EasyCloneYALI: CRISPR/Cas9-Based Synthetic Toolbox for Engineering of the Yeast Yarrowia lipolytica.
Carina HolkenbrinkMarie I DamKanchana R KildegaardJohannes BederJonathan DahlinDavid Doménech BeldaIrina BorodinaPublished in: Biotechnology journal (2018)
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.
Keyphrases
- crispr cas
- genome editing
- gene expression
- dna repair
- genome wide
- saccharomyces cerevisiae
- dna methylation
- cell wall
- fatty acid
- dna damage
- copy number
- single molecule
- circulating tumor
- dna damage response
- computed tomography
- oxidative stress
- magnetic resonance
- loop mediated isothermal amplification
- network analysis
- sensitive detection
- bioinformatics analysis