Multiplexed CRISPR-Cas9-Based Genome Editing of Rhodosporidium toruloides

Otoupal, Peter B.; Ito, Masakazu; Arkin, Adam P.; Magnuson, Jon K.; Gladden, John M.; Skerker, Jeffrey M.; Mitchell, Aaron P.

doi:10.1128/mSphere.00099-19

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

Journal Article · 20 March 2019 · mSphere

DOI: https://doi.org/10.1128/mSphere.00099-19 · OSTI ID:1508520

^[1]; Ito, Masakazu ^[2]; Arkin, Adam P. ^[3]; Magnuson, Jon K. ^[4]; Gladden, John M. ^[1]; Skerker, Jeffrey M. ^[5]; Mitchell, Aaron P. ^[6]

Joint BioEnergy Inst., Emeryville, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States)
Toyota Motor Corp., Aichi (Japan); Energy Biosciences Inst., Berkeley, CA (United States)
Energy Biosciences Inst., Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
Joint BioEnergy Inst., Emeryville, CA (United States); Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Energy Biosciences Inst., Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Carnegie Mellon Univ., Pittsburgh, PA (United States)

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.

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Research Organization:: Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)

Sponsoring Organization:: USDOE

Grant/Contract Number:: AC05-76RL01830

OSTI ID:: 1508520

Report Number(s):: PNNL-SA-142513

Journal Information:: mSphere, Vol. 4, Issue 2; ISSN 2379-5042

Publisher:: American Society for MicrobiologyCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
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URA3
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