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

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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.
Original languageEnglish
Title of host publicationSynthetic Metabolic Pathways
Publication date2018
ISBN (Print)978-1-4939-7294-4
ISBN (Electronic)978-1-4939-7295-1
Publication statusPublished - 2018
SeriesMethods in Molecular Biology


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

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