Cancer Systems Biology Group, Theodor Boveri Institute, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
Mildred Scheel Early Career Center, University of Würzburg, Beethovenstraße 1A, 97080 Würzburg, Germany.
STAR Protoc. 2021 Nov 19;2(4):100949. doi: 10.1016/j.xpro.2021.100949. eCollection 2021 Dec 17.
Targeted protein degradation using degrons, such as the mini-Auxin-inducible degron (mAID), has an advantage over genetic silencing/knockout. However, the efficiency of sgRNA, homologous recombination, tedious expansion, and screening single clones makes the process of tagging endogenous proteins long and laborious. This protocol describes a practical and economical way to obtain AID-tagged endogenous proteins using CRISPR/Cas9-mediated homology-directed repair (HDR). We use the generation of endogenously AID-tagged SPT6 in U2OS cells as an example but provide sufficient details for usage in other cell types. For complete details on the use and execution of this protocol, please refer to Narain et al. (2021).
利用降解标签（如 mini-Auxin-inducible degron，mAID）进行靶向蛋白降解相对于基因沉默/敲除具有优势。然而，sgRNA 的效率、同源重组、繁琐的扩增和筛选单克隆使得标记内源性蛋白的过程既漫长又费力。本方案描述了一种使用 CRISPR/Cas9 介导的同源定向修复（HDR）获得 AID 标记内源性蛋白的实用且经济的方法。我们以 U2OS 细胞中内源性 AID 标记 SPT6 的产生为例，但提供了足够的详细信息，可用于其他细胞类型。有关此方案使用和执行的完整详细信息，请参阅 Narain 等人（2021 年）。
