Conventionally, the standard human genome consists of 23 pairs of chromosomes and several mitochondrial DNA variants. However, it is clear that human cells contain other, unconventional forms of DNA, although a robust census of the identity of such human extrachromosomal (ec)DNAs is missing. Furthermore, there is reason to believe that, like their classical counterparts, these “misfit” DNAs have the potential to orchestrate cellular and organismal function, in health, and disease. We will develop a comprehensive suite of tools combining traditional (e.g. electrophoresis; nuclease digestions) and next-generation (e.g. high-throughput sequencing; in-cell genetic engineering of nucleolytic systems) molecular biology in order to interrogate the existence of the human ecDNA genome; probe its effects on integral and novel cellular processes; and delineate its mechanism of formation. We predict concrete outputs of: 15 publications (all high impact), 20 conference presentations, 45 invited talks, and 5 media events. The Outcomes of our work will produce a novel gene regulatory atlas for biological discovery and pharmacotherapy; in redefining the function of genetic material, it will give revolutionary insight into how cells enforce order in a complex cellular system.
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