ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons
ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons
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DOI：
10.1371/journal.pbio.3000665
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发表时间：
2020-04-01
期刊：
影响因子：
9.8
通讯作者：
MacGillavry, Harold D.
中科院分区：
文献类型：
--
作者：
Willems, Jelmer;de Jong, Arthur P. H.;MacGillavry, Harold D.
蛋白质的正确亚细胞分布建立了神经元的复杂形态和功能。荧光显微镜技术对于研究亚细胞蛋白质分布是非常宝贵的，但是它们受限于用荧光探针有效且可靠地标记内源性蛋白质的能力。我们开发橙子：神经元基因组编辑应用的开放资源，其介导啮齿动物解离神经元培养物、器官型切片和体内表位标签的靶向基因组整合。橙子包括一个用于深入研究内源性蛋白质分布的敲入文库、病毒载体和一个详细的两步克隆方案，以开发针对新靶点的敲入。使用橙子与（活细胞）超分辨率显微镜，我们揭示了内源性神经递质受体和突触支架蛋白，以及以前未知的蛋白质的动态纳米级组织。最后，我们开发了一种机制，在神经元中创建多个敲入，介导内源性蛋白质的多重成像。因此，橙子能够以纳米级分辨率定量神经元中几乎任何蛋白质的表达、分布和动力学。
The correct subcellular distribution of proteins establishes the complex morphology and function of neurons. Fluorescence microscopy techniques are invaluable to investigate subcellular protein distribution, but they suffer from the limited ability to efficiently and reliably label endogenous proteins with fluorescent probes. We developed ORANGE: Open Resource for the Application of Neuronal Genome Editing, which mediates targeted genomic integration of epitope tags in rodent dissociated neuronal culture, in organotypic slices, and in vivo. ORANGE includes a knock-in library for in-depth investigation of endogenous protein distribution, viral vectors, and a detailed two-step cloning protocol to develop knock-ins for novel targets. Using ORANGE with (live-cell) superresolution microscopy, we revealed the dynamic nanoscale organization of endogenous neurotransmitter receptors and synaptic scaffolding proteins, as well as previously uncharacterized proteins. Finally, we developed a mechanism to create multiple knock-ins in neurons, mediating multiplex imaging of endogenous proteins. Thus, ORANGE enables quantification of expression, distribution, and dynamics for virtually any protein in neurons at nanoscale resolution.