Reuben Philip, Amit Sharma, Laura Matellan, Anna C Erpf, Wen-Hsin Hsu, Johnny M Tkach, Haley D M Wyatt, Laurence Pelletier
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引用次数: 0
摘要
内源标记可以在蛋白质的原生调控背景下对其进行研究,通常使用 CRISPR 将标记序列直接插入基因序列中。在这里,我们介绍了qTAG,它是一系列修复盒,使内源标记更容易获得。这些修复盒支持使用常用的可选择标记物进行 N 端和 C 端标记,并具有易于修饰的限制性位点。Lox 位点还能在成功整合后移除标记基因。我们展示了 qTAG 在荧光成像、近距离标记、表位标记和靶向蛋白质降解等应用中的各种标记的实用性。该系统包括 mStayGold 等新型标签,这些标签具有更高的亮度和光稳定性,可用于本地蛋白质动态的活细胞成像。此外,我们还探索了用于条件表达标记、选择性基因敲除标记和安全港表达的替代盒设计。质粒集可通过 Addgene 获得,其中包括用于常见亚细胞标记的即用型构建体和用于靶向感兴趣基因的标记盒。qTAG 系统将作为一种开放资源,供研究人员调整和定制自己的实验。
qTAG: an adaptable plasmid scaffold for CRISPR-based endogenous tagging.
Endogenous tagging enables the study of proteins within their native regulatory context, typically using CRISPR to insert tag sequences directly into the gene sequence. Here, we introduce qTAG, a collection of repair cassettes that makes endogenous tagging more accessible. The cassettes support N- and C-terminal tagging with commonly used selectable markers and feature restriction sites for easy modification. Lox sites also enable the removal of the marker gene after successful integration. We demonstrate the utility of qTAG with a range of diverse tags for applications in fluorescence imaging, proximity labeling, epitope tagging, and targeted protein degradation. The system includes novel tags like mStayGold, offering enhanced brightness and photostability for live-cell imaging of native protein dynamics. Additionally, we explore alternative cassette designs for conditional expression tagging, selectable knockout tagging, and safe-harbor expression. The plasmid collection is available through Addgene, featuring ready-to-use constructs for common subcellular markers and tagging cassettes to target genes of interest. The qTAG system will serve as an open resource for researchers to adapt and tailor their own experiments.
期刊介绍:
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