10 years of CRISPR/CAS genomic engineering in Yarrowia lipolytica.

Yarrowia lipolytica is a versatile cell factory widely used in bioprocesses for producing lipids, organic acids, and other high-value compounds. Historically, its genetic engineering was constrained by low homologous recombination (HR) efficiency and the predominance of non-homologous end joining (NHEJ), limiting strain development and metabolic pathway optimization. The advent of CRISPR-Cas technologies has revolutionized genome editing in Y. lipolytica, enabling precise, efficient, and multiplexed modifications. Innovations such as pCAS1yl and pCRISPRyl plasmids, along with genomic Cas9 integration, have enhanced targeted editing efficiency. CRISPR applications now extend to metabolic engineering for lipids, itaconic acid, erythritol, and other compounds. Beyond canonical Cas9, alternative systems, including CRISPRa, Cas12a, base editors, and sgRNA libraries, provide increased flexibility and functional diversity. Central to these approaches is the rational design of guide RNAs (gRNAs), supported by bioinformatics platforms such as CHOPCHOP v3, CRISPOR, CCTop, and Cas-OFFinder, which assist in target site selection, off-target prediction, and editing optimization. This review summarizes the main CRISPR/Cas9 applications in Y. lipolytica, highlighting key engineered strains and emphasizing the critical role of bioinformatics in improving editing strategies. We also propose a pipeline for systematic gRNA design based on published evidence and discuss future perspectives, including the integration of machine learning, artificial intelligence, and emerging CRISPR variants to further advance yeast metabolic engineering.