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 efficiencies of CAR2 deletion reached roughly 50%. Finally, we demonstrated efficient multiplexed genome editing by disrupting both CAR2 and URA3 in a single transformation. Together, 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.IMPORTANCE Microbial biofuel and bioproduct platforms provide access to clean and renewable carbon sources that are more sustainable and environmentally friendly than petroleum-based carbon sources. Furthermore, they can serve as useful conduits for the synthesis of advanced molecules that are difficult to produce through strictly chemical means. R. toruloides has emerged as a promising potential host for converting renewable lignocellulosic material into valuable fuels and chemicals. However, engineering efforts to improve the yeast's production capabilities have been impeded by a lack of advanced tools for genome engineering. While this is rapidly changing, one key tool remains unexplored in R. toruloides: CRISPR-Cas9. The results outlined here demonstrate for the first time how effective multiplexed CRISPR-Cas9 gene disruption provides a framework for other researchers to utilize this revolutionary genome-editing tool effectively in R. toruloides.

Keywords: CAR2; CRISPR-Cas9; Rhodosporidium toruloides; URA3; genome engineering; multiplexed; tRNA.

FIG 1

Targeted gene disruption using CRISPR-Cas9. (A) Schematic of original CRISPR-Cas9 design for causing indels in R. toruloides. A PCR fragment containing the coding sequences for expressing sgRNA and Cas9 is transformed into competent cells, which take up the DNA into their nucleus and express the machinery from the PCR fragment. (B) Total CFU of R. toruloides under 5-FOA selection with and without application of this CRISPR-Cas9 editing scheme. (C) Partial sequencing of URA3 of each potentially edited colony near the cut site of Cas9. (D) Revised protocol in which the coding sequence for a selectable marker is included in the PCR fragment and an additional selection step for integration of the fragment into the genome is included. (E) Total CFU of R. toruloides under 5-FOA selection with this revised CRISPR-Cas9 editing scheme. Significance was calculated using a two-tailed type II Student t test. (F) Partial sequencing of URA3 of three edited colonies near the cut site of Cas9. All error bars represent the standard deviation of biological triplicates.

FIG 2

Optimization of sgRNA expression and target sequence. (A) Editing efficiency of various sgRNA target sequences. Bars indicate measured CRISPR-Cas9 gene editing efficiency, as a percentage of the total cells in the transformation mixture exhibiting the expected edited phenotype. Significant differences from the original target sequence (highlighted in blue, P < 0.05) are indicated by asterisks and calculated using a two-tailed type II Student t test. The predicted aptitude for a target sequence to achieve successful DNA editing based on the sgRNA Scorer 2.0 algorithm (41) is depicted in parentheses after each target sequence. (B) Measured editing efficiency of sgRNA with and without an HDV ribozyme cleavage site included between the promoter and the 20-nt target sequence. (C) Measured editing efficiency of various promoters used to drive sgRNA expression. All asterisks indicate statistical difference from the original expression design (highlighted in blue, P < 0.05) calculated using a two-tailed type II Student t test. Error bars represent the standard deviation of biological triplicates.

FIG 3

Multiplexed gene disruption using CRISPR-Cas9 in R. toruloides. (A) Editing efficiency of four sgRNAs targeting CAR2. Error bars represent standard deviations of biological triplicates. (B) The design used to express multiple sgRNAs in a single array. The specific cut sites for URA3 and CAR2 are shown below. Guides “a” and “b” correspond to sgRNAs “4” and “3” for URA3, respectively, while guides “c” and “d” correspond to sgRNAs “1” and “3” for CAR2, respectively, based on the sequences presented in Table S3. (C) 5-FOA plate with colonies demonstrating successful simultaneous disruption of the two genes. To the right is shown the editing efficiency of disrupting each gene individually, as well as tandem gene-disruption editing efficiency. (D and E) Gel image showing PCR amplification of genomic DNA of eight unique colonies near the targeted cut site of URA3 (D) or CAR2 (E). (F) Sequencing results near the target cut site of the eight URA3 PCR products from panel D. Cas9 cut sites are indicated, as well as the DNA size of the excised DNA fragment between each cut site. Mutations are highlighted in blue. (G) Sequencing results near the target cut site of the eight CAR2 PCR products from panel E. Mutations caused by Cas9 targeting are highlighted in blue. All error bars represent standard deviation of biological triplicates.