Locus specific engineering of tandem DNA repeats in the genome of Saccharomyces cerevisiae using CRISPR/Cas9 and overlapping oligonucleotides

DNA repeatsconstitute a large part of genomes of multicellular eucaryotes. For a longtime considered as junk DNA, their role in genome organizationand tuning of gene expression is being increasingly documented. Synthetic biologyhas so far largely ignored DNA repeatsas regulatory elements to manipulate functions in engineered genomes. The yeast Saccharomyces cerevisiaehas been a workhorse of synthetic biology, owing to its genetic tractability. Here we demonstrate the ability to synthetize, in a simple manner, tandemDNA repeatsof various size by Cas9-assisted oligonucleotide in vivo assembly in this organism. We show that long tandemDNA repeatsof several kilobases can be assembled in one step for different monomer size and G/C content. The combinatorial nature of the approach allows exploring a wide variety of design for building synthetic tandem repeatedDNA directly at a given locus in the Saccharomyces cerevisiaegenome. This approach provides a simple way to incorporate tandemDNA repeatin synthetic genomedesigns to implement regulatory functions.