Rapid genome editing mediated by the NH-CR system in Yarrowia lipolytica and its applications

Yarrowia lipolytica is an important non-conventional yeast chassis for synthetic biology and metabolic engineering because of its robust lipid metabolism, protein secretion capacity, and industrial application potential. However, genome engineering in this species remains constrained by its strong non-homologous end joining (NHEJ) activity, which limits the efficiency of conventional homologous recombination-based editing strategies and makes iterative strain construction laborious. Although NHEJ can be exploited for homology-independent DNA integration, its random nature and the limited availability of recyclable selectable markers restrict its broader use in multi-round engineering. In this study, we developed the NH-CR system, a CRISPR/Cas9-assisted genome engineering workflow that couples NHEJ-mediated random DNA integration with delayed CRISPR/Cas9-mediated functional inactivation of the selectable marker in Y. lipolytica. The pYl-CRISPR platform enabled rapid target gene knockout within 3 days, with a gene-disruption efficiency exceeding 95%, and further supported random genomic integration of exogenous DNA fragments. To improve its applicability for iterative integration, Cas9 expression was tuned by promoter truncation, which balanced cotransformation efficiency and selectable-marker disruption efficiency. The optimized pYl-CRISPR-Δ75 system increased cotransformation efficiency to approximately 50 CFU/μg DNA while maintaining a 3-day selectable-marker disruption efficiency of approximately 75% after subculturing. The utility of NH-CR was further demonstrated in two metabolic engineering applications. Reinforcement of the native 2-phenylethanol biosynthetic pathway increased 2-phenylethanol production from 1 g/L in the wild-type strain to 2.5 g/L. In addition, iterative integration of heterologous sclareol biosynthetic genes and reinforcement of the endogenous MVA pathway enabled de novo sclareol production, reaching approximately 11 mg/L after 72 h of shake-flask fermentation. The NH-CR system provides a rapid and tunable workflow for iterative genome engineering in Y. lipolytica. By coupling rapid gene disruption and NHEJ-mediated random integration with delayed functional marker inactivation and reuse, the system reduces the operational burden of multi-round strain construction. NH-CR is particularly suited to rapid pathway assembly and phenotype-based screening when precise insertion-site or copy-number control is not required.