Promega Corporation, Madison, Wisconsin.
Boehringer Ingelheim Pharmaceuticals, Ridgefield, Connecticut.
Curr Protoc Pharmacol. 2020 Dec;91(1):e81. doi: 10.1002/cpph.81.
To assess the role of a protein, protein loss phenotypic studies can be used, most commonly through mutagenesis RNAi or CRISPR knockout. Such studies have been critical for the understanding of protein function and the identification of putative therapeutic targets for numerous human disease states. However, these methodological approaches present challenges because they are not easily reversible, and if an essential gene is targeted, an associated loss of cell viability can potentially hinder further studies. Here we present a reversible and conditional live-cell knockout strategy that is applicable to numerous proteins. This modular protein-tagging approach regulates target loss at the protein, rather than the genomic, level through the use of HaloPROTAC3, which specifically degrades HaloTag fusion proteins via recruitment of the VHL E3 ligase component. To enable HaloTag-mediated degradation of endogenous proteins, we provide protocols for HaloTag genomic insertion at the protein N or C terminus via CRISPR/Cas9 and use of HaloTag fluorescent ligands to enrich edited cells via Fluorescence-Activated Cell Sorting (FACS). Using these approaches, endogenous HaloTag fusion proteins present in various subcellular locations can be degraded by HaloPROTAC3. As detecting the degradation of endogenous targets is challenging, the 11-amino-acid peptide tag HiBiT is added to the HaloTag fusion to allows the sensitive luminescence detection of HaloTag fusion levels without the use of antibodies. Lastly, we demonstrate, through comparison of HaloPROTAC3 degradation with that of another fusion tag PROTAC, dTAG-13, that HaloPROTAC3 has a faster degradation rate and similar extent of degradation. © 2020 The Authors. Basic Protocol 1: CRISPR/Cas9 insertion of HaloTag or HiBiT-HaloTag Basic Protocol 2: HaloPROTAC3 degradation of endogenous HaloTag fusions.
为了评估蛋白质的作用，可以使用蛋白质丢失表型研究，最常见的方法是通过诱变 RNAi 或 CRISPR 敲除。这些研究对于理解蛋白质功能和确定许多人类疾病状态的潜在治疗靶点至关重要。然而，这些方法学方法存在挑战，因为它们不容易逆转，如果靶向的是必需基因，细胞活力的丧失可能会潜在地阻碍进一步的研究。在这里，我们提出了一种可逆转和条件性的活细胞敲除策略，该策略适用于许多蛋白质。这种模块化的蛋白质标记方法通过使用 HaloPROTAC3 调节靶蛋白的丢失，而不是通过基因组水平的丢失，HaloPROTAC3 通过募集 VHL E3 连接酶组件特异性降解 HaloTag 融合蛋白。为了使 HaloTag 介导的内源性蛋白质降解，我们提供了通过 CRISPR/Cas9 在蛋白质 N 或 C 末端插入 HaloTag 的方案，并使用 HaloTag 荧光配体通过荧光激活细胞分选（FACS）富集编辑细胞。使用这些方法，可以降解存在于各种亚细胞位置的内源性 HaloTag 融合蛋白。由于检测内源性靶标的降解具有挑战性，因此添加 11 个氨基酸肽标签 HiBiT 到 HaloTag 融合物中，以在不使用抗体的情况下灵敏地检测 HaloTag 融合物的水平。最后，我们通过比较 HaloPROTAC3 与另一种融合标签 PROTAC dTAG-13 的降解，证明了 HaloPROTAC3 具有更快的降解速度和相似的降解程度。© 2020 作者。基本方案 1：CRISPR/Cas9 插入 HaloTag 或 HiBiT-HaloTag 基本方案 2：HaloPROTAC3 降解内源性 HaloTag 融合物。
