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CasPER: A CRISPR/Cas9-Based Method for Directed Evolution in Genomic Loci in Saccharomyces cerevisiae

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Yeast Metabolic Engineering

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2513))

Abstract

Here, in this chapter, we describe a detailed protocol for the method named Cas9-mediated protein evolution reaction or short CasPER. CasPER is based on the generation of large 300–600-bp mutagenized linear DNA fragments by error-prone PCR which are used as a donor for repair of double-strand break mediated by Cas9 and subsequently integrated to the genome. This method can be efficiently used for directed evolution of desired essential or nonessential genes in the genome and most importantly can be multiplexed. Altogether, the described method allows for heterogeneous DNA integration with successful transformation efficiencies of 98–100% for both single and multiplex targeting.

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Author information

Authors and Affiliations

  1. The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Lyngby, Denmark

    Tadas Jakočiūnas, Michael K. Jensen & Jay D. Keasling

  2. Joint BioEnergy Institute, Emeryville, CA, USA

    Jay D. Keasling

  3. Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

    Jay D. Keasling

  4. Department of Chemical and Biomolecular Engineering and Department of Bioengineering, University of California, Berkeley, CA, USA

    Jay D. Keasling

  5. Center for Synthetic Biochemistry, Institute for Synthetic Biology, Shenzhen Institutes for Advanced Technologies, Shenzhen, China

    Jay D. Keasling

Authors
  1. Tadas Jakočiūnas
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  2. Michael K. Jensen
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  3. Jay D. Keasling
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Corresponding author

Correspondence to Michael K. Jensen .

Editor information

Editors and Affiliations

  1. Division of Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden

    Valeria Mapelli

  2. Division of Industrial Biotechnology, Department of Biology and Biological Engineering, Chalmers University of Technology, Gothenburg, Sweden

    Maurizio Bettiga

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Jakočiūnas, T., Jensen, M.K., Keasling, J.D. (2022). CasPER: A CRISPR/Cas9-Based Method for Directed Evolution in Genomic Loci in Saccharomyces cerevisiae. In: Mapelli, V., Bettiga, M. (eds) Yeast Metabolic Engineering. Methods in Molecular Biology, vol 2513. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2399-2_3

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  • DOI: https://doi.org/10.1007/978-1-0716-2399-2_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2398-5

  • Online ISBN: 978-1-0716-2399-2

  • eBook Packages: Springer Protocols

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