An international team of scientists, led by Prof. Roland Rad at the University Hospital Klinikum rechts der Isar of Technical University of Munich (TUM), has developed a multiplexed screening approach together with colleagues of the Wellcome Trust Sanger Institute. The method can be used to mutate simultaneously many different genes in adult mice. This enables scientists to imitate cancer development rapidly and flexibly in adult mice and investigate interactions between mutations. They used the CRISPR/Cas9 system.
Cancer is a disease of DNA, in which hundreds or thousands of mutations develop in a cancer cell, confounding DNA-sequencing efforts to distinguish the cancer-driving mutations from the "passenger" mutations that are side-effects of cancer development. In addition, many cancer genes are difficult to discover by sequencing, for example, because they are not mutated, but dysregulated by other means in cancer.
Fast and flexible testing of mutations
In their study the scientists developed a new approach based on the CRISPR/Cas9 system. With colleagues of the Wellcome Trust Sanger Institute they mutated genes in liver cells in adult mice. CRISPR/Cas9 is a method that enables researchers to target mutations to any position in the genome, switching off one gene or several genes. "Our approach enables us to simultaneously target multiple putative genes in individual cells," says Professor Roland Rad, project leader at the Technical University of Munich and the German Cancer Research Center Heidelberg.
The approach is rapid, highly flexible, efficient and scalable, allowing the team to study many genes simultaneously or to examine large regions of the genome. It engineers mutations directly in adult mice and thus bypasses the need to create transgenic animals. "We decided to add the mutations in adult liver cells. That does more closely mimic the biological processes operating in human cancers: as in most human cancers, the mutations occur in the adult and only affect few cells," explains Julia Weber, who is first author of the publication together with Dr. Rupert Öllinger.
Novel high-throughput approach also identifies dysregulated genes
The team of researchers developed, on the basis of literature search, a list of up to 18 genes with known or unknown evidence for their importance in two forms of liver cancer. They introduced CRISPR/Cas9 molecules targeting various combinations of these genes into mice. The genes were knocked out and the mice developed liver or bile duct cancer within a few months.
This approach confirmed that a set of proteins called ARID, which influence the organization of chromosomes, are important for liver cancer development. Mutations in a second protein, TET2, were found to be causative in bile duct cancer: although TET2 has not been found to be mutated in human biliary cancers, the proteins that it interacts with have been, showing that the CRISPR/Cas9 method can identify human cancer genes that are not mutated, but whose function is disturbed by other events.
Search in "genomic deserts" becomes easier
The team’s CRISPR/Cas9 approach is also promising for the biological exploitation of genomic deserts - regions of the human genome that appear to be bereft of genes. Recent studies, such as the ENCODE Project, suggest that deserts can be populated, if not by genes, then by regulatory DNA regions that influence the activity of genes.
"Liver cancer has many DNA alterations in regions lacking genes: we don’t know which of these might be important for the disease," explains Dr. Rupert Öllinger. "We could show that it is now possible to delete such regions to systematically determine their role in liver cancer development."
Julia Weber, Rupert Öllinger, Mathias Friedrich, Ursula Ehmer, Maxim Barenboima, Katja Steiger, Irina Heid, Sebastian Mueller, Roman Maresch, Thomas Engleitner, Nina Gross, Ulf Geumann, Beiyuan Fu, Angela Segler, Detian Yuan, Sebastian Lange, Alexander Strong, Jorge de la Rosa, Irene Esposito, Pentao Liu, Juan Cadiñanos, George S. Vassiliou, Roland M. Schmid, Günter Schneider, Kristian Unger, Fengtang Yang, Rickmer Braren, Mathias Heikenwälder, Ignacio Varela, Dieter Saur, Allan Bradley, and Roland Rad, CRISPR/Cas9 somatic multiplex-mutagenesis for high-throughput functional cancer genomics in mice, Proceedings of the National Academy of Sciences, October 2015.