own DNA repair mechanisms can lead to retroactively altering or eliminating introduced genes. In their study, the researchers have now developed a variant of the gene scissors whose activity is less susceptible to this DNA repair. They achieved this by replacing the Cas9 enzyme with the Cas9 nickase (Cas9n). These gene scissors are then used by the scientists to equip cattle with a resistance gene against bovine tuberculosis. This infectious disease caused by the pathogen Mycobacterium bovis is transmissible to humans.
More resistant to infection
To give the cattle resistance to the tuberculosis bacterium, Zhang and his colleagues began to use connective tissue cells isolated from bovine fetuses. With the help of their modified gene scissors, they introduce the resistance gene NRAMP1 into the genome of these immature cells. In preliminary experiments, they had clarified which place in the genome is the cheapest. The nuclei of these genetically modified cells were planted in the envelope of a cored bovine egg cell. This so-called somatic nuclear transfer gave rise to a bovine embryo, which in principle was cloned from the manipulated connective tissue cell. In 173 of these cloned embryos, the scientists used surrogate cows. "16 calves were born from this group, 11 of which survived the first few weeks, " Zhang and his colleagues report. Their investigations showed that the resistance gene was present in the genome of the target cells of these calves, but not in unwanted tissues or organs. "Our study demonstrates for the first time that the CRISPR / Cas9n system can be used to produce transgenic livestock without unwanted off-target effects, " says Zhang.
Whether the introduced gene also works as hoped, the scientists tested in an infection test: They administered six genetically manipulated calves and six control animals each one dose of the tuberculosis pathogen Mycobacterium bovis in the lungs. Over the next few weeks, researchers used blood samples to check whether the bacteria in the animals' bodies were able to keep up and multiply. The result: The control animals showed clear signs of tuberculosis infection after three weeks. In the case of genetically manipulated calves, however, significantly fewer pathogens survived in the body. "The cattle showed increased resistance to Mycobacterium bovis, " Zhang and his colleagues report. "In this way we have discovered a technology and a favorable position in the bovine heredity that allows the introduction of beneficial genes into these livestock." However, given the relatively low proportion of successfully born calves, a lot of work is still needed to make such methods practicable for livestock and agriculture close. display
- Yong Zhang (Northwest A & F University, Yangling) et al., Genome Biology, doi: 10.1186 / s13059-016-1144-4