Iterative SCRaMbLE for engineering synthetic genome modules and chromosomes

Saccharomyces cerevisiae is closing-in on the first synthetic eukaryotic genome with genome-wide redesigns, including LoxPsym site insertions that enable inducible genomic rearrangements in vivo via Cre recombinase through SCRaMbLE (Synthetic Chromosome Recombination and Modification by LoxPsym-mediated Evolution). Combined with selection, SCRaMbLE quickly generates phenotype-enhanced strains by diversifying gene arrangement and content. Here, we demonstrate how iterative cycles of SCRaMbLE reorganises synthetic genome modules and chromosomes to improve functions. We introduce SCOUT ( S CRaMbLE C ontinuous O utput and U niversal T racker), a reporter system that allows sorting of SCRaMbLEd cells into high-diversity pools. Paired with long-read sequencing, SCOUT enables high-throughput mapping of genotype abundance and genotype-phenotype relationships. Iterative SCRaMbLE is applied here to yeast strains with a full synthetic chromosome and histidine biosynthesis modules. Five HIS module designs are tested, and SCRaMbLE is used to optimise the poorest performer. Our results highlight iterative SCRaMbLE as a powerful tool for data driven modular genome design.