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Title: Multiplexed CRISPR-Cas9-Based Genome Editing of Rhodosporidium toruloides

Abstract

Microbial production of biofuels and bioproducts offers a sustainable and economic alternative to petroleum-based fuels and chemicals. The basidiomycete yeast Rhodosporidium toruloides is a promising platform organism for generating bioproducts due to its ability to consume a broad spectrum of carbon sources (including those derived from lignocellulosic biomass) and to naturally accumulate high levels of lipids and carotenoids, two biosynthetic pathways that can be leveraged to produce a wide range of bioproducts. While R. toruloides has great potential, it has a more limited set of tools for genetic engineering relative to more advanced yeast platform organisms such as Yarrowia lipolytica and Saccharomyces cerevisiae. Significant advancements in the past few years have bolstered R. toruloides’ engineering capacity. Here we expand this capacity by demonstrating the first use of CRISPR-Cas9-based gene disruption in R. toruloides. Transforming a Cas9 expression cassette harboring nourseothricin resistance and selecting transformants on this antibiotic resulted in strains of R. toruloides exhibiting successful targeted disruption of the native URA3 gene. While editing efficiencies were initially low (0.002%), optimization of the cassette increased efficiencies 364-fold (to 0.6%). Applying these optimized design conditions enabled disruption of another native gene involved in carotenoid biosynthesis, CAR2, with much greater success; editing efficienciesmore » of CAR2 deletion reached roughly 50%. Finally, we demonstrated efficient multiplexed genome editing by disrupting both CAR2 and URA3 in a single transformation. Altogether, our results provide a framework for applying CRISPR-Cas9 to R. toruloides that will facilitate rapid and high-throughput genome engineering in this industrially relevant organism.« less

Authors:
ORCiD logo [1];  [2];  [3];  [4];  [1];  [5];  [6]
  1. Joint BioEnergy Inst., Emeryville, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States)
  2. Toyota Motor Corp., Aichi (Japan); Energy Biosciences Inst., Berkeley, CA (United States)
  3. Energy Biosciences Inst., Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
  4. Joint BioEnergy Inst., Emeryville, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
  5. Energy Biosciences Inst., Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  6. Carnegie Mellon Univ., Pittsburgh, PA (United States)
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1508520
Report Number(s):
PNNL-SA-142513
Journal ID: ISSN 2379-5042
Grant/Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
mSphere
Additional Journal Information:
Journal Volume: 4; Journal Issue: 2; Journal ID: ISSN 2379-5042
Publisher:
American Society for Microbiology
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; CAR2; CRISPR-Cas9; Rhodosporidium toruloides; URA3; genome engineering; multiplexed; tRNA

Citation Formats

Otoupal, Peter B., Ito, Masakazu, Arkin, Adam P., Magnuson, Jon K., Gladden, John M., Skerker, Jeffrey M., and Mitchell, Aaron P.. Multiplexed CRISPR-Cas9-Based Genome Editing of Rhodosporidium toruloides. United States: N. p., 2019. Web. doi:10.1128/mSphere.00099-19.
Otoupal, Peter B., Ito, Masakazu, Arkin, Adam P., Magnuson, Jon K., Gladden, John M., Skerker, Jeffrey M., & Mitchell, Aaron P.. Multiplexed CRISPR-Cas9-Based Genome Editing of Rhodosporidium toruloides. United States. doi:10.1128/mSphere.00099-19.
Otoupal, Peter B., Ito, Masakazu, Arkin, Adam P., Magnuson, Jon K., Gladden, John M., Skerker, Jeffrey M., and Mitchell, Aaron P.. Wed . "Multiplexed CRISPR-Cas9-Based Genome Editing of Rhodosporidium toruloides". United States. doi:10.1128/mSphere.00099-19.
@article{osti_1508520,
title = {Multiplexed CRISPR-Cas9-Based Genome Editing of Rhodosporidium toruloides},
author = {Otoupal, Peter B. and Ito, Masakazu and Arkin, Adam P. and Magnuson, Jon K. and Gladden, John M. and Skerker, Jeffrey M. and Mitchell, Aaron P.},
abstractNote = {Microbial production of biofuels and bioproducts offers a sustainable and economic alternative to petroleum-based fuels and chemicals. The basidiomycete yeast Rhodosporidium toruloides is a promising platform organism for generating bioproducts due to its ability to consume a broad spectrum of carbon sources (including those derived from lignocellulosic biomass) and to naturally accumulate high levels of lipids and carotenoids, two biosynthetic pathways that can be leveraged to produce a wide range of bioproducts. While R. toruloides has great potential, it has a more limited set of tools for genetic engineering relative to more advanced yeast platform organisms such as Yarrowia lipolytica and Saccharomyces cerevisiae. Significant advancements in the past few years have bolstered R. toruloides’ engineering capacity. Here we expand this capacity by demonstrating the first use of CRISPR-Cas9-based gene disruption in R. toruloides. Transforming a Cas9 expression cassette harboring nourseothricin resistance and selecting transformants on this antibiotic resulted in strains of R. toruloides exhibiting successful targeted disruption of the native URA3 gene. While editing efficiencies were initially low (0.002%), optimization of the cassette increased efficiencies 364-fold (to 0.6%). Applying these optimized design conditions enabled disruption of another native gene involved in carotenoid biosynthesis, CAR2, with much greater success; editing efficiencies of CAR2 deletion reached roughly 50%. Finally, we demonstrated efficient multiplexed genome editing by disrupting both CAR2 and URA3 in a single transformation. Altogether, our results provide a framework for applying CRISPR-Cas9 to R. toruloides that will facilitate rapid and high-throughput genome engineering in this industrially relevant organism.},
doi = {10.1128/mSphere.00099-19},
journal = {mSphere},
issn = {2379-5042},
number = 2,
volume = 4,
place = {United States},
year = {2019},
month = {3}
}

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