ArticleMicrobial cell factories2025
Multi-gene precision editing tool using CRISPR-Cas12a/Cpf1 system in Ogataea polymorpha.
Article in Microbial cell factories, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
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5 citing papers in PubMed.
- Engineering an asymmetric cross-feeding consortium inSynthetic and systems biotechnology · 2026Article
- Yeast species resource exploration and application in cell factories.Synthetic and systems biotechnology · 2026Review
- Lignocellulosic biomass fermentation: a roadmap for Candida famata and Ogataea polymorpha.FEMS yeast research · 2025Review
- Engineering recombination machinery facilitates the construction of yeast cell factories.FEMS yeast research · 2025Review
- Integrating yeast biodiversity and machine learning for predictive metabolic engineering.FEMS yeast research · 2025Review
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Abstract
backgroundOgataea 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.
resultsIn 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.
conclusionsThis 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.
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