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CRISPR Interference and Activation to Modulate Transcription in Yarrowia lipolytica

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Yarrowia lipolytica

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2307))

Abstract

Recent developments in RNA-guided nuclease technologies have advanced the engineering of a wide range of organisms, including the nonconventional yeast Yarrowia lipolytica. Y. lipolytica has been the focus of a range of synthetic biology and metabolic engineering studies due to its high capacity to synthesize and accumulate intracellular lipids. The CRISPR-Cas9 system from Streptococcus pyogenes has been successfully adapted and used for genome editing in Y. lipolytica. However, as engineered strains are moved closer to industrialization, the need for finer control of transcription is still present. To overcome this challenge, we have developed CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) systems to allow modulating the transcription of endogenous genes. We begin this protocol chapter by describing how to use the CRISPRi system to repress expression of any gene in Y. lipolytica. A second method describes how to use the CRISPRa system to increase expression of native Y. lipolytica genes. Finally, we describe how CRISPRi or CRISPRa vectors can be combined to enable multiplexed activation or repression of more than one gene. The implementation of CRISPRi and CRISPRa systems improves our ability to control gene expression in Y. lipolytica and promises to enable more advanced synthetic biology and metabolic engineering studies in this host.

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Acknowledgments

This work was supported by the National Science Foundation (NSF CBET-1706545) and the University of California, Riverside Chancellor’s Research Fellowship. The protocols included here are based on work that has been previously published [14, 16, 18]. The authors declare no conflict of interest.

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Authors and Affiliations

  1. Chemical and Environmental Engineering, University of California Riverside, Riverside, CA, USA

    Joshua Misa & Cory Schwartz

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  1. Joshua Misa
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  2. Cory Schwartz
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Correspondence to Cory Schwartz .

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Editors and Affiliations

  1. Chemical and Environmental Engineering, University of California Riverside, Riverside, CA, USA

    Ian Wheeldon

  2. Department of Chemical and Biomolecular Engineering, Clemson University, Clemson, SC, USA

    Mark Blenner

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Misa, J., Schwartz, C. (2021). CRISPR Interference and Activation to Modulate Transcription in Yarrowia lipolytica. In: Wheeldon, I., Blenner, M. (eds) Yarrowia lipolytica. Methods in Molecular Biology, vol 2307. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1414-3_6

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  • DOI: https://doi.org/10.1007/978-1-0716-1414-3_6

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1413-6

  • Online ISBN: 978-1-0716-1414-3

  • eBook Packages: Springer Protocols

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