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EasyCloneYALI: CRISPR/Cas9‐Based Synthetic Toolbox for Engineering of the Yeast Yarrowia lipolytica
Author(s) -
Holkenbrink Carina,
Dam Marie I.,
Kildegaard Kanchana R.,
Beder Johannes,
Dahlin Jonathan,
Doménech Belda David,
Borodina Irina
Publication year - 2018
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201700543
Subject(s) - yarrowia , crispr , genome editing , cas9 , biology , genome engineering , computational biology , genome , synthetic biology , metabolic engineering , gene , yeast , transformation (genetics) , genetics , toolbox , computer science , programming language
The 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.