| 引用本文: | 王淑瑞,郇丽,顾文辉,王旭雷,牛建峰,王广策.基于CRISPR/Cas12a基因转化的条斑紫菜基因编辑底盘藻株构建[J].海洋科学,2025,49(5):34-46. |
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| 基于CRISPR/Cas12a基因转化的条斑紫菜基因编辑底盘藻株构建 |
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王淑瑞1,2,3, 郇丽1,2, 顾文辉1,2, 王旭雷1,2, 牛建峰1,2, 王广策1,4,2
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1.中国科学院海洋研究所中国科学院暨山东省实验海洋生物学重点实验室, 山东 青岛 266404;2.水产品种创制与高效养殖重点实验室(中国科学院), 山东 青岛 266404;3.中国科学院大学, 北京 100049;4.青岛海洋科技中心海洋生物学与生物技术功能实验室, 山东 青岛 266237
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| 摘要: |
| CRISPR/Cas系统是新一代基因编辑技术的代表, CRISPR/Cas12a系统具有蛋白分子量小、组装简单、脱靶率低等优势。本研究首先根据条斑紫菜密码子偏好性优化获得了PyCas12a基因序列, 并在潮霉素抗性标记基因PyAph7前添加了可被CRISPR/Cas12a识别的PAM位点, 借助原核表达获得Cas12a蛋白, 通过细胞外切割实验验证了其核酸内切酶对潮霉素抗性基因的剪切活性。之后以条斑紫菜(Pyropia yezoensis)叶状体为材料,利用基因枪转化, 筛选获得阳性藻株。经基因组鉴定, 获得转入了Cas12a核酸酶基因的条斑紫菜基因转化藻株4株, 转录组水平鉴定其中3株存在稳定表达。本研究不仅为条斑紫菜功能基因的探索提供了理想材料, 也为基于CRISPR/Cas12a基因编辑系统的条斑紫菜良种培育奠定了基础, 在基础研究与应用研究方面均具有重要意义及广阔应用前景。 |
| 关键词: CRISPR/Cas12a 条斑紫菜(Pyropia yezoensis) 原核表达 细胞外切割 细胞内表达 |
| DOI：10.11759/hykx20250213001 |
| 分类号:Q539;Q51 |
| 基金项目:山东省重点研发计划(重大科技创新工程)项目(2022LZGC004);山东省“泰山学者”工程专项经费资助项目(tspd20210316); 青岛市自然科学基金项目(24-4-4-zrjj-145-jch);山东省自然科学基金项目(ZR2024MD011);国家藻类产业技术体系项目(CARS-50) |
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| Construction of a Pyropia yezoensis strain as a gene-editing chassis using CRISPR/Cas12a |
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WANG Shurui1,2,3, HUAN Li1,2, GU Wenhui1,2, WANG Xulei1,2, NIU Jianfeng1,2, WANG Guangce1,4,2
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1.CAS and Shandong Province Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266404, China;2.Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture(CAS), Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266404, China;3.University of Chinese Academy of Sciences, Beijing 100049, China;4.Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, Qingdao 266237, China
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| Abstract: |
| The CRISPR/Cas system represents a new generation of gene editing technology. The CRISPR/Cas12a system has the advantages of small protein molecular weight, simple assembly, and a low off-target rate. In this study, we sequence-optimized PyCas12a from Pyropia yezoensis based on the codon usage bias. We also added a PAM site that can be recognized by the CRISPR/Cas12a system upstream of the hygromycin B resistance marker gene PyAph7. The Cas12a protein was obtained by prokaryotic expression, and its PyAph7 cleavage activity was verified by an extracellular experiment. Then, the thallus of P. yezoensis was used as receptor material and transformed with PyCas12a employing a gene gun; hygromycin B-resistant thalli were selected. Genome identification was employed to confirm four transgenic alga strains. Transcriptomics confirmed that PyCas12a was stably expressed in three of them. Our work not only identifies P. yezoensis as an ideal material for the functional exploration of genes but also lays a foundation for excellent P. yezoensis germplasms amenable to the CRISPR/Cas12a gene editing system. These findings are of great significance and have broad application prospects in basic and applied research. |
| Key words: CRISPR/Cas12a Pyropia yezoensis prokaryotic expression extracellular cleavage intracellular expression |
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