RNA sequence (RNA-seq) profiling of SynXI strain

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 synthesized 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 removal of 200 kilobases of unexpected 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 activity 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 targeted insertion of these sites can be used to create extrachromosomal circular DNA on demand, allowing direct study of the effects and propagation of these important molecules. Construction and characterization 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. We used RNAseq analysis to compare the transcriptional profiles of the parental strain to the initial synXI assembly strain ysXIb01 and to ysXIb16, which contains the debugged synXI_9.11. We selected BY4742 as our parental control as its auxotrophic profile most closely matched the synXI strains. For the purposes of our analysis, we eliminated mating-type specific genes to compensate for mating type differences between the parental and synXI strains. This included transcripts of the dubious ORF YKL177W as it almost entirely overlaps STE3, which encodes the receptor for a factor pheromone in MATα cells. The CDS of FLO10 was entirely recoded by an early version of REPEATSMASHER due to the presence of highly repetitive sequence (Richardson et al., 2017).