| Summary: | The oleaginous yeast <i>Yarrowia lipolytica</i> is a prominent subject of biorefinery research due to its exceptional performance in oil production, exogenous protein secretion, and utilization of various inexpensive carbon sources. Many CRISPR/Cas9 genome-editing systems have been developed for <i>Y. lipolytica</i> to meet the high demand for metabolic engineering studies. However, these systems often necessitate an additional outgrowth step to achieve high gene editing efficiency. In this study, we introduced the eSpCas9 protein, derived from the <i>Streptococcus pyogenes</i> Cas9(SpCas9) protein, into the <i>Y. lipolytica</i> genome to enhance gene editing efficiency and fidelity, and subsequently explored the optimal expression level of <i>eSpCas9</i> gene by utilizing different promoters and selecting various growth periods for yeast transformation. The results demonstrated that the integrated <i>eSpCas9</i> gene editing system significantly enhanced gene editing efficiency, increasing from 16.61% to 86.09% on <i>TRP1</i> and from 33.61% to 95.19% on <i>LIP2</i>, all without the need for a time-consuming outgrowth step. Furthermore, growth curves and dilution assays indicated that the consistent expression of eSpCas9 protein slightly suppressed the growth of <i>Y. lipolytica</i>, revealing that strong inducible promoters may be a potential avenue for future research. This work simplifies the gene editing process in <i>Y. lipolytica</i>, thus advancing its potential as a natural product synthesis chassis and providing valuable insights for other comparable microorganisms.
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