Protein labeling approaches are important to study proteins in living cells, and genome editing tools make it possible to tag endogenous proteins to address the concerns associated with overexpression. Here we established RNA editing-mediated noncanonical amino acids (ncAAs) protein tagging (RENAPT) to site-specifically label endogenous proteins with ncAAs in living cells. RENAPT labels protein in a temporary and nonheritable manner and is not restricted by protospacer adjacent motif sequence. Using a fluorescent ncAA or ncAA with a bio-orthogonal reaction handle for subsequent dye labeling, we demonstrated that a variety of endogenous proteins can be imaged at their specific subcellular locations. In addition, two proteins can be tagged individually and simultaneously using two different ncAAs. Furthermore, endogenous ion channels and neuron-specific proteins can be real-time labeled in primary neurons. Thus, RENAPT presents a promising platform with broad applicability for tagging endogenous proteins in living cells to study their localization and functions.

蛋白质标记方法对于研究活细胞中的蛋白质非常重要，基因组编辑工具可以标记内源蛋白质，以解决与过度表达相关的问题。在这里，我们建立了 RNA 编辑介导的非规范氨基酸 (ncAA) 蛋白标签 (RENAPT)，以在活细胞中用 ncAA 进行位点特异性标记内源蛋白。 RENAPT 以临时且不可遗传的方式标记蛋白质，并且不受原型间隔子相邻基序序列的限制。使用荧光 ncAA 或带有生物正交反应手柄的 ncAA 进行后续染料标记，我们证明可以对各种内源蛋白在其特定的亚细胞位置进行成像。此外，可以使用两种不同的 ncAA 单独并同时标记两种蛋白质。此外，可以在原代神经元中实时标记内源离子通道和神经元特异性蛋白质。因此，RENAPT 提供了一个有前景的平台，具有广泛的适用性，可用于标记活细胞中的内源蛋白以研究其定位和功能。