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在人类细胞中使用CRISPR-Cas9对内源基因进行双标记的协议

Nannan Kong¹, Ying Wai Chan¹

¹School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China.

|May 30, 2023

概括

这项研究详细介绍了CRISPR-Cas9协议,用于人类细胞中的双基因标记. 该方法可以同时标记内源基因与降解蛋白和光蛋白进行先进研究.

科学领域:

分子生物学分子生物学
遗传学是一个遗传学.
细胞生物学细胞生物学

背景情况:

CRISPR-Cas9基因编辑提供了精确的基因组修改能力.
标记内源基因对于研究蛋白质功能和本地化在生物体中至关重要.
开发强大的双标记协议对于全面的遗传分析至关重要.

研究的目的:

介绍人类细胞内源性基因双标记的详细协议.
以RIF1基因为例来演示该协议的应用.
通过与降解蛋白和光蛋白同时标记C端子来促进基因功能的研究.

主要方法:

利用CRISPR-Cas9基因编辑技术进行精确的DNA修饰.
设计和准备的单导向RNA (sgRNA) 和同类修复模板.
采用了双标记的策略,结合了迷你辅酶诱导降解子和绿色光蛋白 (GFP).
包括克隆选择和验证步骤,以确保成功编辑.

主要成果:

成功建立了一个对内源基因双标记的协议.
证明了RIF1基因的标记,其C终端具有迷你辅酶诱导降解子和GFP.
为sgRNA设计,修复模板准备和克隆验证提供了详细的方法.

结论:

提出的协议使人类细胞内源基因的有效双标记成为可能.
这种方法允许精确的时间空间控制蛋白质水平使用auxin诱导降解系统.
开发的技术是功能性基因组学和细胞生物学研究的宝贵工具.

关键词:

克里斯普尔是什么意思?克里斯普尔是什么意思?细胞生物学细胞生物学

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