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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| Format: | Article |
| Language: | English |
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Elsevier
2025-06-01
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| Series: | Engineering Microbiology |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2667370325000050 |
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| author | Jianhui Liu Yamin Zhu Jin Hou |
| author_facet | Jianhui Liu Yamin Zhu Jin Hou |
| author_sort | Jianhui Liu |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-a5d499c593204e6ca86de67d5c9eeb87 |
| institution | Kabale University |
| issn | 2667-3703 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Engineering Microbiology |
| spelling | doaj-art-a5d499c593204e6ca86de67d5c9eeb872025-08-20T03:30:30ZengElsevierEngineering Microbiology2667-37032025-06-015210019310.1016/j.engmic.2025.100193Optimizing the CRISPR/Cas9 system for gene editing in Yarrowia lipolyticaJianhui Liu0Yamin Zhu1Jin Hou2State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, ChinaState Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, ChinaCorresponding author.; State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, ChinaYarrowia 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.http://www.sciencedirect.com/science/article/pii/S2667370325000050CRISPR/Cas9 technologyYarrowia lipolyticasgRNA promoterCas9 expression strategyMultiplex gene editingGenome integration |
| spellingShingle | Jianhui Liu Yamin Zhu Jin Hou Optimizing the CRISPR/Cas9 system for gene editing in Yarrowia lipolytica Engineering Microbiology CRISPR/Cas9 technology Yarrowia lipolytica sgRNA promoter Cas9 expression strategy Multiplex gene editing Genome integration |
| title | Optimizing the CRISPR/Cas9 system for gene editing in Yarrowia lipolytica |
| title_full | Optimizing the CRISPR/Cas9 system for gene editing in Yarrowia lipolytica |
| title_fullStr | Optimizing the CRISPR/Cas9 system for gene editing in Yarrowia lipolytica |
| title_full_unstemmed | Optimizing the CRISPR/Cas9 system for gene editing in Yarrowia lipolytica |
| title_short | Optimizing the CRISPR/Cas9 system for gene editing in Yarrowia lipolytica |
| title_sort | optimizing the crispr cas9 system for gene editing in yarrowia lipolytica |
| topic | CRISPR/Cas9 technology Yarrowia lipolytica sgRNA promoter Cas9 expression strategy Multiplex gene editing Genome integration |
| url | http://www.sciencedirect.com/science/article/pii/S2667370325000050 |
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