Yeast Oligo-Mediated Genome Engineering (YOGE)

Abstract

High-frequency oligonucleotide-directed recombination engineering (recombineering) has enabled rapid modification of several prokaryotic genomes to date. Here, we present a method for oligonucleotide-mediated recombineering in the model eukaryote and industrial production host S. cerevisiae, which we call Yeast Oligo-mediated Genome Engineering (YOGE). Through a combination of overexpression and knockouts of relevant genes and optimization of transformation and oligonucleotide designs, we achieve high gene modification frequencies at levels that only require screening of dozens of cells. We demonstrate the robustness of our approach in three divergent yeast strains, including those involved in industrial production of bio-based chemicals. Furthermore, YOGE can be iteratively executed via cycling to generate genomic libraries up to 105 individuals at each round for diversity generation. YOGE cycling alone, or in combination with phenotypic selections or endonuclease-based negative genotypic selections, can be used to easily generate modified alleles in yeast populations with high frequencies.

A highly precise and portable genome engineering method allows comparison of mutational effects across bacterial species

“…In fact, even the human mismatchrepair protein MutL homolog 1 (hMLH1) functionally interacts with the E. coli MMR machinery and was able to induce a dominant mutator state in E. coli (39). Because inactivation of the MMR machinery greatly improves allele replacement efficiency in organisms ranging from yeast (40) to human cell lines (41,42), pORTMAGE could inspire future development of general genome editing tools.…”

Efficient Multiplexed Integration of Synergistic Alleles and Metabolic Pathways in Yeasts via CRISPR-Cas

“…Both of these methods are problematic for essential genes, require at least two rounds of genetic engineering, and are not amenable to multiplexing. Finally, although MAGE (multiplex automated genome engineering) is an excellent option for multiplex introduction of point mutations in E. coli, the yeast equivalent (YOGE) functions at a much lower efficiency (Wang et al, 2009;DiCarlo et al, 2013b). There are several considerations for the introduction of a point mutation using CRISPR-Cas.…”

Perspectives and Prospects for Whole Cell Catalysis

“…Methods have since been developed that can be directed towards the larger goal of genomescale editing. The new methods include so-called ''clustered regularly interspaced short palindromic repeats'' (CRISPR) technology [73], multiplex automated genome engineering [74], and yeast oligo-mediated genome engineering [75]. The prospect of acellular synthesis can be viewed as the other end of the spectrum that will exist.…”