A groundbreaking genetic screening tool for human organoids

Date:
October 29, 2020
Source:
IMBA- Institute of Molecular Biotechnology of the Austrian Academy
of Sciences
Summary:
Researchers have developed CRISPR-LICHT, a revolutionary
technology that allows genetic screens in human tissues such
as brain organoids. By applying the novel technology to brain
organoids, the ER-stress pathway was identified to play a major
role in regulating the size of the human brain.



FULL STORY ==========================================================================
Many of the fundamental principles in biology and essentially all pathways regulating development were identified in so-called genetics screens.

Originally pioneered in the fruit fly Drosophila and the nematode
C. elegans, genetic screens involve inactivation of many genes one by
one. By analyzing the consequences of gene loss, scientists can draw conclusions about its function.

This way, for example, all genes required for formation of a brain can
be identified.


========================================================================== Genetic screens can routinely be carried out in flies and worms. In
humans, a wealth of knowledge exists about genetic disorders and
the consequences of disease-relevant mutations, but their systematic
analysis was impossible. Now, the Knoblich lab at IMBA has developed a groundbreaking technique allowing 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.

By using cerebral organoids, a 3D cell culture model for the human brain developed in Ju"rgen Knoblich's group at IMBA, hundreds of mutations
can now be analyzed for their role in the human brain using CRISPR-LICHT.

"The basis of the technique is a combination of the well-known CRISPR-Cas9 technology, which was awarded the Nobel Prize in October 2020, and a
dual- barcoding method. The key trick was to apply a guide RNA, but also
a genetic barcode, a piece of DNA that we add to the genome of the cells
we use to grow organoids. This allows us to see the entire cell lineage
of each organoid, while a second barcode lets us to count the number of
cells generated by each starting cell. This reduces noise and so we can determine the effect of each guide RNA on the number of cells produced
during organoid growth. To describe our approach, we termed the method CRIPSR-Lineage Tracing at Cellular resolution in Heterogenous Tissue (CRISPR-LICHT)," explains co-first author Dominik Lindenhofer, PhD
student at IMBA.

The researchers applied CRISPR-LICHT to microcephaly, a genetic disorder
caused by a reduction in brain size and severe mental impairment in
patients. By means of this revolutionary new technology, the scientists screened through all genes suspected to play a role in the disease.

"Not only were we able to identify microcephaly genes with CRISPR-LICHT,
but we also pinpointed a specific mechanism involved in controlling the
size of the brain," says IMBA postdoc and co-first author Christopher
Esk. The endoplasmic reticulum (ER) was identified as main hub in
controlling extracellular matrix protein secretion. This mechanism affects
the integrity of tissue, and thus the brain size and was identified as
one cause of microcephaly.

Genetic screens in Drosophila have long been an established tool in
genome-wide screenings and have a long tradition in Vienna. The "Vienna Drosophila Research Center" (VDRC), co-developed by scientists from IMBA,
is the only Drosophila stock center in Europe and boasts one of the
worldwide largest fly collections for functional gene studies. Ju"rgen Knoblich, IMBA Scientific Director and group leader, also has his roots
in fly genetics and, thanks to the fruit fly, gained important insights
into the role of stem cells for brain development.

"We are very excited to report that we now can routinely do genetic
screens in complex organoid systems. The method can be applied to other organoid models and to any disease affecting organ formation. It is a completely new approach to analyzing brain disorders, and bears incredible future potential, as it can be applied to any brain disease, including
autism. Our work was only possible due to the collaborative spirit at the Vienna BioCenter, as groups from our neighboring institutes of the Max
Perutz Labs and the Institute of Molecular Pathology also substantially contributed to the ideas that helped developing the novel technology,"
says Ju"rgen Knoblich, last author of the study.


========================================================================== Story Source: Materials provided by IMBA-_Institute_of_Molecular_Biotechnology_of_the
Austrian_Academy_of_Sciences. Note: Content may be edited for style
and length.


========================================================================== Journal Reference:
1. Esk, Lindenhofer et al. A human tissue screen identifies a
regulator of
ER secretion as a brain size determinant. Science, 2020 DOI:
10.1126/ science.abb5390 ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2020/10/201029142004.htm

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