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

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. In this paper, 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%. Finally, this tool can be used to accelerate future metabolic engineering work in this yeast.

Authors:
 [1];  [1]; ORCiD logo [1]
  1. Univ. of Illinois at Urbana‐Champaign, Urbana, IL (United States)
Publication Date:
Research Org.:
Center for Advanced Bioenergy and Bioproducts Innovation (CABBI), Urbana, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
1542927
Alternate Identifier(s):
OSTI ID: 1515089
Grant/Contract Number:  
SC0018420; SC0018260
Resource Type:
Accepted Manuscript
Journal Name:
Biotechnology and Bioengineering
Additional Journal Information:
Journal Volume: 116; Journal Issue: 8; Journal ID: ISSN 0006-3592
Publisher:
Wiley
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 09 BIOMASS FUELS; CRISPR/Cas9; genome editing; metabolic engineering; multiplex; Rhodosporidium toruloides

Citation Formats

Schultz, J. Carl, Cao, Mingfeng, and Zhao, Huimin. Development of a CRISPR/Cas9 system for high efficiency multiplexed gene deletion in Rhodosporidium toruloides. United States: N. p., 2019. Web. doi:10.1002/bit.27001.
Schultz, J. Carl, Cao, Mingfeng, & Zhao, Huimin. Development of a CRISPR/Cas9 system for high efficiency multiplexed gene deletion in Rhodosporidium toruloides. United States. doi:10.1002/bit.27001.
Schultz, J. Carl, Cao, Mingfeng, and Zhao, Huimin. Tue . "Development of a CRISPR/Cas9 system for high efficiency multiplexed gene deletion in Rhodosporidium toruloides". United States. doi:10.1002/bit.27001. https://www.osti.gov/servlets/purl/1542927.
@article{osti_1542927,
title = {Development of a CRISPR/Cas9 system for high efficiency multiplexed gene deletion in Rhodosporidium toruloides},
author = {Schultz, J. Carl and Cao, Mingfeng and Zhao, Huimin},
abstractNote = {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. In this paper, 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%. Finally, this tool can be used to accelerate future metabolic engineering work in this yeast.},
doi = {10.1002/bit.27001},
journal = {Biotechnology and Bioengineering},
number = 8,
volume = 116,
place = {United States},
year = {2019},
month = {4}
}

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Works referenced in this record:

Self‐processing of ribozyme‐flanked RNAs into guide RNAs in vitro and in vivo for CRISPR‐mediated genome editing
journal, February 2014

  • Gao, Yangbin; Zhao, Yunde
  • Journal of Integrative Plant Biology, Vol. 56, Issue 4
  • DOI: 10.1111/jipb.12152

Auxotrophic Yeast Strains in Fundamental and Applied Research
journal, May 2002


Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system
journal, November 2017


Rhodosporidium toruloides: a new platform organism for conversion of lignocellulose into terpene biofuels and bioproducts
journal, October 2017


DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways
journal, December 2008

  • Shao, Zengyi; Zhao, Hua; Zhao, Huimin
  • Nucleic Acids Research, Vol. 37, Issue 2
  • DOI: 10.1093/nar/gkn991

Modular Ligation Extension of Guide RNA Operons (LEGO) for Multiplexed dCas9 Regulation of Metabolic Pathways in Saccharomyces cerevisiae
journal, April 2018

  • Deaner, Matthew; Holzman, Allison; Alper, Hal S.
  • Biotechnology Journal, Vol. 13, Issue 9
  • DOI: 10.1002/biot.201700582

Development and Applications of CRISPR-Cas9 for Genome Engineering
journal, June 2014


Synthetic RNA Polymerase III Promoters Facilitate High-Efficiency CRISPR–Cas9-Mediated Genome Editing in Yarrowia lipolytica
journal, January 2016

  • Schwartz, Cory M.; Hussain, Murtaza Shabbir; Blenner, Mark
  • ACS Synthetic Biology, Vol. 5, Issue 4
  • DOI: 10.1021/acssynbio.5b00162

Metabolic engineering of the oleaginous yeast Rhodosporidium toruloides IFO0880 for lipid overproduction during high-density fermentation
journal, September 2016

  • Zhang, Shuyan; Ito, Masakazu; Skerker, Jeffrey M.
  • Applied Microbiology and Biotechnology, Vol. 100, Issue 21
  • DOI: 10.1007/s00253-016-7815-y

Functional integration of multiple genes into the genome of the oleaginous yeast Rhodosporidium toruloides
journal, February 2014


Multiplexed CRISPR-Cas9-Based Genome Editing of Rhodosporidium toruloides
journal, March 2019


Combinatorial optimization of CRISPR/Cas9 expression enables precision genome engineering in the methylotrophic yeast Pichia pastoris
journal, October 2016


Developing a flippase-mediated maker recycling protocol for the oleaginous yeast Rhodosporidium toruloides
journal, March 2018


Engineering Rhodosporidium toruloides for increased lipid production : Engineering
journal, November 2015

  • Zhang, Shuyan; Skerker, Jeffrey M.; Rutter, Charles D.
  • Biotechnology and Bioengineering, Vol. 113, Issue 5
  • DOI: 10.1002/bit.25864

Fatty alcohols production by oleaginous yeast
journal, August 2015

  • Fillet, Sandy; Gibert, Jordi; Suárez, Beatriz
  • Journal of Industrial Microbiology & Biotechnology, Vol. 42, Issue 11
  • DOI: 10.1007/s10295-015-1674-x

Standardized Markerless Gene Integration for Pathway Engineering in Yarrowia lipolytica
journal, November 2016

  • Schwartz, Cory; Shabbir-Hussain, Murtaza; Frogue, Keith
  • ACS Synthetic Biology, Vol. 6, Issue 3
  • DOI: 10.1021/acssynbio.6b00285

Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae.
journal, July 1995


Engineering the oleaginous red yeast Rhodotorula glutinis for simultaneous β-carotene and cellulase production
journal, July 2018


5S rRNA Promoter for Guide RNA Expression Enabled Highly Efficient CRISPR/Cas9 Genome Editing in Aspergillus niger
journal, April 2018