Hou Senqin, Yang Shibin, Bai Wenqin
National Center of Technology Innovation for Synthetic Biology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
Key Laboratory of Engineering Biology for Low-Carbon Manufacturing, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China.
Microb Cell Fact. 2025 Jan 21;24(1):28. doi: 10.1186/s12934-025-02654-8.
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.
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.
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.
多形汉逊酵母是一种非常规甲基营养型酵母，在异源蛋白表达以及高价值化学品和生物制药生产方面显示出巨大潜力。然而，缺乏精确高效的基因组编辑工具严重阻碍了细胞工厂的构建。尽管已在多形汉逊酵母中建立了CARISP-Cas9系统，但基因编辑效率，尤其是多基因编辑效率，仍需进一步提高。
在本研究中，我们在多形汉逊酵母中开发了一种高效的CRISPR-Cpf1介导的基因组编辑系统，该系统对单基因（98.1±1.7%）、双基因（93.9±2.4%）和三基因（94.0±6.0%）均表现出高编辑效率。此外，通过敲除非同源末端连接（NHEJ）相关基因，同源重组（HR）效率从不到30%提高到90%至100%，显著增强了精确基因组编辑能力。HR率的提高使三基因的整合效率超过90%，以及长达20 kb的大DNA片段的缺失率超过90%。此外，使用这种开发的CRISPR-Cpf1系统，三基因可一步精确整合到基因组中，从而使多形汉逊酵母能够生产番茄红素。
这种由CRISPR-Cpf1介导的新型多重基因组编辑工具能够实现多个基因的缺失和整合，这对于加速这种非常规甲基营养型酵母在代谢工程和基因组进化方面的工程努力，使其成为工业细胞工厂具有巨大的前景。
