Assembly and Multiplex Genome Integration of Metabolic Pathways in Yeast Using CasEMBLR

  • Tadas Jakočiūnas
  • Emil D. Jensen
  • Michael K. JensenEmail author
  • Jay D. Keasling
Part of the Methods in Molecular Biology book series (MIMB, volume 1671)


Genome integration is a vital step for implementing large biochemical pathways to build a stable microbial cell factory. Although traditional strain construction strategies are well established for the model organism Saccharomyces cerevisiae, recent advances in CRISPR/Cas9-mediated genome engineering allow much higher throughput and robustness in terms of strain construction. In this chapter, we describe CasEMBLR, a highly efficient and marker-free genome engineering method for one-step integration of in vivo assembled expression cassettes in multiple genomic sites simultaneously. CasEMBLR capitalizes on the CRISPR/Cas9 technology to generate double-strand breaks in genomic loci, thus prompting native homologous recombination (HR) machinery to integrate exogenously derived homology templates. As proof-of-principle for microbial cell factory development, CasEMBLR was used for one-step assembly and marker-free integration of the carotenoid pathway from 15 exogenously supplied DNA parts into three targeted genomic loci. As a second proof-of-principle, a total of ten DNA parts were assembled and integrated in two genomic loci to construct a tyrosine production strain, and at the same time knocking out two genes. This new method complements and improves the field of genome engineering in S. cerevisiae by providing a more flexible platform for rapid and precise strain building.

Key words

Genome engineering CRISPR/Cas9 Metabolic engineering In vivo assembly DNA assembly CasEMBLR Homologous recombination 


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Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Tadas Jakočiūnas
    • 1
  • Emil D. Jensen
    • 1
  • Michael K. Jensen
    • 1
    Email author
  • Jay D. Keasling
    • 1
    • 2
    • 3
    • 4
    • 5
  1. 1.The Novo Nordisk Foundation Center for BiosustainabilityTechnical University of DenmarkKgs. LyngbyDenmark
  2. 2.Joint BioEnergy InstituteEmeryvilleUSA
  3. 3.Physical Biosciences DivisionLawrence Berkeley National LaboratoryBerkeleyUSA
  4. 4.Department of Chemical and Biomolecular EngineeringUniversity of California, BerkeleyBerkeleyUSA
  5. 5.Department of BioengineeringUniversity of California, BerkeleyBerkeleyUSA

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