Researchers at ETH Zurich have created a technique that enables them to genetically alter every animal cell in a unique way. This enables them to investigate something that previously required several animal experiments in a single experiment. Researchers have identified genes that are important for severe, uncommon genetic diseases using the new methodology.
One proven way of determining the genetic etiology of diseases is to remove a single gene from animals and observe the effects on the organism. The issue is that the pathogenesis of many diseases is dictated by several genes.
This makes determining the extent to which any of the genes is implicated in the disease exceedingly challenging for scientists. To do this, they would need to conduct several animal examinations, one for each targeted gene modification.
Researchers led by Randall Platt, Professor of Biological Engineering at the Department of Biosystems Science and Engineering at ETH Zurich in Basel, have produced a method that will significantly streamline and speed up research with laboratory animals: they use the CRISPR-Cas gene scissors to make several dozen gene changes in the cells of a single animal, similar to a mosaic.
While just one gene is mutated in each cell, the cells within an organ are transformed in diverse ways. Individual cells can then be examined in great detail. This allows researchers to evaluate the effects of several gene modifications in a single experiment.
First Time in Adult Animals
The ETH Zurich researchers have successfully employed this strategy in living animals — adult mice — for the first time, detailed in their recent Nature paper. Other researchers have previously devised a similar method for cultured cells or animal embryos.
The researchers employed adeno-associated virus (AAV), a delivery technique that can target any organ, to “inform” the mice’s cells on which genes the CRISPR- Cas gene scissors should delete.
They modified the viruses so that each viral particle carried the information to kill a certain gene, and then infected the mice with a mix of viruses carrying different gene-destroying instructions. With this method, the team were able to shut off different genes in the cells of one organ, the brain.
New Pathogenic Genes Discovered
Using this technology, researchers from ETH Zurich and the University of Geneva discovered fresh clues regarding a rare genetic condition in humans known as 22q11.2 deletion syndrome. Patients with the disease exhibit a wide range of symptoms and are frequently diagnosed with additional disorders such as schizophrenia and autism spectrum disorder.
Previously, it was thought that this condition was caused by a chromosomal region comprising 106 genes. It was also recognized that the disease was linked to several genes, but it was unclear which genes performed various roles in the condition.
The researchers focused their investigation in mice on 29 genes from this chromosomal region that are also active in the mouse brain. They changed one of these 29 genes in each individual mouse brain cell and then examined the RNA profiles of those brain cells. The researchers were able to demonstrate that three of these genes are substantially responsible for brain cell malfunction.
Furthermore, scientists discovered patterns in mouse cells that are similar to schizophrenia and autism spectrum diseases. One of the three genes was already recognized, but the other two had not previously received much scientific attention.
If we know which genes in a disease have abnormal activity, we can try to develop drugs that compensate for that abnormality.”
António Santinha, Study Lead Author and Doctoral Student, ETH Zurich
The method could potentially be applied to investigate other genetic disorders.
Santinha added, “In many congenital diseases, multiple genes play a role, not just one. This is also the case with mental illnesses such as schizophrenia. Our technique now lets us study such diseases and their genetic causes directly in fully grown animals.
The number of changed genes per experiment could have expanded from the current 29 to several hundred.
“It is a big advantage that we can now do these analyses in living organisms, because cells behave differently in culture to how they do as part of a living body.”
Another advantage is that the AAVs can be easily injected into the animals' bloodstreams. There are many AAVs with varying functional features. In this investigation, researchers employed a virus that penetrates the brains of the animals.
“Depending on what you are trying to investigate, though, you could also use AAVs that target other organs,” Santinha added.
The technique has been patented by ETH Zurich. The researchers now intend to utilize it as part of a spin-off company they are launching.
Perturbing the Genome
The method described here is one of several novel genetic editing techniques being utilized to modify cell genomes in a mosaic-like fashion. The precise word for this research strategy, which includes altering the genome using CRISPR-Cas gene scissors, is “CRISPR perturbation.” The study of the biological sciences is now being revolutionized by this method.
It enables the collection of a significant amount of data from a single scientific experiment. The method might thereby hasten biomedical research, for example in the look for the molecular origins of genetically complicated disorders.
Another research group from the Department of Biosystems Science and Engineering at ETH Zurich in Basel, in collaboration with a team from Vienna, presented a study using CRISPR perturbation in organoids a week ago. Organoids are microtissue spheroids generated from stem cells that have a structure comparable to genuine organs - in other words, they are small organs.
Organoids are an animal-free research approach that supplements animal research. Since both approaches—CRISPR perturbation in animals and in organoids—can yield more information with fewer experiments, they have the potential to minimize the number of animal experiments in the long run.
Santinha, A. J., et al. (2023). Transcriptional linkage analysis with in vivo AAV-Perturb-seq. Nature. doi.org/10.1038/s41586-023-06570-y