海洋科学2025，Vol.49Issue(5)：34-46,13.DOI:10.11759/hykx20250213001
基于CRISPR/Cas12a基因转化的条斑紫菜基因编辑底盘藻株构建
Construction of a Pyropia yezoensis strain as a gene-editing chassis using CRISPR/Cas12a
摘要
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 trans-formed 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 ex-pressed in three of them.Our work not only identifies P.yezoensis as an ideal material for the functional explora-tion 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 ap-plied research.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Key words
CRISPR/Cas12a/Pyropia yezoensis/prokaryotic expression/extracellular cleavage/intracellular expression分类
生物学引用本文复制引用
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):34-46,13.基金项目
山东省重点研发计划(重大科技创新工程)项目(2022LZGC004) （重大科技创新工程）
山东省"泰山学者"工程专项经费资助项目(tspd20210316) （tspd20210316）
青岛市自然科学基金项目(24-4-4-zrjj-145-jch) （24-4-4-zrjj-145-jch）
山东省自然科学基金项目(ZR2024MD011) （ZR2024MD011）
国家藻类产业技术体系项目(CARS-50)[Key Research and Development Program of Shandong Prov-ince,Major Scientific and Technological Innovation Project,No.2022LZGC004 （CARS-50）
The Research Fund for the Taishan Scholar Project of Shandong Province,No.tspd20210316 （）
Qingdao Natural Science Founda-tion,No.24-4-4-zrjj-145-jch （）
Shandong Provincial Natural Science Foun-dation,No.ZR2024MD011 （）
Earmarked Fund for China Agriculture Re-search System,No.CARS-50] （）
