Optimizing the CRISPR/Cas9 system for gene editing in Yarrowia lipolytica
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 con...
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| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2025-06-01
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| Series: | Engineering Microbiology |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2667370325000050 |
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| Summary: | 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 Cas9D147Y, 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. |
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| ISSN: | 2667-3703 |