Optimizing the CRISPR/Cas9 system for gene editing in Yarrowia lipolytica

Abstract

Yarrowia lipolytica is a promising host for producing valuable chemicals owing to its robustness and metabolic versatility. Efficient genome editing tools are essential for advancing its biotechnological applications. Although CRISPR/Cas9 technology has been applied in Y. lipolytica, achieving a consistently high editing performance remains challenging owing to the low homologous recombination efficiency and variability in system components. In this study, we optimized CRISPR/Cas9-mediated genome editing in Y. lipolytica to enhance its editing efficiency. Using the RNA polymerase III promoter SCR1-tRNA for sgRNA expression, we achieved a gene disruption efficiency of 92.5 %. The tRNA-sgRNA architecture enabled a dual gene disruption efficiency of 57.5 %. KU70 deletion in the Cas9 system increased the integration efficiency to 92.5 %, and Rad52 and Sae2 overexpression boosted homologous recombination. The introduction of Cas9^{D147Y, P411T} (iCas9) enhanced the efficiency of both gene disruption and genome integration. This study provides a powerful tool for efficient gene editing in Y. lipolytica, which will accelerate the construction of yeast cell factories.

Keywords

CRISPR/Cas9 technology / Yarrowia lipolytica / sgRNA promoter / Cas9 expression strategy / Multiplex gene editing / Genome integration

Data Availability Statement

All relevant data supporting the findings of this study are available in this manuscript and the supplementary materials.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Jianhui Liu: Writing - original draft, Methodology, Investigation, Formal analysis, Data curation. Yamin Zhu: Writing - original draft, Methodology, Investigation, Formal analysis, Data curation. Jin Hou: Writing - review & editing, Writing - original draft, Resources, Project administration, Funding acquisition.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (U23A20268), the National Natural Science Foundation of Shandong Province (ZR2022ZD24), and the Taishan Scholar Project of Shandong Province (tsqn202312061).

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