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Prof. Dr. Stefan Mundlos
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Original publication

Katerina Kraft, Sinje Geuer, Anja J. Will, Wing Lee Chan, Christina Paliou, Marina Borschiwer, Izabela Harabula, Lars Wittler, Martin Franke, Daniel M. Ibrahim, Bjørt K. Kragesteen, Malte Spielmann, Stefan Mundlos, Darío G. Lupiáñez, and Guillaume Andrey
Deletions, Inversions, Duplications: Engineering of Structural Variants Using CRISPR/Cas in Mice

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Every person is unique – as is his or her genome. Changes in the genome, known as mutations, also vary from one person to the next. Some mutations alter the letter sequence of the genetic code. Others affect the DNA structure, where the number or position of letters is altered across large segments of the DNA molecule. These so called structural variations are a common cause of disease. Until now, such rearrangements were very difficult or impossible to model in mice. Researchers from the Max Planck Institute for Molecular Genetics in Berlin in collaboration with colleagues from Charité - Universitätsmedizin Berlin have now developed a new method to reproduce large genomic rearrangements quickly and efficiently in mice. Using this method the scientists have engineered a deletion of over 300,000 base pairs in mice, thus creating a model for the associated disease.

Mutations often involve the replacement of individual letters of the genetic code by others. However, larger structural changes are also possible: for example, entire DNA segments may be inserted into the genome. Sometimes, long segments containing many genes are copied and duplicated. Others are lost or are moved to incorrect positions. Such changes can have highly variable effects on an organism. Some reduce or increase gene activity, leading to, for example, cancer or so-called “rare” diseases.

At least 26 to 30 million people in Europe suffer from these diseases, which are rare only in relative terms. Often they reduce life quality and expectancy of those affected and their families. Because they are uncommon and the symptoms can vary greatly from one patient to the next, specific testing for rare diseases is difficult. Moreover, many mutations exert their pathogenic effects during embryonic development, so that scientists are unable to characterize them.

Using a modification of the CRISPR/Cas method, the Berlin-based researchers have reproduced large structural rearrangements of the human genome in mice. Cas proteins are enzymes produced by bacteria to fend off pathogens such as viruses. In the technique used, the enzymes specifically cut the DNA molecule in embryonic mice stem cells at two predefined locations. During the repair process, a DNA section is inserted or omitted between the two cleavage points. In this way, researchers can effect mutations in the mouse genome at precisely the same site as they occur in genetic diseases in humans.

Using this technique, stem cells with the desired mutations can be produced in just a few weeks. The animals that develop from those stem cells carry the exact same genetic change in their genome as affected humans. This is the only way for scientists to learn more about such diseases and possibly develop effective treatments.

In a first application of the method, the scientists altered a section of the genome in embryonic stem cells to reproduce a mutation that occurs in patients with extremely short leg bones. The mice then also developed shortened leg bones. The scientists can now use the animals to investigate more precisely what lies at the root of this disorder.

The Research Group of Stefan Mundlos is supported by a private donor of the Max Planck Foundation.

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