CRISPR-Cas9基因编辑技术对细胞内源蛋白进行荧光标记的实验操作
收稿日期: 2022-12-02
修回日期: 2023-01-06
网络出版日期: 2023-01-09
基金资助
国家自然科学基金项目(91957106);国家自然科学基金项目(31970659);国家重点研发计划(2021YFA0804802);国家重点研发计划(2022YFA1304500)
The protocol of tagging endogenous proteins with fluorescent tags using CRISPR-Cas9 genome editing
Received date: 2022-12-02
Revised date: 2023-01-06
Online published: 2023-01-09
Supported by
the National Natural Science Foundation of China(91957106);the National Natural Science Foundation of China(31970659);the Ministry of Science and Technology of the People’s Republic of China(2021YFA0804802);the Ministry of Science and Technology of the People’s Republic of China(2022YFA1304500)
CRISPR-Cas9是目前广泛应用的基因编辑技术，可对目的基因进行高效精准编辑，快速实现目的基因的敲除或敲入。Cas9蛋白在sgRNA引导下对靶序列进行剪切并造成DNA双链断裂，在与剪切位点两端同源的DNA模板序列存在时，可通过同源重组修复方式引入外源序列，实现荧光蛋白或其他标签在基因组上的精准敲入，进而实现对内源蛋白进行荧光标签的融合标记。通过基因编辑技术对内源目的蛋白进行标记，可避免由于过表达造成蛋白质定位、动力学或功能等的潜在影响，可显著提升细胞成像实验的稳定性和可重复性。本文重点介绍了利用CRISPR-Cas9基因编辑系统对目的蛋白进行荧光蛋白或自标记蛋白标签标记的方法与操作流程，为构建内源蛋白荧光标记的哺乳动物细胞系提供参考。
关键词： CRISPR-Cas9基因编辑; 活细胞; 内源蛋白; 荧光蛋白; 自标记蛋白标签
吴仲胜, 高誉, 杜勇涛, 党颂, 何康敏 . CRISPR-Cas9基因编辑技术对细胞内源蛋白进行荧光标记的实验操作[J]. 遗传, 2023 , 45(2) : 165 -175 . DOI: 10.16288/j.yczz.22-395
The currently widely used CRISPR-Cas9 genome editing technology enables the editing of target genes (knock-out or knock-in) with high accuracy and efficiency. Guided by the small guide RNA, the Cas9 nuclease induces a DNA double-strand break at the targeted genomic locus. The DNA double-strand break can be repaired by the homology-directed repair pathway in the presence of a repair template. With the repair template containing the coding sequence of a fluorescent tag, the targeted gene can be inserted with the sequence of a fluorescent tag at the designed position. The genome editing mediated labeling of endogenous proteins with fluorescent tags avoids the potential artifacts caused by gene overexpression and substantially improves the reproductivity of imaging experiments. This protocol focuses on creating mammalian cell lines with endogenous proteins tagged with fluorescent proteins or self-labeling protein tags using CRISPR-Cas9 genome editing.
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