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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) (SC-23)
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.
@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}
}

Journal Article:
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This content will become publicly available on April 30, 2020
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