New Technique Modifies Single Cells To Create Genetic “Mosaic” Organs

A new technique has been developed that allows different genes to be “switched off” in individual cells within an organ to create a “mosaic” of genetically modified cells. The research, which shed light on the origins of a rare genetic disorder, is published in Nature.

Understanding the genetic origins of disease

Animals such as mice are commonly used in research to understand the genetic basis of disease. Genes can be removed, or “knocked out”, to investigate the biological consequences and tease out the gene’s function.

However, many diseases are not caused by one faulty gene alone – several genetic factors are typically at play. This makes exploring how individual genes are involved in disease very difficult for researchers, often necessitating a different group of experimental animals for each gene to be studied.

Now, researchers from ETH Zurich and the University of Geneva have developed a technique that can make many simultaneous genetic changes in animal cells, in which cells in individual organs can be modified to create a “mosaic”-like animal. This builds upon previous studies, which have been able to achieve this in lab-grown cells or embryos, but not in live adult mice.

Technique enables “mosaic”-like modifications

Their CRIPSR-based technique involved using an adeno-associated virus (AAV) to direct the CRISPR-Cas genetic “scissors” toward their genes of interest, infecting the mice with a mixture of viruses that target a specific gene. This enabled multiple different genes to be switched off in the cells of a single organ.

For this study, the researchers chose to target the brain. Specifically, they investigated the possible genetic causes underlying a rare disorder called 22q11.2 deletion syndrome (22q11.2DS). The features of the condition vary greatly but often include heart defects, delays in growth and speech development or learning disabilities. 22q11.2DS is also associated with conditions such as schizophrenia and autism spectrum disorders.

The genetic cause of 22q11.2DS originates in a region of chromosome 22 that contains 106 genes. It was understood that multiple genes are involved in the disease, but not which ones. The researchers focused on 29 genes in this region that are active in both mice and humans, modifying 1 of their 29 genes of interest in each individual mouse brain cell.

By analyzing the subsequent changes in gene expression in these cells, the researchers identified three genes that affected brain cell function. One had previously been described, though the other two are relatively under-researched.

“If we know which genes in a disease have abnormal activity, we can try to develop drugs that compensate for that abnormality,” explained the study’s lead author António Santinha, a PhD candidate at ETH Zurich.

Potential applications for other genetic diseases

ETH Zurich has now applied for a patent on the technology, and the researchers explain that the technique would also be suitable for studying other genetic disorders. The number of genes modified using the technique could also be expanded to several hundred per experiment.

“In many congenital diseases, multiple genes play a role, not just one,” Santinha explains. “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.”

“It’s 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,” Santinha continues. “Depending on what you’re trying to investigate, though, you could also use AAVs that target other organs.”

Reference: Santinha AJ, Klingler E, Kuhn M, et al. Transcriptional linkage analysis with in vivo AAV-Perturb-seq. Nature. 2023:1-9. doi: 10.1038/s41586-023-06570-y

This article is a rework of a press release issued by ETH Zurich. Material has been edited for length and content.