Improving CRISPR/Cas9-mediated genome editing efficiency in Yarrowia lipolytica using direct tRNA-sgRNA fusions

• Published in: Metabolic engineering 2020-11, Vol.62 (C), p.106-115
• Main Authors: Abdel-Mawgoud, A.M., Stephanopoulos, G.
• Format: Article
• Language: English
• Subjects: CRISPR-Cas Systems - genetics; CRISPR-Cas9; Gene Editing; Genome editing; Knock out/in; RNA, Guide, CRISPR-Cas Systems - genetics; RNA, Transfer - genetics; Secondary structure; tRNA-sgRNA fusion; Yarrowia - genetics; Yarrowia lipolytica
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2020.07.008

Yarrowia lipolytica is an important oleaginous yeast currently used in the production of specialty chemicals and has a great potential for further applications in lipid biotechnology. Harnessing the full potential of Y. lipolytica is, however, limited by its inherent recalcitrance to genetic manipulation. In contrast to Saccharomyces cerevisiae, Y. lipolytica is poor in homology-mediated DNA repair and thus in homologous recombination, which limits site-specific gene editing in this yeast. Recently developed CRISPR/Cas9-based methods using tRNA-sgRNA fusions succeeded in editing some genomic loci in Y. lipolytica. Nonetheless, the majority of other tested loci either failed editing or editing was achieved but at very low efficiency using these methods. Using tools of secondary RNA structure prediction, we were able to improve the design of the tRNA-sgRNA fusions used for the expression of single guide RNA (sgRNA) in such methods. This resulted in high efficiency CRISPR/cas9 gene editing at chromosomal loci that failed gene editing or were edited at very low efficiencies with previous methods. In addition, we characterized the gene editing performance of our newly designed tRNA-sgRNA fusions for both chromosomal gene integration and deletion. As such, this study presents an efficient CRISPR/Cas9-mediated gene-editing tool for efficient genetic engineering of Yarrowia lipolytica. •Direct tRNA-sgRNA fusions demonstrate better sgRNA function.•Combined perturbations of sgRNA 2ry structure drasrically reduce its activity.•Direct tRNA-sgRNA fusions enable recombinations using donors with 50-bp-long homology arms.•CRISPR/Cas9 gene editing efficiencies close to 100% were achieved in Y. lipolytica.