Development of a CRISPR/Cas9 system for high efficiency multiplexed gene deletion in Rhodosporidium toruloides

J. Carl Schultz, Mingfeng Cao, Huimin Zhao

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)2103-2109
Number of pages7
JournalBiotechnology and bioengineering
Volume116
Issue number8
DOIs
StatePublished - Aug 2019

Keywords

  • CRISPR/Cas9
  • Rhodosporidium toruloides
  • genome editing
  • metabolic engineering
  • multiplex

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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