Sequencing S. cerevisiae with synthetic chromosome XI

We describe construction of the 660 kilobase synthetic yeast chromosome XI (synXI) and reveal how synthetic redesign of non-coding DNA elements impact the cell. To aid construction from synthesised 5 to 10 kilobase DNA fragments, we implemented CRISPR-based methods for synthetic crossovers in vivo and used these methods in an extensive process of bug discovery, redesign and chromosome repair, including for the precise condensation of 200 kilobases of repeated sequence. In synXI, the underlying causes of several fitness defects were identified as modifications to non-coding DNA, including defects related to centromere function and mitochondrial health that were subsequently corrected. As part of synthetic yeast chromosome design, loxPsym sequences for Cre-mediated recombination are inserted between most genes. Using the GAP1 locus from chromosome XI, we show here that these inserted sites can be used to generate extrachromosomal circular DNA on demand, allowing direct study of the effects and propagation of these important molecules. Construction and characterisation of synXI has uncovered effects of non-coding and extrachromosomal circular DNA, contributing to better understanding of these elements and informing future synthetic genome design.