Development of a CRISPR/Cas9 System for High-Efficiency Multiplexed Gene Deletion in Rhodosporidium toruloides

Themes: Conversion

Keywords: Genome Engineering, Genomics, Transcriptomics


Schultz, J.C., Cao, M.Zhao H. April 30, 2019. “Development of a CRISPR/Cas9 System for High-Efficiency Multiplexed Gene Deletion in Rhodosporidium toruloides.” Biotechnology and Bioengineering. DOI: 10.1002/bit.27001.


gRNA expression driven by a 5S-tRNA fusion promoter (b) yielded higher knockout rate of the CRTYB gene, as indicated by albiono colonies, than gRNA expression driven by 5S rRNA(a).

The oleaginous yeast Rhodosporidium toruloides is considered a promising candidate for production of chemicals and biofuels thanks to its ability to grow on lignocellulosic biomass, and its high production of lipids and carotenoids. However, efforts to engineer this organism are hindered by a lack of suitable genetic tools. Here we report the development of a CRISPR/Cas9 system for genome editing in R. toruloides based on a fusion 5S rRNA–tRNA promoter for guide RNA (gRNA) expression, capable of greater than 95% gene knockout for various genetic targets. Additionally, multiplexed double‐gene knockout mutants were obtained using this method with an efficiency of 78%. This tool can be used to accelerate future metabolic engineering work in this yeast.


Download (6.5 KB) includes:

  • Knockout efficiencies
  • NLS sequences
  • Primers, plasmids, and gRNAs used

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