Improving and Streamlining Gene Editing in <i>Yarrowia lipolytica</i> via Integration of Engineered Cas9 Protein
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 devel...
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| Format: | Article |
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MDPI AG
2024-01-01
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| Series: | Journal of Fungi |
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| Online Access: | https://www.mdpi.com/2309-608X/10/1/63 |
| _version_ | 1797343282136612864 |
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| author | Baixi Zhang Jiacan Cao |
| author_facet | Baixi Zhang Jiacan Cao |
| author_sort | Baixi Zhang |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-08T10:45:26Z |
| format | Article |
| id | doaj.art-916317441e71442cbe85bec38a15cebf |
| institution | Directory Open Access Journal |
| issn | 2309-608X |
| language | English |
| last_indexed | 2024-03-08T10:45:26Z |
| publishDate | 2024-01-01 |
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| record_format | Article |
| series | Journal of Fungi |
| spelling | doaj.art-916317441e71442cbe85bec38a15cebf2024-01-26T17:19:08ZengMDPI AGJournal of Fungi2309-608X2024-01-011016310.3390/jof10010063Improving and Streamlining Gene Editing in <i>Yarrowia lipolytica</i> via Integration of Engineered Cas9 ProteinBaixi Zhang0Jiacan Cao1School of Food Science and Technology, Jiangnan University, Wuxi 214122, ChinaSchool of Food Science and Technology, Jiangnan University, Wuxi 214122, ChinaThe 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.https://www.mdpi.com/2309-608X/10/1/63<i>Yarrowia lipolytica</i>genome editingCRISPR/Cas9 |
| spellingShingle | Baixi Zhang Jiacan Cao Improving and Streamlining Gene Editing in <i>Yarrowia lipolytica</i> via Integration of Engineered Cas9 Protein Journal of Fungi <i>Yarrowia lipolytica</i> genome editing CRISPR/Cas9 |
| title | Improving and Streamlining Gene Editing in <i>Yarrowia lipolytica</i> via Integration of Engineered Cas9 Protein |
| title_full | Improving and Streamlining Gene Editing in <i>Yarrowia lipolytica</i> via Integration of Engineered Cas9 Protein |
| title_fullStr | Improving and Streamlining Gene Editing in <i>Yarrowia lipolytica</i> via Integration of Engineered Cas9 Protein |
| title_full_unstemmed | Improving and Streamlining Gene Editing in <i>Yarrowia lipolytica</i> via Integration of Engineered Cas9 Protein |
| title_short | Improving and Streamlining Gene Editing in <i>Yarrowia lipolytica</i> via Integration of Engineered Cas9 Protein |
| title_sort | improving and streamlining gene editing in i yarrowia lipolytica i via integration of engineered cas9 protein |
| topic | <i>Yarrowia lipolytica</i> genome editing CRISPR/Cas9 |
| url | https://www.mdpi.com/2309-608X/10/1/63 |
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