CRISPR/Cas9 used for rapid functional study of cancer-causing genes
Novel high-throughput approach finds combinations of genes that induce cancer
In a novel use of the CRISPR/Cas9 system, which can be deployed to switch genes off, researchers from Germany, the UK and Spain have developed a multiplexed screening approach to study and model cancer development in mice. The scientists mutated genes in the adult mouse liver uncovering their cancer-causing roles and determining which combinations of genes cooperate to cause liver cancer.
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. By engineering mutations directly in adult mice and thus bypassing the need to create transgenic animals, the method can more rapidly deliver results and, in some instances, more closely mimic the biological processes operating in human cancers.
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. Another major challenge is to determine the function of mutated genes in normal cells and understand how their mutation causes cancer.
CRISPR/Cas9 is a method that enables researchers to target mutations to any position in the genome, switching off one gene or several genes. The application of this technology in model organisms is however still in its infancy. In this study, the team used CRISPR/Cas9 to cause mutations in liver cells of adult mice in order to trigger liver cancer development. With approximately 800,000 new cases per year, liver cancer is the sixth most-prevalent cancer in the world.
“We reasoned that, by targeting mutations directly to adult liver cells using CRISPR/Cas9, we could better study and understand the biology of this important cancer. Other approaches to engineer mutations in mice, such as stem cell manipulation, are limited by the laborious process, the long time frames and large numbers of animals needed. And, our method better mimics important aspects of human cancer biology than many ‘classic’ mouse models: as in most human cancers, the mutations occur in the adult and only affect few cells.”
Dr Mathias Friedrich An author on the paper from the Wellcome Trust Sanger Institute
The team of researchers developed a list of up to eighteen 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 mice developed liver or bile duct cancer within a few months.
“Our approach enables us to simultaneously target multiple putative genes in individual cells. We can now rapidly and efficiently screen which genes are cancer-causing and which ones are not. And, we can study how genes work together to cause cancers – a crucial piece of the puzzle we must solve to understand and tackle the disease.”
Professor Roland Rad Project leader at the Technical University of Munich and the German Cancer Research Center Heidelberg
This approach confirmed that a set of proteins called ARID, which influence the organisation 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.
“The new tools of targeting genes in combination and inducing insertions or deletions in chromosomes change our ability to identify new cancer-causing genes and to understand their role in cancer. Our results show that this approach is feasible and productive in liver cancer; we will now continue to study our new findings and try to extend the approach to other cancer types.”
Professor Allan Bradley Senior group leader and Director Emeritus from the Sanger Institute
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. We could show that it is now possible to delete such regions to systematically determine their role in liver cancer development.”
The team’s new approach will allow them to study the importance of other deserts by removing them using CRISPR/Cas9 and studying the consequences for cell, tissue and animal biology.
The work was supported by the German Cancer Consortium Joint Funding Program and the Helmholtz Gemeinschaft (Preclinical Comprehensive Cancer Center).
Department of Medicine II, Klinikum Rechts der Isar, Technische Universität München, Munich, Germany; German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany; The Wellcome Trust Sanger Institute, Hinxton/Cambridge, United Kingdom; Department of Pathology, Klinikum Rechts der Isar, Technische Universität München, Munich, Germany; Institute of Radiology, Klinikum Rechts der Isar, Technische Universität München, Munich, Germany; Institute of Virology, Klinikum Rechts der Isar, Technische Universität München, Munich, Germany; Institute of Pathology, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany; Instituto de Medicina Oncológica y Molecular de Asturias, Oviedo, Spain; Research Unit Radiation Cytogenetics, Helmholtz Zentrum München, München/Neuherberg, Germany; and Instituto de Biomedicina y Biotecnología de Cantabria, Santander, Spain
The university hospital Klinikum rechts der Isar (on the right side of the river Isar) serves its patients with a highly skilled team of dedicated doctors, nurses, research scientists, and technical assistants. The hospital rechts der Isar is the university hospital of the Technische Universität München (TUM).
The Science of Healing
With a workforce of over 5,000 personnel, the university hospital is a renowned center for the care of the sick, for medical research, and for the teaching of medicine. The hospital rechts der Isar is composed of more than 30 separate clinics and departments treating some 60,000 in-house patients and 245,000 out-patients yearly. The 1,100-bed hospital covers the entire spectrum of modern medicine with state-of-the-art efficiency. Through the close cooperation between health care and research, the latest advances in medical techniques can be quickly integrated into patient treatment procedures.
The German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ) with its more than 3,000 employees is the largest biomedical research institute in Germany. At DKFZ, more than 1,000 scientists investigate how cancer develops, identify cancer risk factors and endeavor to find new strategies to prevent people from getting cancer. They develop novel approaches to make tumor diagnosis more precise and treatment of cancer patients more successful. The staff of the Cancer Information Service (KID) offers information about the widespread disease of cancer for patients, their families, and the general public. Jointly with Heidelberg University Hospital, DKFZ has established the National Center for Tumor Diseases (NCT) Heidelberg, where promising approaches from cancer research are translated into the clinic.
In the German Consortium for Translational Cancer Research (DKTK), one of six German Centers for Health Research, DKFZ maintains translational centers at seven university partnering sites. Combining excellent university hospitals with high-profile research at a Helmholtz Center is an important contribution to improving the chances of cancer patients. DKFZ is a member of the Helmholtz Association of National Research Centers, with ninety percent of its funding coming from the German Federal Ministry of Education and Research and the remaining ten percent from the State of Baden-Württemberg.
The Wellcome Trust Sanger Institute is one of the world’s leading genome centres. Through its ability to conduct research at scale, it is able to engage in bold and long-term exploratory projects that are designed to influence and empower medical science globally. Institute research findings, generated through its own research programmes and through its leading role in international consortia, are being used to develop new diagnostics and treatments for human disease.
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