Cancer is one of the three leading causes of death in industrialized countries. The development of neoplastic diseases is caused by the unrestricted growth of cells that have been transformed into a malignant state. Genetic defects – mainly caused by single nucleotide variants (SNPs) in tumour suppressor genes or in proto-oncogenes – allow cancer cells to acquire essential biological properties and deregulate several cellular processes, ensuring their survival and efficient growth. To model these SNPs, the application of the recently described CRISPR base editing technology can be employed.
In a first approach, we are establishing experimental procedures in vitro using the CRISPR base editing technology together with single guide (sg)RNA libraries specifically designed to introduce the most frequently found mutations in human cancer-causing genes into the mouse genome. Therefore, tumour prone EµMyc cell lines will be transduced with lentiviral base editor plasmids able to either introduce C-to-T (Cytosine base editor, CBE) or A-to-G (Adenine base editor, ABE) base changes. EµMyc cells which carry CBE or ABE will then additionally be transduced with a corresponding sgRNA library for introducing the individual mutations into the genes. This pool of engineered cell lines will then be treated with diverse chemotherapeutic drugs, such as DNA damaging agents or BCL-2 family inhibitors (e.g., etoposide or BH3 mimetics, respectively). In a second approach, we are using newly developed CBE transgenic mice which are crossed to the tumour prone EµMyc transgenic animals (a model of B cell lymphoma) and isolate haematopoietic stem and progenitor cells (HSPCs) from these animals. These EµMyc/CBE double transgenic cells will then be transduced with sgRNA libraries and used to reconstitute lethally irradiated mice. Tumours that arise at an accelerated pace will be isolated and the tumour promoting sgRNA and genetic mutation identified.
Using this approach will reveal critical tumour suppressor pathways and oncogenes involved in the transformation of haematological malignancies. This will enhance our understanding of malignant transformation and provide novel targets for anti-cancer therapies.