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Genome Engineering of Yarrowia lipolytica with the PiggyBac Transposon System

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Yarrowia lipolytica

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

Abstract

A mutant excision+/integration− piggyBac transposase can be used to seamlessly excise a chromosomally integrated, piggyBac-compatible selection marker cassette from the Yarrowia lipolytica genome. This piggyBac transposase-based genome engineering process allows for both positive selection of targeted homologous recombination events and scarless or footprint-free genome modifications after precise marker recovery. Residual non-native sequences left in the genome after marker excision can be minimized (0–4 nucleotides) or customized (user-defined except for a TTAA tetranucleotide). Both of these options reduce the risk of unintended homologous recombination events in strains with multiple genomic edits. A suite of dual positive/negative selection marker pairs flanked by piggyBac inverted terminal repeats (ITRs) have been constructed and are available for precise genome engineering in Y. lipolytica using this method. This protocol specifically describes the split marker homologous recombination-based disruption of Y. lipolytica ADE2 with a piggyBac ITR-flanked URA3 cassette, followed by piggyBac transposase-mediated excision of the URA3 marker to leave a 50 nucleotide synthetic barcode at the ADE2 locus. The resulting ade2 strain is auxotrophic for adenine, which enables the use of ADE2 as a selectable marker for further strain engineering.

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Acknowledgments

This work was funded through the Office of Naval Research (ONR) under grant N00014-15-1-2785. A portion was also funded by Undergraduate Research Fellowships (URF) awarded to M.V.V. and E.V.W. by the University of Texas at Austin. J.M.W. acknowledges additional support from the National Science Foundation (NSF) Graduate Research Fellowship Program (DGE-1110007).

We would like to thank Kelly Markham for helpful discussions and general Yarrowia lipolytica strain engineering expertise. We would also like to thank Dr. Yuki Naito for updating the CRISPRdirect interface to include sgRNA specificity checks for the Y. lipolytica genome.

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Authors and Affiliations

  1. McKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, TX, USA

    James M. Wagner, Maya V. Venkataraman, Lars H. Lauffer, Joshua M. Wiggers, Eden V. Williams & Hal S. Alper

  2. Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA

    Claire M. Palmer, Xiunan Yi & Hal S. Alper

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  1. James M. Wagner
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  2. Claire M. Palmer
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  3. Maya V. Venkataraman
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  4. Lars H. Lauffer
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  5. Joshua M. Wiggers
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  6. Eden V. Williams
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  7. Xiunan Yi
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  8. Hal S. Alper
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Corresponding author

Correspondence to Hal S. Alper .

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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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Wagner, J.M. et al. (2021). Genome Engineering of Yarrowia lipolytica with the PiggyBac Transposon System. 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_1

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  • DOI: https://doi.org/10.1007/978-1-0716-1414-3_1

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1413-6

  • Online ISBN: 978-1-0716-1414-3

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