title: CrEdit: CRISPR mediated multi-loci gene integration in Saccharomyces cerevisiae.
abstract: BACKGROUND
  One of the bottlenecks in production of biochemicals and pharmaceuticals in Saccharomyces cerevisiae is stable and homogeneous expression of pathway genes. Integration of genes into the genome of the production organism is often a preferred option when compared to expression from episomal vectors. Existing approaches for achieving stable simultaneous genome integrations of multiple DNA fragments often result in relatively low integration efficiencies and furthermore rely on the use of selection markers.
  
  
  RESULTS
  Here, we have developed a novel method, CrEdit (CRISPR/Cas9 mediated genome Editing), which utilizes targeted double strand breaks caused by CRISPR/Cas9 to significantly increase the efficiency of homologous integration in order to edit and manipulate genomic DNA. Using CrEdit, the efficiency and locus specificity of targeted genome integrations reach close to 100% for single gene integration using short homology arms down to 60 base pairs both with and without selection. This enables direct and cost efficient inclusion of homology arms in PCR primers. As a proof of concept, a non-native β-carotene pathway was reconstructed in S. cerevisiae by simultaneous integration of three pathway genes into individual intergenic genomic sites. Using longer homology arms, we demonstrate highly efficient and locus-specific genome integration even without selection with up to 84% correct clones for simultaneous integration of three gene expression cassettes.
  
  
  CONCLUSIONS
  The CrEdit approach enables fast and cost effective genome integration for engineering of S. cerevisiae. Since the choice of the targeting sites is flexible, CrEdit is a powerful tool for diverse genome engineering applications.
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark. carro@biosustain.dtu.dk.
authors.forename: Carlotta
authors.initials: C
authors.lastname: Ronda
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark. jmau@biosustain.dtu.dk.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Maury
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark. tajak@biosustain.dtu.dk.
authors.forename: Tadas
authors.initials: T
authors.lastname: Jakočiunas
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark. simo@biosustain.dtu.dk.
authors.forename: Simo Abdessamad Baallal
authors.initials: SA
authors.lastname: Jacobsen
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark. sumge@biosustain.dtu.dk.
authors.forename: Susanne Manuela
authors.initials: SM
authors.lastname: Germann
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark. S.Harrison@BHAM.AC.UK.
authors.forename: Scott James
authors.initials: SJ
authors.lastname: Harrison
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark. irbo@biosustain.dtu.dk.
authors.forename: Irina
authors.initials: I
authors.lastname: Borodina
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark. JDKeasling@lbl.gov.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark. mije@biosustain.dtu.dk.
authors.forename: Michael Krogh
authors.initials: MK
authors.lastname: Jensen
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Allé 6, 2970, Hørsholm, Denmark. atn@biosustain.dtu.dk.
authors.forename: Alex Toftgaard
authors.initials: AT
authors.lastname: Nielsen
mesh_terms: D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D060847:Metabolic Engineering / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism