title: EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9.
abstract: Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker-free overexpression cassettes in the genome of S. cerevisiae, we have developed a vector toolkit EasyClone-MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained with 90-100% and 60-70% targeting efficiency, respectively. We demonstrate application of the vector toolkit by constructing a haploid laboratory strain (CEN.PK113-7D) and a diploid industrial strain (Ethanol Red) for production of 3-hydroxypropionic acid, where we tested three different acetyl-CoA supply strategies, requiring overexpression of three to six genes each. Among the tested strategies was a bacterial cytosolic pyruvate dehydrogenase complex, which was integrated into the genome in a single transformation. The publicly available EasyClone-MarkerFree vector suite allows for facile and highly standardized genome engineering, and should be of particular interest to researchers working on yeast chassis with limited markers available.
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
authors.forename: Mathew M
authors.initials: MM
authors.lastname: Jessop-Fabre
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
authors.forename: Tadas
authors.initials: T
authors.lastname: Jakočiūnas
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
authors.forename: Vratislav
authors.initials: V
authors.lastname: Stovicek
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, Gothenburg, Sweden.|Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden.
authors.forename: Zongjie
authors.initials: Z
authors.lastname: Dai
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.
authors.forename: Michael K
authors.initials: MK
authors.lastname: Jensen
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark.|Joint BioEnergy Institute, Emeryville, CA, USA.|Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.|Department of Chemical and Biomolecular Engineering & Department of Bioengineering University of California, Berkeley, CA, USA.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark. irbo@biosustain.dtu.dk.
authors.forename: Irina
authors.initials: I
authors.lastname: Borodina
mesh_terms: D064113:CRISPR-Cas Systems; D005818:Genetic Engineering / Q000379:methods*; D056426:Genetic Loci; D005822:Genetic Vectors / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: 3-hydroxypropionic acid; CRISPR-Cas9; Metabolic engineering; Saccharomyces cerevisiae