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ACS Synth Biol. 2013 Dec 20;2(12):741-9. doi: 10.1021/sb400117c. Epub 2013 Nov 18.

Yeast oligo-mediated genome engineering (YOGE).

Author information

1
Department of Genetics, Harvard Medical School , Boston, Massachusetts 02115, United States.

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 Saccharomyces 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 biobased chemicals. Furthermore, YOGE can be iteratively executed via cycling to generate genomic libraries up to 10 (5) 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 generate modified alleles easily in yeast populations with high frequencies.

PMID:
24160921
PMCID:
PMC4048964
DOI:
10.1021/sb400117c
[Indexed for MEDLINE]
Free PMC Article

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