We describe complete design of a synthetic eukaryotic genome, Sc2.0, a highly modified Saccharomyces cerevisiae genome reduced in size by nearly 8%, with 1.1 megabases of the synthetic genome deleted, inserted, or altered. Sc2.0 chromosome design was implemented with BioStudio, an open-source framework developed for eukaryotic genome design, which coordinates design modifications from nucleotide to genome scales and enforces version control to systematically track edits. To achieve complete Sc2.0 genome synthesis, individual synthetic chromosomes built by Sc2.0 Consortium teams around the world will be consolidated into a single strain by "endoreduplication intercross. " Chemically synthesized genomes like Sc2.0 are fully customizable and allow experimentalists to ask otherwise intractable questions about chromosome structure, function, and evolution with a bottom-up design strategy.

Rapid advances in DNA synthesis techniques have made it possible to engineer viruses, biochemical pathways and assemble bacterial genomes. Here, we report the synthesis of a functional 272,871 bp designer eukaryotic chromosome, synIII, which is based on the 316,617 bp native Saccharomyces cerevisiae chromosome III. Changes to synIII include TAG/TAA stop-codon replacements, deletion of subtelomeric regions, introns, tRNAs, transposons and silent mating loci as well as insertion of loxPsym sites to enable genome scrambling. SynIII is functional in S. cerevisiae. Scrambling of the chromosome in a heterozygous diploid reveals a large increase in “a mater” derivatives resulting from loss of the MATα allele on synIII. The total synthesis of synIII represents the first complete design and synthesis of a eukaryotic chromosome, establishing S. cerevisiae as the basis for designer eukaryotic genome biology.

We describe design, rapid assembly, and characterization of synthetic yeast Sc2.0 chromosome VI (synVI). A mitochondrial defect in the synVI strain mapped to synonymous coding changes within (), encoding an essential proteasome subunit; Sc2.0 coding changes reduced Pre4 protein accumulation by half. Completing Sc2.0 specifies consolidation of 16 synthetic chromosomes into a single strain. We investigated phenotypic, transcriptional, and proteomewide consequences of Sc2.0 chromosome consolidation in poly-synthetic strains. Another "bug" was discovered through proteomic analysis, associated with alteration of the transcription start due to transfer RNA deletion and loxPsym site insertion. Despite extensive genetic alterations across 6% of the genome, no major global changes were detected in the poly-synthetic strain "omics" analyses. This work sets the stage for completion of a designer, synthetic eukaryotic genome.