Many of the fundamental principles in biology and essentially all pathways regulating development were identified in genetic screens. Originally pioneered in the fruit fly Drosophila and the nematode Caenorhabditis 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, with the CRISPR-LICHT technique, the Knoblich lab has shown that CRISPR screening can allow hundreds of genes to be analyzed in parallel in human tissue.
“The basis of the technique is a combination of the well-known CRISPR-Cas9 technology … and a dual-barcoding method,” detailed Dominik Lindenhofer, a PhD student at IMBA and co-first author of the current study. “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.”
“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,” stressed IMBA postdoc and co-first author Christopher Esk. In this mechanism, the endoplasmic reticulum serves a control element in the secretion of extracellular matrix protein. When the mechanism fails, tissue integrity suffers, affecting brain size and contributing to microcephaly.
