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

Area of Science:

• Synthetic Biology
• Molecular Biology
• Genetics

Background:

• The Synthetic Yeast Genome (Sc2.0) project introduces numerous loxPsym sites for genome shuffling.
• Cre-loxP recombination is a powerful tool for DNA manipulation and genome engineering.
• Existing inducible systems, like estradiol-dependent SCRaMbLE, offer limited control over recombination events.

Purpose of the Study:

• To develop a novel, light-inducible Cre recombinase system for precise control of recombination in Saccharomyces cerevisiae.
• To provide an improved tool for genome engineering applications within the Sc2.0 project and beyond.
• To enhance the scope and variety of recombination events achievable in yeast.

Main Methods:

• Development and characterization of L-SCRaMbLE, a light-controlled Cre recombinase system.
• Utilizing red light to induce Cre recombinase activity, regulated by the chromophore phycocyanobilin (PCB).
• Assessing recombination efficiency and control by varying light exposure time and PCB concentration.

Main Results:

• L-SCRaMbLE demonstrates tight regulation of Cre recombinase activity with up to 179-fold induction by red light.
• Recombination extent is tunable based on induction duration and PCB concentration.
• L-SCRaMbLE offers superior recombination control compared to previous systems, enabling a wider range of recombination outcomes.

Conclusions:

• L-SCRaMbLE provides a facile and highly controllable method for light-induced recombination in yeast.
• This system significantly advances the potential for genome customization and engineering in Saccharomyces cerevisiae.
• L-SCRaMbLE is a valuable tool for the Sc2.0 genome re-engineering project and other recombination-based applications.