The Human Mini-Cell

Is it possible to derivatize a human mini-cell with a size-reduced genome which can survive in culture? To answer this question, we will start-off with a human haploid cell line (1n) which, compared to a diploid cell line (2n), has only one copy of a single genome. More precisely, we will be working with the cancer cell line HAP1 which, as common for cancer cells, has a reduced and fragmented chromosome set, hence having a smaller genome. And in this project, we intend to make its genome even smaller. We will be doing so by utilizing methods and tools of biochemistry and molecular biology that generate deletions. Certain drugs, for example, lead to instability of chromosomes and deletions of parts of chromosomes. We will further express nucleases in the cells, including nuclease from the CRISPR/Cas system, to generate deletions in chromosomes. Nucleases, more generally speaking, can cut DNA and upon repairwhich the cells provide by themselvesdeletions can happen. In this project we hence use dedicated drugs as well as modern molecular biology tools and the cells very own error-prone repair mechanisms to our advantage. The loss of genetic material we will make visible and quantifiable by staining the DNA directly with fluorescent dyes. Using an appropriate technique, fluorescence-activated cell sorting (FACS), we will be able to collect the desired mini-cells. Once selected they will be sequenced, and their genome will be analysed to find out what is and what is not there anymore in comparison to the progenitor cells we started with. Thereby we hope to learn basic concepts as to what parts of the chromosome and which genes are essential for life. Further, those mini-cells will allow us to investigate the metabolism with a potentially reduced redundancy and complexity. With these cells we hope to provide a simplified system for studying cellular processes and metabolism. This can have implications on our current understanding of cell biology in general as well as to cancer biology more specifically. Cancer cells often have reduced genomes and it would be interesting to find new handles how to control their growth and thereby not effecting healthy cells. We further envision that the mini-cells will be of interest to systems biologists and genome engineers.