Genome evolution represents the basis of species adaptation to changing environments and habitats. Recent breakthroughs in sequencing technologies resulted in the acquisition of complete genome information for an increasing number of animal species, propelling the field of evolutionary genomics into a new era of discovery. Yet, our limited capacity to interpret genome variation hinders our understanding on how phenotypical changes drive adaptation. This urges the development of novel strategies to reconcile genomic sequence and function, for which a proper integration of cell-specific gene programs, non-coding regulation and 3D chromatin organization becomes essential. Further, establishing causal relationships between genome mutations and phenotypes still remains a major challenge in the field. Within this context, novel synthetic biology approaches are emerging as a means to understand developmental processes in the context of evolution. The aim of this EMBO workshop is to bring together international scientists with distinct, but complementary expertises on interpreting genome variation, on mechanisms of gene regulation and on in vivo synthetic biology approaches. This allows a comprehensive overview that goes from fundamental principles encoded in genomes to their ultimate biological significance on the formation of living, evolving organisms.