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Method for Multiplexed Integration of Synergistic Alleles and Metabolic Pathways in Yeasts via CRISPR-Cas9

  • Jessica M. WalterEmail author
  • Max G. Schubert
  • Stephanie H. Kung
  • Kristy Hawkins
  • Darren M. Platt
  • Aaron D. Hernday
  • Tina Mahatdejkul-Meadows
  • Wayne Szeto
  • Sunil S. Chandran
  • Jack D. Newman
  • Andrew A. Horwitz
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2049)

Abstract

CRISPR-Cas has proven to be a powerful tool for precision genetic engineering in a variety of difficult genetic systems. In the highly tractable yeast S. cerevisiae, CRISPR-Cas can be used to conduct multiple engineering steps in parallel, allowing for engineering of complex metabolic pathways at multiple genomic loci in as little as 1 week. In addition, CRISPR-Cas can be used to consolidate multiple causal alleles into a single strain, bypassing the laborious traditional methods using marked constructs, or mating. These tools compress the engineering timeline sixfold or more, greatly increasing the productivity of the strain engineer.

Key words

CRISPR Cas9 gRNA Yeast Multiplex Allele Metabolic engineering Synthetic biology 

Notes

Acknowledgments

This method, which is patented under U.S. Patent No. 9,476,065 and other patents pending, was developed with Government support under Agreement HR0011-12-3-0006, awarded by DARPA. We thank the many members of the Amyris community who supported this effort, including Joel Cherry, the Amyris Automated Strain Engineering group for assembly of DNA constructs, and Amyris Lab Services group for reagents, media, and strain banking. At the time this method was developed, the authors were employees and shareholders of Amyris, Inc.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Jessica M. Walter
    • 1
    Email author
  • Max G. Schubert
    • 1
  • Stephanie H. Kung
    • 1
  • Kristy Hawkins
    • 1
  • Darren M. Platt
    • 1
  • Aaron D. Hernday
    • 1
  • Tina Mahatdejkul-Meadows
    • 1
  • Wayne Szeto
    • 1
  • Sunil S. Chandran
    • 1
  • Jack D. Newman
    • 1
  • Andrew A. Horwitz
    • 1
  1. 1.Amyris, Inc.EmeryvilleUSA

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