Microbial production of biofuels and bioproducts offers a sustainable and economic alternative to petroleum-based fuels and chemicals. The basidiomycete yeastis 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. Whilehas great potential, it has a more limited set of tools for genetic engineering relative to more advanced yeast platform organisms such asandSignificant advancements in the past few years have bolstered' engineering capacity. Here we expand this capacity by demonstrating the first use of CRISPR-Cas9-based gene disruption inTransforming a Cas9 expression cassette harboring nourseothricin resistance and selecting transformants on this antibiotic resulted in strains ofexhibiting successful targeted disruption of the nativegene. 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,, with much greater success; editing efficiencies ofdeletion reached roughly 50%. Finally, we demonstrated efficient multiplexed genome editing by disrupting bothandin a single transformation. Together, our results provide a framework for applying CRISPR-Cas9 tothat will facilitate rapid and high-throughput genome engineering in this industrially relevant organism.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.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: 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. Rhodosporidium toruloides R. toruloides Yarrowia lipolytica Saccharomyces cerevisiae R. toruloides R. toruloides R. toruloides URA3CAR2CAR2CAR2URA3R. toruloides R. toruloides R. toruloides R. toruloides IMPORTANCE
