Unlock the power of SCRaMbLE with ReSCUeS

New technologies to synthesize DNA, facilitated by methods exploiting synthesis of oligonucleotides made on microarrays for example, provides a great opportunity to completely redesign the entire genome of an organism. A group of researchers worldwide has teamed up to re-synthesize a designer eukaryotic genome, Sc2.0. The aim of the Sc2.0 project is to build a synthetic genome which will allow us not only to test our ability to construct a yeast with multiple synthetic chromosomes, but also to answer a series of important biological questions. One important feature integrated into the synthetic genome was the inducible genome rearrangement system named SCRaMbLE. However, the power of SCRaMbLE has not been fully explored because an efficient selection method for SCRaMbLEd cells is still missing.

We designed ReSCuES (Reporter of SCRaMbLEd Cells using Efficient Selection) to isolate SCRaMbLEd strains based on the loxP-mediated switch of two auxotrophic markers, which happens only in the presence of an active recombinase. We showed that all randomly isolated clones contained rearrangements within the synthetic chromosome (synXII in this study), demonstrating high efficiency of selection. To our surprise, given the presence of near 300 loxPsym sites in synXII, only a small number of rearrangements were identified in each strain, with the majority as inversions and deletions. The largest structural variant was an inversion of 325,571bp DNA flanking the centromere of synXII, which is longer than the entire chromosome III, demonstrating the power of SCRaMbLE to generate long-range chromosome rearrangement. Furthermore, using ReSCuES we illustrated, for the first time, the ability of SCRaMbLE to generate strains with increased tolerance to several stress factors, such as ethanol, heat and acetic acid. In addition, by analysing these tolerant strains, we were able to identify Ace2p, a transcription factor required for septum destruction after cytokinesis, as a negative regulator of ethanol tolerance, and first reported the importance of its 3’UTR for maintaining the normal mRNA level. Collectively, this work not only established a generic platform to rapidly identify strains of interest from a SCRaMbLEd synthetic yeast population, but also provides methods to dissect the underlying mechanisms of resistance.