Multi-gene precision editing tool using CRISPR-Cas12a/Cpf1 system in Ogataea polymorpha
Abstract Background Ogataea polymorpha, a non-conventional methylotrophic yeast, has demonstrated significant potential for heterologous protein expression and the production of high-value chemicals and biopharmaceuticals. However, the lack of precise and efficient genome editing tools severely hind...
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BMC
2025-01-01
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| Series: | Microbial Cell Factories |
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| Online Access: | https://doi.org/10.1186/s12934-025-02654-8 |
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| author | Senqin Hou Shibin Yang Wenqin Bai |
| author_facet | Senqin Hou Shibin Yang Wenqin Bai |
| author_sort | Senqin Hou |
| collection | DOAJ |
| description | Abstract Background Ogataea polymorpha, a non-conventional methylotrophic yeast, has demonstrated significant potential for heterologous protein expression and the production of high-value chemicals and biopharmaceuticals. However, the lack of precise and efficient genome editing tools severely hinders the construction of cell factories. Although the CARISP-Cas9 system has been established in Ogataea polymorpha, the gene editing efficiency, especially for multiple genes edition, needs to be further improved. Results In this study, we developed an efficient CRISPR-Cpf1-mediated genome editing system in O. polymorpha that exhibited high editing efficiency for single gene (98.1 ± 1.7%), duplex genes (93.9 ± 2.4%), and triplex genes (94.0 ± 6.0%). Additionally, by knocking out non-homologous end joining (NHEJ) related genes, homologous recombination (HR) efficiency was increased from less than 30% to 90 ~ 100%, significantly enhancing precise genome editing capabilities. The increased HR rates enabled over 90% integration efficiency of triplex genes, as well as over 90% deletion rates of large DNA fragments up to 20 kb. Furthermore, using this developed CRISPR-Cpf1 system, triple genes were precisely integrated into the genome by one-step, enabling lycopene production in O. polymorpha. Conclusions This novel multiplexed genome-editing tool mediated by CRISPR-Cpf1 can realize the deletion and integration of multiple genes, which holds great promise for accelerating engineering efforts on this non-conventional methylotrophic yeast for metabolic engineering and genomic evolution towards its application as an industrial cell factory. |
| format | Article |
| id | doaj-art-c20fab54cdbf4f1f8daafc220ae413d2 |
| institution | Kabale University |
| issn | 1475-2859 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | BMC |
| record_format | Article |
| series | Microbial Cell Factories |
| spelling | doaj-art-c20fab54cdbf4f1f8daafc220ae413d22025-01-26T12:58:53ZengBMCMicrobial Cell Factories1475-28592025-01-0124111310.1186/s12934-025-02654-8Multi-gene precision editing tool using CRISPR-Cas12a/Cpf1 system in Ogataea polymorphaSenqin Hou0Shibin Yang1Wenqin Bai2National Center of Technology Innovation for Synthetic Biology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesNational Center of Technology Innovation for Synthetic Biology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesNational Center of Technology Innovation for Synthetic Biology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of SciencesAbstract Background Ogataea polymorpha, a non-conventional methylotrophic yeast, has demonstrated significant potential for heterologous protein expression and the production of high-value chemicals and biopharmaceuticals. However, the lack of precise and efficient genome editing tools severely hinders the construction of cell factories. Although the CARISP-Cas9 system has been established in Ogataea polymorpha, the gene editing efficiency, especially for multiple genes edition, needs to be further improved. Results In this study, we developed an efficient CRISPR-Cpf1-mediated genome editing system in O. polymorpha that exhibited high editing efficiency for single gene (98.1 ± 1.7%), duplex genes (93.9 ± 2.4%), and triplex genes (94.0 ± 6.0%). Additionally, by knocking out non-homologous end joining (NHEJ) related genes, homologous recombination (HR) efficiency was increased from less than 30% to 90 ~ 100%, significantly enhancing precise genome editing capabilities. The increased HR rates enabled over 90% integration efficiency of triplex genes, as well as over 90% deletion rates of large DNA fragments up to 20 kb. Furthermore, using this developed CRISPR-Cpf1 system, triple genes were precisely integrated into the genome by one-step, enabling lycopene production in O. polymorpha. Conclusions This novel multiplexed genome-editing tool mediated by CRISPR-Cpf1 can realize the deletion and integration of multiple genes, which holds great promise for accelerating engineering efforts on this non-conventional methylotrophic yeast for metabolic engineering and genomic evolution towards its application as an industrial cell factory.https://doi.org/10.1186/s12934-025-02654-8Ogataea polymorphaCRISPR-Cpf1Homologous recombination ratesLarge DNA fragment deletionMultiple genes integration |
| spellingShingle | Senqin Hou Shibin Yang Wenqin Bai Multi-gene precision editing tool using CRISPR-Cas12a/Cpf1 system in Ogataea polymorpha Microbial Cell Factories Ogataea polymorpha CRISPR-Cpf1 Homologous recombination rates Large DNA fragment deletion Multiple genes integration |
| title | Multi-gene precision editing tool using CRISPR-Cas12a/Cpf1 system in Ogataea polymorpha |
| title_full | Multi-gene precision editing tool using CRISPR-Cas12a/Cpf1 system in Ogataea polymorpha |
| title_fullStr | Multi-gene precision editing tool using CRISPR-Cas12a/Cpf1 system in Ogataea polymorpha |
| title_full_unstemmed | Multi-gene precision editing tool using CRISPR-Cas12a/Cpf1 system in Ogataea polymorpha |
| title_short | Multi-gene precision editing tool using CRISPR-Cas12a/Cpf1 system in Ogataea polymorpha |
| title_sort | multi gene precision editing tool using crispr cas12a cpf1 system in ogataea polymorpha |
| topic | Ogataea polymorpha CRISPR-Cpf1 Homologous recombination rates Large DNA fragment deletion Multiple genes integration |
| url | https://doi.org/10.1186/s12934-025-02654-8 |
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