Genome Engineering of Yarrowia lipolytica with the PiggyBac Transposon System

James M Wagner; Claire M Palmer; Maya V Venkataraman; Lars H Lauffer; Joshua M Wiggers; Eden V Williams; Xiunan Yi; Hal S Alper

doi:10.1007/978-1-0716-1414-3_1

Genome Engineering of Yarrowia lipolytica with the PiggyBac Transposon System

Methods Mol Biol. 2021:2307:1-24. doi: 10.1007/978-1-0716-1414-3_1.

Authors

James M Wagner¹, Claire M Palmer², Maya V Venkataraman¹, Lars H Lauffer¹, Joshua M Wiggers¹, Eden V Williams¹, Xiunan Yi², Hal S Alper^{3

4}

Affiliations

¹ McKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, TX, USA.
² Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA.
³ McKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, TX, USA. halper@che.utexas.edu.
⁴ Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX, USA. halper@che.utexas.edu.

PMID: 33847979
DOI: 10.1007/978-1-0716-1414-3_1

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.

Keywords: Genome engineering; Homologous recombination; PiggyBac transposon; Transposase; Yarrowia lipolytica.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

DNA Transposable Elements*
Genetic Engineering / methods*
Genetic Vectors
Genome, Fungal
Homologous Recombination
Transposases / metabolism*
Workflow
Yarrowia / genetics*

Substances

DNA Transposable Elements
Transposases