Novel Method Genetically Modifies Individual Cells in Mice

Researchers led by Randall Platt, PhD, professor of biological engineering at the department of biosystems science and engineering at ETH Zurich in Basel, have developed a method to genetically modify individual cells in animals. Their method will enable researchers to study the ramifications of many different gene changes in a single experiment.

The findings are published in Nature in an article titled, “Transcriptional linkage analysis with in vivo AAV-Perturb-seq.”

“The ever-growing compendium of genetic variants associated with human pathologies demands new methods to study genotype–phenotype relationships in complex tissues in a high-throughput manner,” wrote the researchers. “Here we introduce adeno-associated virus (AAV)-mediated direct in vivo single-cell CRISPR screening, termed AAV-Perturb-seq, a tuneable and broadly applicable method for transcriptional linkage analysis as well as high-throughput and high-resolution phenotyping of genetic perturbations in vivo.”

To “inform” the mice’s cells as to which genes the CRISPR-Cas gene scissors should destroy, the researchers used the AAV. They prepared the viruses so that each virus particle carried the information to destroy a particular gene, then infected the mice with a mixture of viruses carrying different instructions for gene destruction. In this way, they were able to switch off different genes in the cells of one organ. For this study, they chose the brain.

Using this method, the researchers from ETH Zurich, together with colleagues from the University of Geneva, obtained new clues to a rare genetic disorder in humans, known as 22q11.2 deletion syndrome. Before now, it was known that a chromosomal region containing 106 genes is responsible for this disease. It was also known that the disease was associated with multiple genes, however, it was not known which of the genes played which part in the disease.

The researchers focused on 29 genes of this chromosomal region that are also active in the mouse brain. In each individual mouse brain cell, they modified one of these 29 genes and then analyzed the RNA profiles of those brain cells. The scientists were able to show that three of these genes are largely responsible for the dysfunction of brain cells. In addition, they found patterns in the mouse cells that are reminiscent of schizophrenia and autism spectrum disorders. Among the three genes, one was already known, but the other two had not previously been the focus of 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,” explained António Santinha, a doctoral student in Platt’s group and lead author of the study.

“In many congenital diseases, multiple genes play a role, not just one, Santinha said. “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 continued. Another advantage is that the scientists can simply inject the AAVs into the animals’ bloodstreams. There are various different AAVs with different functional properties. In this study, researchers used a virus that enters the animals’ brains. “Depending on what you’re trying to investigate, though, you could also use AAVs that target other organs,” Santinha concluded.