Tuberculosis-resistant cows have been created
Julia Korowski, XX2 century
Scientists from the Chinese Northwest Scientific and Technical University of Agriculture and Forestry have bred transgenic cows with increased resistance to tuberculosis. They used a modified version of CRISPR, thanks to which they managed to avoid a common problem of the original technology – the appearance of unplanned mutations. The results of the work are published in the journal Genome Biology (Gao et al., Single Cas9 nickase induced generation of NRAMP1 knockin cattle with reduced off-target effects).
"We have successfully integrated a tuberculosis resistance gene called NRAMP1 into the cow genome thanks to a new version of the CRISPR/Cas9n system," says lead author of the study, Dr. Yong Zhang. – Then we managed to breed cows with increased resistance to tuberculosis. It is important to note that our method does not lead to the appearance of non-targeted effects, which means that the CRISPR technology we used may be better suited for creating transgenic cattle with a purposefully altered genome."
Guess which of these cows is genetically modified? The correct answer is both.
Recently, CRISPR/Cas9 technology has been actively used in genetic engineering. It is relatively simple, but periodically leads to unexpected mutations. Preventing their occurrence is one of the most important tasks of modern genetics. "When inserting a new gene into the mammalian genome, the difficulty is to find the best place to insert it,– Zhang explains. – You need to dig through the genome in search of the region that you think has the least effect on other genes that are in close proximity. We were able to identify the most suitable site for inserting the gene, which, as we were able to demonstrate, does not lead to non-targeted effects."
About ten years ago it became known that the development and course of tuberculosis is regulated by the NRAMP1 gene. About every third inhabitant of the planet is infected with Mycobacterium tuberculosis, but most of the pathogens are in a "dormant state". Alleles (variants) of the NRAMP1 gene affect whether a person (or animal) will develop tuberculosis and how fast the process will proceed if it does happen. The researchers embedded the desired variant of the NRAMP1 gene into the genome of fibroblasts obtained from fetuses aged 35-40 days, and then transferred the nuclei of these cells into cow eggs. From them, embryos were obtained in the laboratory, which were placed in the uterus of cows. Similar experiments were conducted using the classic CRISPR/Cas9 technology to compare the results.
In total, scientists received 20 calves from 4819 embryos, while 11 of them lived longer than three months. The genome of the surviving calves was checked for unforeseen changes using PCR and Southern blotting is a method that allows you to detect specific forms of DNA in cells. In the process of Southern blotting, DNA molecules are removed from cells and divided into small fragments by special enzymes. These fragments are separated from each other, and with the help of a gene probe (a DNA section labeled with a radioactive substance), identical DNA sections are searched. None of the nine calves obtained with CRISPR/Cas9n could detect any non-targeted genome changes. The built-in NRAMP1 did not affect the work of other genes, and the protein encoded by it did not get into the skin, muscles, heart, liver, kidneys or lungs – and therefore scientists concluded that the gene fell exactly into place. But in two animals created with CRISPR, unplanned mutations were found.
Scientists took blood samples from transgenic and ordinary cows of the same breed and exposed them to the pathogens of "bovine tuberculosis" – Mycobacteria Mycobacterium bovis. It turned out that the white blood cells of GM cows fight infection better. In particular, once infected, the cells of transgenic animals self–destruct twice as often - this process, called "apoptosis", helps prevent the spread of infection. The researchers then conducted an in vivo experiment: they randomly selected six transgenic and the same number of normal animals and forced them to inhale M. bovis. After that, blood tests were taken from the calves at certain intervals. By measuring the level of standard infection markers in a blood sample, the scientists concluded that GM cows are indeed more resistant to the disease.
"Our study has shown for the first time that the CRISP/Cas9n system can be used to create transgenic cattle without receiving non–targeted effects," says Zhang. "During the experiments, we discovered a site in the genome of cows in which, thanks to this technology, it will be possible to insert new genes useful for farm animals."
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02.02.2017