Synthetic chromosomes for 3D functional genomics: from principles to AI-guided design

The role of genome 3D organization for fundamental chromatin processes, such as long-range promoter-activator regulatory interactions, remains ambiguous. Synthetic genomics, defined as the rational design of kilobase-scale genomic sequences and their assembly into chromosomal segments spanning up to hundreds of kb (and ultimately, entire genomes), provides powerful solutions to lift this ambiguity and directly test structure-function relationships through controlled perturbations. In this review, we highlight how synthetic genomics approaches overcome traditional experimental constraints by creating simplified but functional systems that isolate specific organizational principles, so far mostly in budding yeast and mammalian cells. Combined with AI-based sequence design, these approaches not only provide unprecedented insight into the organizational logic governing genome function but also open new avenues for therapeutic genomic engineering.