An Optimized Genotyping Workflow for Identifying Highly SCRaMbLEd Synthetic Yeasts

Synthetic Sc2.0 yeast
strains contain hundreds to thousands
of loxPsym recombination sites that allow restructuring
of
the Saccharomyces cerevisiae genome
by SCRaMbLE. Thus, a highly diverse yeast population can arise from
a single genotype. The selection of genetically diverse candidates
with rearranged synthetic chromosomes for downstream analysis requires
an efficient and straightforward workflow. Here we present loxTags,
a set of qPCR primers for genotyping across loxPsym sites to detect not only deletions but also inversions and translocations
after SCRaMbLE. To cope with the large number of amplicons, we generated
qTagGer, a qPCR genotyping primer prediction tool. Using loxTag-based
genotyping and long-read sequencing, we show that light-inducible
Cre recombinase L-SCRaMbLE can efficiently generate diverse recombination
events when applied to Sc2.0 strains containing a linear or a circular
version of synthetic chromosome III.