title: CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae.
abstract: Homologous recombination (HR) in Saccharomyces cerevisiae has been harnessed for both plasmid construction and chromosomal integration of foreign DNA. Still, native HR machinery is not efficient enough for complex and marker-free genome engineering required for modern metabolic engineering. Here, we present a method for marker-free multiloci integration of in vivo assembled DNA parts. By the use of CRISPR/Cas9-mediated one-step double-strand breaks at single, double and triple integration sites we report the successful in vivo assembly and chromosomal integration of DNA parts. We call our method CasEMBLR and validate its applicability for genome engineering and cell factory development in two ways: (i) introduction of the carotenoid pathway from 15 DNA parts into three targeted loci, and (ii) creation of a tyrosine production strain using ten parts into two loci, simultaneously knocking out two genes. This method complements and improves the current set of tools available for genome engineering in S. cerevisiae.
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , 2800 Kongens Lyngby, Denmark.
authors.forename: Tadas
authors.initials: T
authors.lastname: Jakočiūnas
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , 2800 Kongens Lyngby, Denmark.
authors.forename: Arun S
authors.initials: AS
authors.lastname: Rajkumar
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , 2800 Kongens Lyngby, Denmark.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , 2800 Kongens Lyngby, Denmark.
authors.forename: Dushica
authors.initials: D
authors.lastname: Arsovska
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , 2800 Kongens Lyngby, Denmark.
authors.forename: Angelica
authors.initials: A
authors.lastname: Rodriguez
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , 2800 Kongens Lyngby, Denmark.
authors.forename: Christian Bille
authors.initials: CB
authors.lastname: Jendresen
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , 2800 Kongens Lyngby, Denmark.
authors.forename: Mette L
authors.initials: ML
authors.lastname: Skjødt
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , 2800 Kongens Lyngby, Denmark.
authors.forename: Alex T
authors.initials: AT
authors.lastname: Nielsen
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , 2800 Kongens Lyngby, Denmark.
authors.forename: Irina
authors.initials: I
authors.lastname: Borodina
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , 2800 Kongens Lyngby, Denmark.
authors.forename: Michael K
authors.initials: MK
authors.lastname: Jensen
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , 2800 Kongens Lyngby, Denmark.|Joint BioEnergy Institute , Emeryville, California 94608, United States.|Physical Biosciences Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.|Department of Chemical and Biomolecular Engineering & Department of Bioengineering, University of California , Berkeley, California 94720, United States.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
mesh_terms: D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004271:DNA, Fungal / Q000235:genetics*; D016681:Genome, Fungal; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D058615:Synthetic Biology
chemical_list: D004271:DNA, Fungal
keywords: CRISPR/Cas9; DNA assembly; double-strand break; metabolic engineering