title: Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae.
abstract: CRISPR/Cas9 is a simple and efficient tool for targeted and marker-free genome engineering. Here, we report the development and successful application of a multiplex CRISPR/Cas9 system for genome engineering of up to 5 different genomic loci in one transformation step in baker's yeast Saccharomyces cerevisiae. To assess the specificity of the tool we employed genome re-sequencing to screen for off-target sites in all single knock-out strains targeted by different gRNAs. This extensive analysis identified no more genome variants in CRISPR/Cas9 engineered strains compared to wild-type reference strains. We applied our genome engineering tool for an exploratory analysis of all possible single, double, triple, quadruple and quintuple gene disruption combinations to search for strains with high mevalonate production, a key intermediate for the industrially important isoprenoid biosynthesis pathway. Even though we did not overexpress any genes in the mevalonate pathway, this analysis identified strains with mevalonate titers greater than 41-fold compared to the wild-type strain. Our findings illustrate the applicability of this highly specific and efficient multiplex genome engineering approach to accelerate functional genomics and metabolic engineering efforts.
authors: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark. Electronic address: tajak@biosustain.dtu.dk.
authors: Tadas
authors: T
authors: Jakočiūnas
authors: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark. Electronic address: idab@biosustain.dtu.dk.
authors: Ida
authors: I
authors: Bonde
authors: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark. Electronic address: herrgard@biosustain.dtu.dk.
authors: Markus
authors: M
authors: Herrgård
authors: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark. Electronic address: sjha@biosustain.dtu.dk.
authors: Scott J
authors: SJ
authors: Harrison
authors: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark. Electronic address: metk@biosustain.dtu.dk.
authors: Mette
authors: M
authors: Kristensen
authors: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark. Electronic address: laeb@biosustain.dtu.dk.
authors: Lasse E
authors: LE
authors: Pedersen
authors: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark. Electronic address: mije@biosustain.dtu.dk.
authors: Michael K
authors: MK
authors: Jensen
authors: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, 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. Electronic address: jdkeasling@lbl.gov.
authors: Jay D
authors: JD
authors: Keasling
mesh_terms: D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D005818:Genetic Engineering / Q000379:methods*; D016681:Genome, Fungal; D012441:Saccharomyces cerevisiae / Q000235:genetics*
keywords: CRISPR/Cas9; Mevalonate; Multiplex genome editing; Off-target analysis; Yeast