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A groundbreaking new genetic screening tool for human organs



LIGHT CRISPR

Shines light on human brain development. Credit: © Dexheimer / IMBA

Many fundamentals in biology and fundamentally all developmental pathways have been identified in the so-called genetic screens. Initially pioneered in the Drosophila fruit fly and the nematode C. elegans, the genetic display is involved in inactivating many genes one by one. By analyzing the consequences of gene loss, scientists can draw conclusions about its function. In this way, for example, all of the genes required to form the brain can be identified.

Genetic screening can be done on a regular basis in flies and worms. In humans, there is a lot of knowledge about genetic disorders and the consequences of disease-related mutations, but systematic analysis is not possible. Now, the Knoblich lab at IMBA has developed a groundbreaking technique that allows hundreds of genes to be analyzed in parallel in human tissue. They named the new technology CRISPR-LICHT and published their findings in the journal Science.

Using brain organoids, a 3D human brain cell culture model developed in Jürgen Knoblich’s group at IMBA, hundreds of mutations can now be analyzed for their role in the human brain using CRISPR-LICHT.

Organoid immunofluorescence

Immunofluorescent imaging of the entire organoid. Red stem cells, green nerve cells and blue nucleus. Credit: © IMBA

“The basis of this technique is a combination of the famous CRISPR-Cas9 technology that was awarded the Nobel Prize in October 2020 and the dual barcode method. The main trick is to apply a guideline RNA, but also genetic barcode, part of DNA that we add to the genomes of the cells we use to grow organic matter. This allows us to see the entire cell line of each organoid, while the second barcode allows us to count the number of cells created by each starting cell. This reduced the noise and thus we were able to determine the effect of each of the pathway RNAs on the number of cells produced during organoid growth. To describe our approach, we call the CRIPSR-Traceability method at cellular resolution in heterologous tissue (CRISPR-LICHT), ”first co-author Dominik Lindenhofer, study born at IMBA, explained.

The researchers applied CRISPR-LICHT to microcephaly, an inherited disorder caused by reduced brain size and severe mental impairment in patients. Using this revolutionary new technology, the scientists screened all the genes suspected to be responsible for the disease.

“Not only were we able to identify microcephaly genes using CRISPR-LICHT, but we were also able to precisely identify a specific mechanism involved in controlling brain size,” said IMBA postdoc and co-author Christopher Esk. know. The endoplasmic reticulum (ER) has been identified as the primary hub for controlling secretion of extracellular matrix proteins. This mechanism affects tissue integrity, and therefore brain size and is identified as one of the causes of microcephaly.

Genetic displays in Drosophila have long been an established tool in whole genome screening and have a long tradition in Vienna. The “Vienna Drosophila Research Center” (VDRC), co-developed by scientists from the IMBA, is the only Drosophila stock exchange in Europe and boasts one of the largest fly collections in the world. on functional gene research. Jürgen Knoblich, IMBA Chief Scientist and team leader, is also rooted in fly genetics and thanks to fruit flies, he gained important insights into the role of stem cells in brain development. .

“We’re delighted to announce that we can now routinely perform genetic screening in complex organoid systems. This method can be applied to other organoid models and to any disease that affects organ formation. This is a whole new approach to the analysis of brain disorders and has incredible potential for the future, as it can be applied to any brain disease, including autism. Our work is only possible through the spirit of cooperation at Vienna BioCenter, as teams from our neighboring institutes Max Perutz Laboratory and Institute of Molecular Pathology also contribute significantly to This idea will help develop new technology, ”said Jürgen Knoblich, the study’s final author.

Reference: October 29, 2020, Science.




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