CRISPR-Cas9-Mediated Knock-In Approach to Insert the GFP11 Tag into the Genome of a Human Cell Line.
CRISPR-Cas9-Mediated Knock-In Approach to Insert the GFP11 Tag into the Genome of a Human Cell Line.
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CRISPR-Cas9 介导的敲入方法将 GFP11 标签插入人类细胞系的基因组中。
DOI：
10.1007/978-1-0716-2667-2_8
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Kamiyama,Daichi
中科院分区：
文献类型：
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作者：
Tamura,Ryo;Kamiyama,Daichi
本章中的方案描述了在人细胞系中使用自互补分裂绿色荧光蛋白（分裂GFP 1 -10/11）标记内源性蛋白的方法。通过核转染直接递送Cas9/sgRNA核糖核蛋白（RNP）复合物，该方案允许通过CRISPR-Cas9介导的同源定向修复（HDR）将GFP 11有效整合到特定的基因组基因座中。我们使用GFP 11序列以单链DNA（ssDNA）的形式作为HDR模板。因为这里使用的少于200个核苷酸的ssDNA是商业合成的，所以这种方法仍然是无克隆的。GFP 11的整合在稳定表达GFP 1 -10的细胞中进行，从而诱导荧光重建。随后，使用荧光流式细胞术分析这样的重构信号，以估计敲入效率并富集GFP阳性细胞群。最后，可以使用荧光显微镜观察富集的细胞。
The protocol in this chapter describes a method to label endogenous proteins using a self-complementing split green fluorescent protein (split GFP1-10/11) in a human cell line. By directly delivering Cas9/sgRNA ribonucleoprotein (RNP) complexes through nucleofection, this protocol allows for the efficient integration ofGFP11into a specific genomic locus via CRISPR-Cas9-mediated homology-directed repair (HDR). We use theGFP11sequence in the form of a single-stranded DNA (ssDNA) as an HDR template. Because the ssDNA with less than 200 nucleotides used here is commercially synthesized, this approach remains cloning-free. The integration ofGFP11is performed in cells stably expressingGFP1-10, thereby inducing fluorescence reconstitution. Subsequently, such a reconstituted signal is analyzed using fluorescence flow cytometry for estimating knock-in efficiencies and enriching the GFP-positive cell population. Finally, the enriched cells can be visualized using fluorescence microscopy.