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

J Carl Schultz; Mingfeng Cao; Huimin Zhao

doi:10.1002/bit.27001

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

Biotechnol Bioeng. 2019 Aug;116(8):2103-2109. doi: 10.1002/bit.27001. Epub 2019 May 21.

Authors

J Carl Schultz¹, Mingfeng Cao¹, Huimin Zhao^{1

2}

Affiliations

¹ Department of Chemical and Biomolecular Engineering, Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois.
² Departments of Chemistry, Biochemistry, and Bioengineering, University of Illinois at Urbana-Champaign, Urbana, Illinois.

PMID: 31038202
DOI: 10.1002/bit.27001

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.

Keywords: CRISPR/Cas9; Rhodosporidium toruloides; genome editing; metabolic engineering; multiplex.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

Basidiomycota / genetics*
CRISPR-Cas Systems*
Clustered Regularly Interspaced Short Palindromic Repeats
Gene Deletion*
Gene Editing / methods
Genes, Fungal
Metabolic Engineering / methods
RNA, Guide, CRISPR-Cas Systems / genetics

Substances

RNA, Guide, CRISPR-Cas Systems