基于CRISPR/Cas12a基因转化的条斑紫菜基因编辑底盘藻株构建

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Cas12a核酸内切酶对潮霉素抗性基因的剪切活性。之后以条斑紫菜叶状体为材料，利用基因枪转化，筛选获得阳性藻株。经DNA与RNA水平的鉴定，获得转入了Cas12a核酸酶基因的条斑紫菜基因转化藻株。

The PyCas12a gene sequence was obtained through codon optimization based on the codon bias of Pyropia yezoensis. A PAM site recognizable by CRISPR/Cas12a was added upstream of the hygromycin resistance marker gene PyAph7. Cas12a protein was acquired via prokaryotic expression, and the cleavage activity of Cas12a endonuclease against the hygromycin resistance gene was verified through in vitro cleavage assays. Subsequently, using Pyropia yezoensis thalli as experimental materials, biolistic transformation was conducted, and positive algal strains were screened. Following identification at both DNA and RNA levels, Pyropia yezoensis transgenic algal strains integrated with the Cas12a endonuclease gene were successfully obtained.

搜集汇总

背景与挑战

背景概述

该数据集聚焦于利用CRISPR/Cas12a基因编辑技术构建条斑紫菜的基因编辑底盘藻株，通过优化基因序列、原核表达蛋白和细胞外切割验证活性，最终利用基因枪转化获得阳性藻株。研究在2023年09月至2025年01月期间于青岛进行，涉及基因编辑、荧光定量等关键要素，属于海洋生物学领域，数据以申请共享方式提供。