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

Schultz, J. Carl; Cao, Mingfeng; Zhao, Huimin

doi:10.1002/bit.27001

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:

Schultz, J. Carl ^[1]; Cao, Mingfeng ^[1];

^[1]

Univ. of Illinois at Urbana‐Champaign, Urbana, IL (United States)

Publication Date:: Tue Apr 30 00:00:00 EDT 2019

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:: Journal Article: 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.

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                    Schultz, J. Carl, Cao, Mingfeng, & Zhao, Huimin. Development of a CRISPR/Cas9 system for high efficiency multiplexed gene deletion in Rhodosporidium toruloides.  United States.  https://doi.org/10.1002/bit.27001

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                    Schultz, J. Carl, Cao, Mingfeng, and Zhao, Huimin. 2019.  
        "Development of a CRISPR/Cas9 system for high efficiency multiplexed gene deletion in Rhodosporidium toruloides".  United States.  https://doi.org/10.1002/bit.27001.  https://www.osti.gov/servlets/purl/1542927.

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@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},

  url          = {https://www.osti.gov/biblio/1542927},
  journal      = {Biotechnology and Bioengineering},

  issn         = {0006-3592},

  number       = 8,

  volume       = 116,

  place        = {United States},

  year         = {Tue Apr 30 00:00:00 EDT 2019},

  month        = {Tue Apr 30 00:00:00 EDT 2019}

}

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Works referencing / citing this record:

Dual sgRNA‐directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system
journal, August 2019

Liu, Ke; Sun, Bin; You, Hao
Microbial Biotechnology, Vol. 13, Issue 2
https://doi.org/10.1111/1751-7915.13534

CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing in Yarrowia lipolytica
journal, June 2020

Yang, Zhiliang; Edwards, Harley; Xu, Peng
Metabolic Engineering Communications, Vol. 10
https://doi.org/10.1016/j.mec.2019.e00112

Dual sgRNA‐directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system
journal, August 2019

Liu, Ke; Sun, Bin; You, Hao
Microbial Biotechnology, Vol. 13, Issue 2
https://doi.org/10.1111/1751-7915.13534

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Similar Records

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

Abstract

Citation Formats

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

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

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

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

Cloning and evaluation of different constitutive promoters in the oleaginous yeast Rhodosporidium toruloides : Evaluation of five constitutive promoters in journal, January 2016

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

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

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

Fatty alcohols production by oleaginous yeast journal, August 2015

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

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

Molecular characterization of KU70 and KU80 homologues and exploitation of a KU70-deficient mutant for improving gene deletion frequency in Rhodosporidium toruloides journal, January 2014

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

Dual sgRNA‐directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system journal, August 2019

CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing in Yarrowia lipolytica journal, June 2020

Dual sgRNA‐directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system journal, August 2019

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

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

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

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

Cloning and evaluation of different constitutive promoters in the oleaginous yeast Rhodosporidium toruloides : Evaluation of five constitutive promoters in
journal, January 2016

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

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

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

Fatty alcohols production by oleaginous yeast
journal, August 2015

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

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

Molecular characterization of KU70 and KU80 homologues and exploitation of a KU70-deficient mutant for improving gene deletion frequency in Rhodosporidium toruloides
journal, January 2014

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

Dual sgRNA‐directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system
journal, August 2019

CRISPR-Cas12a/Cpf1-assisted precise, efficient and multiplexed genome-editing in Yarrowia lipolytica
journal, June 2020

Dual sgRNA‐directed gene deletion in basidiomycete Ganoderma lucidum using the CRISPR/Cas9 system
journal, August 2019