开发一种基于CRISPR的胞嘧啶碱基编辑器用于限制修饰系统失活，以提高弧菌属dhg中的转化效率
Development of a CRISPR-based cytosine base editor for restriction-modification system inactivation to enhance transformation efficiency in Vibrio Sp. dhg.
作者信息
Shon Yang Jun, Baek Dongyeop, Jin Su Bin, Kim Woo Jae, Jung Gyoo Yeol, Lim Hyun Gyu
机构信息
Department of Chemical Engineering, Pohang University of Science and Technology, 77 Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk, 37673, Korea.
Department of Biological Sciences and Bioengineering, Inha University, 100 Inha-Ro, Michuhol-Gu, Incheon, 22212, Korea.
出版信息
J Biol Eng. 2025 Apr 9;19(1):30. doi: 10.1186/s13036-025-00500-4.
BACKGROUND
Vibrio sp. dhg is a fast-growing, alginate-utilizing, marine bacterium being developed as a platform host for macroalgae biorefinery. To maximize its potential in the production of various value-added products, there is a need to expand genetic engineering tools for versatile editing.
RESULTS
The CRISPR-based cytosine base editing (CBE) system was established in Vibrio sp. dhg, enabling C: G-to-T: A point mutations in multiple genomic loci. This CBE system displayed high editing efficiencies for single and multiple targets, reaching up to 100%. The CBE system efficiently introduced premature stop codons, inactivating seven genes encoding putative restriction enzymes of the restriction-modification system in two rounds. A resulting engineered strain displayed significantly enhanced transformation efficiency by up to 55.5-fold.
CONCLUSIONS
Developing a highly efficient CBE system and improving transformation efficiency enable versatile genetic manipulation of Vibrio sp. dhg for diverse engineering in brown macroalgae bioconversion.
背景
弧菌属dhg是一种生长迅速、利用藻酸盐的海洋细菌，正被开发为大型藻类生物炼制的平台宿主。为了最大限度地发挥其在生产各种增值产品方面的潜力，需要扩展用于通用编辑的基因工程工具。
结果
在弧菌属dhg中建立了基于CRISPR的胞嘧啶碱基编辑（CBE）系统，可在多个基因组位点实现C:G到T:A的点突变。该CBE系统对单个和多个靶点均显示出高编辑效率，最高可达100%。CBE系统有效地引入了提前终止密码子，两轮操作就使七个编码限制修饰系统推定限制酶的基因失活。由此产生的工程菌株转化效率显著提高，最高可达55.5倍。
结论
开发高效的CBE系统并提高转化效率，可实现对弧菌属dhg进行通用基因操作，用于褐藻生物转化中的多种工程应用。
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