An Optimized Genotyping Workflow for Identifying Highly SCRaMbLEd Synthetic Yeasts.

Area of Science:

• Synthetic biology
• Genomics
• Molecular biology

Background:

• Synthetic yeast strains (Sc2.0) possess numerous recombination sites (loxPsym) enabling genome restructuring via SCRaMbLE.
• SCRaMbLE technology can generate highly diverse yeast populations from a single genotype.

Purpose of the Study:

• To develop an efficient workflow for selecting genetically diverse yeast candidates with rearranged synthetic chromosomes.
• To introduce loxTags, qPCR primers for genotyping loxPsym sites, and qTagGer, a primer prediction tool.

Main Methods:

• Developed loxTags, a set of qPCR primers for genotyping across loxPsym sites.
• Created qTagGer, a qPCR genotyping primer prediction tool to manage numerous amplicons.
• Utilized loxTag-based genotyping and long-read sequencing.

Main Results:

• loxTags can detect deletions, inversions, and translocations after SCRaMbLE.
• Light-inducible Cre recombinase (L-SCRaMbLE) efficiently generated diverse recombination events in Sc2.0 strains.
• Demonstrated successful application on synthetic chromosome III (linear and circular versions).

Conclusions:

• loxTags and qTagGer provide an efficient workflow for analyzing SCRaMbLE-induced genomic diversity in synthetic yeast.
• L-SCRaMbLE is effective in generating complex genomic rearrangements in Sc2.0 yeast strains.