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


CABBI Theme: 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.



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Table S1: Sequences of codon-optimized SpCas9-NLS3, 5S-tRNA promoter

Table S2: NLS sequences used in this study

Table S3: CRTYB knockout efficiency for N-terminal NLS-Cas9-NLS3

Table S4: CRTYB knockout efficiency for pGPD1-, pFBA1-, pPGI1-, pPGK1-, and pTEF1-driven Cas9 expression

Table S5: gRNAs used in this study

Table S6:  Comparison of cell death and editing efficiency effected by different Cas9 promoters in combination with 5S-tRNA-CRTYB Guide 1 gRNA

Table S7: Primers used in this study

Table S8: Plasmids used in this study