Project summary Eukaryotic genomes contain arrays of tandemly repeated non-coding sequences that we currently know little about—satellite DNAs. Typically found near centromeres, telomeres, and on Y chromosomes, satellite DNAs can comprise over 50% of some eukaryotic genomes. They are known to change rapidly in sequence and genomic location, which can cause genetic incompatibilities between closely related species. The misregulation of satellite DNA can have serious consequences for genomic stability and cancer formation. Despite being a ubiquitous part of genomes and having important effects on cellular functions, the lack of genetic, genomic, and molecular tools to study tandemly repeated sequences has stymied progress towards understanding satellite DNA evolution and function. For example, satellite DNAs are particularly challenging to sequence, assemble, and manipulate. Recent developments in sequencing and genome editing technologies circumvent some of these problems. This proposal integrates genomic, molecular, and cytological methods to study the evolutionary and functional genomics of satellite DNA in Drosophila genomes. The PI has developed new genomic approaches and resources to study satellite DNA evolution with unprecedented resolution. The PI will use a comparative genomics approach to study changes in satellite DNA sequence, abundance and organization over evolutionary time and to determine the evolutionary forces driving these changes. This proposal also aims to develop comprehensive population genetic models of satellite DNA evolution that take into consideration different types of natural selection based on functional aspects of satellite DNAs studied in this proposal and leverage empirical data about satellite DNA organization generated by the PI. Little is currently known of satellite DNA function: the precise genetic manipulation of satellite DNAs with site-specific gene editing approaches had not been possible in the past due to a lack of unique target sites. The PI has used their assemblies of satellite loci to identify target sites and has successfully manipulated satellite DNA loci using CRISPR/Cas9-based genome editing techniques. They have made precise genomic deletions and duplications of satellite DNA in Drosophila melanogaster that they will use to test specific hypotheses about the regulation, fitness effects, and selfish genetic behavior of satellite DNA. This proposal will also leverage the new molecular genetic resources the PI created to manipulate satellite DNA expression to ask questions about their functions in chromosome segregation and chromatin organization. These experiments will have broad implications not only for genome evolution and speciation, but also for understanding the regulation of satellite DNA in cancer and aging.