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

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.

[1]  Xuya Yu,et al.  Dual sgRNA‐directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system , 2020, Microbial biotechnology.

[2]  Harley Edwards,et al.  CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing in Yarrowia lipolytica , 2019, bioRxiv.

[3]  Jibin Sun,et al.  5S rRNA Promoter for Guide RNA Expression Enabled Highly Efficient CRISPR/Cas9 Genome Editing in Aspergillus niger. , 2019, ACS synthetic biology.

[4]  Huimin Zhao,et al.  Development of a CRISPR/Cas9-Based Tool for Gene Deletion in Issatchenkia orientalis , 2019, mSphere.

[5]  Jeffrey M. Skerker,et al.  Multiplexed CRISPR-Cas9-Based Genome Editing of Rhodosporidium toruloides , 2019, mSphere.

[6]  H. Alper,et al.  Modular Ligation Extension of Guide RNA Operons (LEGO) for Multiplexed dCas9 Regulation of Metabolic Pathways in Saccharomyces cerevisiae. , 2018, Biotechnology journal.

[7]  Wen-Hsiung Li,et al.  Engineering the oleaginous red yeast Rhodotorula glutinis for simultaneous β-carotene and cellulase production , 2018, Scientific Reports.

[8]  Sufang Zhang,et al.  Developing a flippase-mediated maker recycling protocol for the oleaginous yeast Rhodosporidium toruloides , 2018, Biotechnology Letters.

[9]  Huimin Zhao,et al.  Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system , 2017, Nature Communications.

[10]  Jeffrey M. Skerker,et al.  Rhodosporidium toruloides: a new platform organism for conversion of lignocellulose into terpene biofuels and bioproducts , 2017, bioRxiv.

[11]  I. Wheeldon,et al.  Standardized Markerless Gene Integration for Pathway Engineering in Yarrowia lipolytica. , 2017, ACS synthetic biology.

[12]  S. Aves,et al.  Four Inducible Promoters for Controlled Gene Expression in the Oleaginous Yeast Rhodotorula toruloides , 2016, Front. Microbiol..

[13]  E. Hopmans,et al.  Abundance and Diversity of Denitrifying and Anammox Bacteria in Seasonally Hypoxic and Sulfidic Sediments of the Saline Lake Grevelingen , 2016, Front. Microbiol..

[14]  T. Vogl,et al.  Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris. , 2016, Journal of biotechnology.

[15]  C. V. Rao,et al.  Metabolic engineering of the oleaginous yeast Rhodosporidium toruloides IFO0880 for lipid overproduction during high-density fermentation , 2016, Applied Microbiology and Biotechnology.

[16]  C. V. Rao,et al.  Engineering Rhodosporidium toruloides for increased lipid production , 2016, Biotechnology and bioengineering.

[17]  Sufang Zhang,et al.  Cloning and evaluation of different constitutive promoters in the oleaginous yeast Rhodosporidium toruloides , 2016, Yeast.

[18]  Ian Wheeldon,et al.  Synthetic RNA Polymerase III Promoters Facilitate High-Efficiency CRISPR-Cas9-Mediated Genome Editing in Yarrowia lipolytica. , 2016, ACS synthetic biology.

[19]  J. Adrio,et al.  Fatty alcohols production by oleaginous yeast , 2015, Journal of Industrial Microbiology & Biotechnology.

[20]  E. Lander,et al.  Development and Applications of CRISPR-Cas9 for Genome Engineering , 2014, Cell.

[21]  Z. Zhao,et al.  Functional integration of multiple genes into the genome of the oleaginous yeast Rhodosporidium toruloides. , 2014, FEMS yeast research.

[22]  Yunde Zhao,et al.  Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. , 2014, Journal of integrative plant biology.

[23]  Yanbin Liu,et al.  Molecular characterization of KU70 and KU80 homologues and exploitation of a KU70-deficient mutant for improving gene deletion frequency in Rhodosporidium toruloides , 2014, BMC Microbiology.

[24]  J. Pronk.  Auxotrophic Yeast Strains in Fundamental and Applied Research , 2002, Applied and Environmental Microbiology.

[25]  P. Hooykaas,et al.  Trans‐kingdom T‐DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. , 1995, The EMBO journal.

[26]  Zengyi Shao,et al.  Iowa State University From the SelectedWorks of Zengyi Shao 2009 DNA assembler , an in vivo genetic method for rapid construction of biochemical pathways , 2016 .