Centromeres are essential to genome inheritance, serving as the site of kinetochore assembly and coordinating chromosome segregation during cell division. Abnormal centromere function is associated with birth defects, infertility, and cancer. Most human centromeres are consistently formed at regions containing highly repetitive alpha satellite DNA. However, in cancer cells, new, ectopic centromeres (neocentromeres) frequently arise on intact chromosomes or small supernumerary marker chromosomes (sSMCs) at genomic regions that lack alpha satellite DNA. Studies in model organisms (Drosophila, yeast, chicken cells) have suggested that certain regions of the genome, including pericentromeres, heterochromatin, and regions of open chromatin or high transcription, are particularly amenable to neocentromere activation. Little is known about the mechanisms of human neocentromere formation or the role of neocentromeres in neoplasia. This is largely due to the lack of experimental systems to study ectopic centromere formation in human cells. In this proposal, we propose to establish CRISPR-based assays to induce neocentromeres on specific human chromosomes, compare their (epi)genomic organization and functional efficiency, and define DNA and chromatin features at sites of formation. In Aim 1, we will use CRISPR-based approaches to independently trigger neocentromere formation on individual human chromosomes after removal or suppression of the native centromere. Our rationale is that by removing or incapacitating the largest concentration of CENP-A, neocentromeres will arise at ectopic sites that harbor low concentrations of CENP-A and/or are vulnerable to CENP-A invasion. In Aim 2, we will define the functional consequences of neocentromere formation by mapping chromatin structure at ectopic centromere regions and measuring the effect of neocentromere formation on gene expression and the surrounding chromatin environment. The technological and intellectual outcomes of this study will fundamentally transform our ability to create and study human chromosome abnormalities and expand our view of genome function and plasticity. Our proposed experiments will establish new methods to induce ectopic centromere assembly on specific human chromosomes to better understand the clinical consequences of neocentromere formation.