L-SCRaMbLE as a tool for light-controlled Cre-mediated recombination in yeast

Abstract

The synthetic yeast genome constructed by the International Synthetic Yeast Sc2.0 consortium adds thousands of loxPsym recombination sites to all 16 redesigned chromosomes, allowing the shuffling of Sc2.0 chromosome parts by the Cre-loxP recombination system thereby enabling genome evolution experiments. Here, we present L-SCRaMbLE, a light-controlled Cre recombinase for use in the yeast Saccharomyces cerevisiae. L-SCRaMbLE allows tight regulation of recombinase activity with up to 179-fold induction upon exposure to red light. The extent of recombination depends on induction time and concentration of the chromophore phycocyanobilin (PCB), which can be easily adjusted. The tool presented here provides improved recombination control over the previously reported estradiol-dependent SCRaMbLE induction system, mediating a larger variety of possible recombination events in SCRaMbLE-ing a reporter plasmid. Thereby, L-SCRaMbLE boosts the potential for further customization and provides a facile application for use in the S. cerevisiae genome re-engineering project Sc2.0 or in other recombination-based systems.

Synthetic yeast genome reveals its versatility

“…This in turn allows a greater number of beneficial rearrangements to be manifested. Although it is premature to claim that SCRaMbLE is a universal tool for engineering yeast, taken together, the various findings [3][4][5][6][7] certainly show that it has great potential for generating yeasts for a wide range of purposes.…”

“…He recognized that this was a hereditary syndrome, but the underlying cause was not obvious. Little research into XP was then done until the 1960s, when a process called nucleotide excision repair was discovered in bacteria [3][4][5] . In this process, enzymes clip out segments of DNA that have been damaged by light and replace them with fresh, undamaged DNA.…”

“…In this process, enzymes clip out segments of DNA that have been damaged by light and replace them with fresh, undamaged DNA. Mutant bacterial strains were isolated that could be killed by low doses of ultraviolet radiation, and some of these were found to be unable to carry out excision repair 4,5 . These concepts of DNA repair were then extended to human cells.…”

“…Sc2.0 is therefore a versatile platform that can be easily modified and evolved to produce yeasts that have desired attributes 2 . A collection of seven papers [3][4][5][6][7][8][9] published in Nature Communications demonstrates the immense potential of Sc2.0 for engineering and understanding yeast.…”

“…Jia et al 4 have developed a SCRaMbLE variant in which both oestradiol and galactose molecules are required to activate rearrangement, also reducing background rearrangement. Hochrein et al 5 have engineered Cre recombinase so that it is activated by red light, providing a new way to control SCRaMbLE. And Luo et al 6 have introduced a reporter DNA sequence into a synthetic yeast strain, which allows cells that have undergone SCRaMbLE-induced genome rearrangement to be easily distinguished from those that have not.…”