Centromeres are essential to genome inheritance. Abnormal centromere function is associated with birth defects, infertility, and cancer. Human centromeric (CEN) chromatin typically forms on alpha satellite DNA, a 171bp monomeric sequence that is organized into tandem arrays extending for several megabases. The Telomere-to- Telomere (T2T) Consortium that recently produced the first complete human genome assembly revealed that nearly all endogenous human chromosomes contain multiple alpha satellite arrays. We have shown that on some chromosomes, multiple arrays are competent for centromere assembly. Using Homo sapiens chromosome 17 (HSA17) as a model, we demonstrated that centromere location is dictated by genomic variation within alpha satellite DNA. On HSA17 that has three distinct higher order repeat (HOR) unit arrays, when the largest array contains size and sequence variation, centromere assembly shifts to a nearby array. If the centromere forms at a highly variant array, fewer centromere proteins are present and the chromosome experiences instability. We hypothesize that placement/organization of variant HORs within an alpha satellite array influences centromere location and kinetochore assembly. Since most human chromosomes must choose between two (or more) sites at which to build a stable centromere, our work addresses a fundamental gap in the knowledge of basic processes that influence centromere location, competence, and long-term stability. However, we still lack a comprehensive view of the extent of alpha satellite variation within the population and thus the range of functional centromere outcomes. In this competing renewal application, the proposed work builds on our classification of specific alpha satellite variants identified in diverse human populations to assemble stable, de novo centromeres. We will also systematically test centromere competency of long-range alpha satellite organization, coupled with variant content and proximity to mobile elements. Our project goals are to: 1) produce new genomic assemblies of functionally characterized centromeres using targeted long read sequencing approaches, 2) use human artificial chromosome assays to assess competency of different alpha satellite DNA arrangements for de novo centromere formation, 3) explore the molecular basis for variant centromere defects, and 4) use genome engineering to rehabilitate and/or rescue defective variant centromeres. Successful completion of this work will result in major advancement of our basic understanding of genomic variation within large repetitive DNA arrays in humans and its link to specific centromere outcomes and long-term chromosome maintenance and stability.