CRISPR/Cas9 Genome Engineering in Non-Conventional Oleaginous Yeasts: Applications, Challenges, and Prospects.

Given the biotechnological potential of yeast-derived oils for oleochemical production, genes encoding lipid metabolism enzymes are key targets for metabolic engineering. Genetic engineering tools such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, Transcription Activator-Like Effector Nucleases (TALENs), Zinc-Finger Nucleases (ZFNs), RNA interference (RNAi), and integrative plasmids can be used to modulate fatty acid biosynthesis and optimize lipid production. Among them, the CRISPR/Cas9 system, recognized for its simplicity and efficiency, has been deployed as a tool to create oleaginous yeast strains with high lipid productivity and features suitable for application in biorefineries. Species such as Cutaneotrichosporon oleaginosus, Rhodotorula toruloides, Candida spp., and Yarrowia lipolytica have already been engineered using CRISPR/Cas9 to enhance the production of fatty acids and their derivatives. However, designing and constructing an efficient CRISPR/Cas9 platform for oleaginous yeasts faces several hurdles, including low transformation efficiency, difficulties in expressing Cas9 and sgRNAs efficiently and consistently, the lack of well-characterized promoters, limited availability of PAM sequences, and poorly understood DNA repair mechanisms. Here, we address the application of the CRISPR/Cas9 system in oleaginous yeasts, laying out the challenges to developing efficient platforms and highlighting key trends in the field. We compare and discuss alternative CRISPR-Cas9 expression strategies to provide an overview of the current landscape and support the development of new approaches.