• Carina Holkenbrink, Marie I Dam, Kanchana R Kildegaard, Johannes Beder, Jonathan Dahlin, David Doménech Belda, and Irina Borodina.
• The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, DK-2800 Kongens Lyngby, Denmark.
• Biotechnol J. 2018 Sep 1; 13 (9): e1700543.

AbstractThe oleaginous yeast Yarrowia lipolytica is an emerging host for production of fatty acid-derived chemicals. To enable rapid iterative metabolic engineering of this yeast, there is a need for well-characterized genetic parts and convenient and reliable methods for their incorporation into yeast. Here, the EasyCloneYALI genetic toolbox, which allows streamlined strain construction with high genome editing efficiencies in Y. lipolytica via the CRISPR/Cas9 technology is presented. The toolbox allows marker-free integration of gene expression vectors into characterized genome sites as well as marker-free deletion of genes with the help of CRISPR/Cas9. Genome editing efficiencies above 80% were achieved with transformation protocols using non-replicating DNA repair fragments (such as DNA oligos). Furthermore, the toolbox includes a set of integrative gene expression vectors with prototrophic markers conferring resistance to hygromycin and nourseothricin.© 2018 The Authors. Biotechnology Journal Published by Wiley-VCH Verlag GmbH & Co. KGaA.

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