Rapid advances in DNA synthesis techniques have made it possible to engineer diverse genomic elements, pathways, and whole genomes, providing new insights into design and analysis of systems. In a major genome engineering effort, the synthetic yeast genome project, Sc2.0, is well on its way with the 16 synthetic Saccharomyces cerevisiae chromosomes now 99% completed by a global team. A hallmark of the synthetic genome is a set of strategically located loxP sites that enable genome restructuring using an inducible evolution system termed Synthetic Chromosome Rearrangement and Modification by LoxP-mediated Evolution (SCRaMbLE). SCRaMbLE can generate millions of derived variant genomes with predictable structures leading to complex genotypes and phenotypes.  Remarkably, the 3D structures of synthetic and native chromosomes are very similar. In a second experimental effort, the yeast karyotype was recently completely engineered, by systematically fusing pairs of telomeres and deleting single centromeres, thus generating an isogenic series of yeast ranging from n=16 to n=2. These strains show reproductive isolation and a massively altered 3D genome structure, yet they are surprisingly “Normal” and show high fitness. Finally, the DNA synthesis pipeline has been automated (the GenomeFoundry@ISG), opening the door to parallelized big DNA assembly, including assembly of human genomic regions of 100 kb along with multiple designer synthetic variants thereof. Such segments can be precisely delivered to stem cells to dissect genomic “dark matter”, to perform transplants of specific human genomic regions to animal genomes, and to endow human cells with new capabilities.

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