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

Abdel-Mawgoud, A. M.; Stephanopoulos, G.

doi:10.1016/j.ymben.2020.07.008

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

Journal Article · Mon Aug 03 00:00:00 EDT 2020 · Metabolic Engineering

DOI:https://doi.org/10.1016/j.ymben.2020.07.008· OSTI ID:1851146

Abdel-Mawgoud, A. M. ^[1]; Stephanopoulos, G. ^[2]

Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States); Laval University, Quebec (Canada); Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States)
Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States)

We report 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.

View Accepted Manuscript (DOE)

Research Organization:: Massachusetts Inst. of Technology (MIT), Cambridge, MA (United States)

Sponsoring Organization:: Natural Sciences and Engineering Research Council of Canada (NSERC); USDOE; USDOE Office of Science (SC)

Grant/Contract Number:: SC0008744

OSTI ID:: 1851146

Alternate ID(s):: OSTI ID: 1811082

Journal Information:: Metabolic Engineering, Journal Name: Metabolic Engineering Journal Issue: C Vol. 62; ISSN 1096-7176

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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

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References (34)

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