Utilizing synthetic yeast chromosomes to map and phenotype large scale-deletions in high-throughput.

Question: The Synthetic Yeast Genome Project (Sc2.0) aims to produce the first synthetic eukaryotic genome. All synthetic chromosomes have been synthesised and characterised in individual strains. The sixteen synthetic chromosomes and the tRNA neochromosome are being consolidated in a single cell.1 All chromosomes have been redesigned according to strict principles. One notable feature is the insertion of symmetrical loxP sites downstream of almost every non-essential gene. Upon activation of Cre recombinase, this leads to highly complex structural variations, turning a single genotype into a population. Here we present our strategy and technology for using synthetic yeast to systematically study the effects of large-scale deletions, complementing our knowledge of single gene deletion libraries.

Methods: The loxP sites are 34 nucleotides in length. We use this as a landing pad to randomly integrate a modified marker by homologous recombination (HR). The resulting population is transformed with a plasmid overexpressing a system to introduce a double-strand break in the marker. The DNA is degraded in vivo until HR occurs at loxP sites. Counter selection ensures that only candidates that have lost the marker are obtained. We use laboratory automation to select and characterise large numbers of candidates. Based on our high-throughput, low-cost end-point genotyping, we can rapidly identify the deletion size for each strain.2,3 This is followed by phenotyping of the relevant strains.

Results: We have developed and established a strategy for targeted introduction of large-scale deletions into synthetic yeast chromosomes. The large deletions will be characterised using molecular and phenotyping techniques to understand their impact on cell viability.

References

(1) Richardson S, et al. (2017) Design of a synthetic yeast genome. Science, 355(6329), 1040-1044.(2) Mitchell LA, et al. (2015) qPCRTag Analysis--A high throughput, real time PCR assay for Sc2.0 genotyping. J Vis Exp 25:(99):e52941.(3) Lindeboom TA, Sánchez Olmos MdC, et al. (2022) L-SCRaMbLE creates large-scale genome rearrangements in synthetic Sc2.0 chromosomes. bioRxiv.