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

Abstract

DNA repeats constitute a large part of genomes of multicellular eucaryotes. For a longtime considered as junk DNA, their role in genome organization and tuning of gene expression is being increasingly documented. Synthetic biology has so far largely ignored DNA repeats as regulatory elements to manipulate functions in engineered genomes. The yeast Saccharomyces cerevisiae has been a workhorse of synthetic biology, owing to its genetic tractability. Here we demonstrate the ability to synthetize, in a simple manner, tandem DNA repeats of various size by Cas9-assisted oligonucleotide in vivo assembly in this organism. We show that long tandem DNA repeats of 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 repeated DNA directly at a given locus in the Saccharomyces cerevisiae genome. This approach provides a simple way to incorporate tandem DNA repeat in synthetic genome designs to implement regulatory functions.

Glucocorticoid exposure during hippocampal neurogenesis primes future stress response by inducing changes in DNA methylation

“…Previously identified genetic variants associated with differential susceptibility and resilience to environmental adversities in human cohorts will be used to generate cell lines with a combination of risk versus protective genotypes. CRISPR-Cas9 tools using homology-directed repair (HDR) for polymorphic repeats 2,3 and single nucleotide polymorphism (SNP) 4 will be used. We will combine risk and protective alleles to create cell lines with an index of risk (from low to high) according to haplotypes observed in the human population.…”

Nucleosome Positioning on Large Tandem DNA Repeats of the ‘601’ Sequence Engineered in Saccharomyces cerevisiae

“…Specifically, we engineered three designs of overlapping oligonucleotides to partially replace the non-essential YMR262 gene and its promoter, located in chromosome XIII, to give rise to arrays of tandem repeats of 167, 197 or 237 bp long monomers (Figure 1 and supplementary Figure S1, S2). Integration at the YMR262 locus resulted in the removal of 129 base pairs upstream the start codon and 231 bp downstream the start codon [21]. Sizes of arrays obtained by this approach ranged from one repeat to arrays longer than 15 kb, as estimated by gel electrophoresis, corresponding to more than 50 repeats (Figure 2).…”

“…We used a strategy that we developed recently [21] to assemble tandem DNA repeats containing the 601 sequence in the genome of Saccharomyces cerevisiae. Specifically, we engineered three designs of overlapping oligonucleotides to partially replace the non-essential YMR262 gene and its promoter, located in chromosome XIII, to give rise to arrays of tandem repeats of 167, 197 or 237 bp long monomers (Figure 1 and supplementary Figure S1, S2).…”

“…Our goal here was to assess the ability of 601 sequence to position nucleosomes in vivo on long tandem arrays using three different linker lengths of 20, 50 and 90 bp, that would respectively constrain the NRL respectively to 167, 197 or 237 bp. Using a genome editing approach that we recently developed to engineer long DNA tandem repeats in yeast chromosomal DNA [21], we assembled tens of 601 repeats and analyzed the statistical positioning of nucleosomes on these arrays. Our results show that, in vivo, the 601 sequence is not able to position the nucleosomes on the 601 core, yielding NRL which are different from the ones we designed, except for the control 167 bp tandem array which correspond to the naturally observed yeast NRL.…”

Nucleosome positioning on large tandem DNA repeats of the ‘601’ sequence engineered in Saccharomyces cerevisiae

“…The full method can be found in the Supplementary Material and Methods. Direct in vivo assembly using CRISPR/Cas9 and overlapping oligonucleotides in the Chromosome XIII of S. cerevisiae strain YPH499 was performed as previously described [21]. Briefly, donor S3.…”