Targeted Protein Degradation Phenotypic Studies Using HaloTag CRISPR/Cas9 Endogenous Tagging Coupled with HaloPROTAC3.
Targeted Protein Degradation Phenotypic Studies Using HaloTag CRISPR/Cas9 Endogenous Tagging Coupled with HaloPROTAC3.
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DOI：
10.1002/cpph.81
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发表时间：
2020-12
影响因子：
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通讯作者：
Daniels DL
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文献类型：
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作者：
Caine EA;Mahan SD;Johnson RL;Nieman AN;Lam N;Warren CR;Riching KM;Urh M;Daniels DL
为了评估蛋白质的作用，可以使用蛋白质丢失表型研究，最常见的是通过诱变 RNAi 或 CRISPR 敲除。此类研究对于理解蛋白质功能和确定多种人类疾病状态的假定治疗靶点至关重要。然而，这些方法学方法面临着挑战，因为它们不易逆转，并且如果以必需基因为目标，相关的细胞活力丧失可能会阻碍进一步的研究。在这里，我们提出了一种可逆的、有条件的活细胞敲除策略，适用于多种蛋白质。这种模块化蛋白质标记方法通过使用 HaloPROTAC3 来调节蛋白质水平而非基因组水平的靶标丢失，HaloPROTAC3 通过招募 VHL E3 连接酶成分来特异性降解 HaloTag 融合蛋白。为了实现 HaloTag 介导的内源蛋白降解，我们提供了通过 CRISPR/Cas9 在蛋白质 N 或 C 末端插入 HaloTag 基因组的方案，并通过荧光激活细胞分选 (FACS) 使用 HaloTag 荧光配体富集编辑的细胞。使用这些方法，HaloPROTAC3 可以降解存在于各个亚细胞位置的内源 HaloTag 融合蛋白。由于检测内源靶标的降解具有挑战性，因此将 11 个氨基酸肽标签 HiBiT 添加到 HaloTag 融合中，以便在不使用抗体的情况下灵敏地发光检测 HaloTag 融合水平。最后，通过将 HaloPROTAC3 降解与另一种融合标签 PROTAC dTAG-13 的降解进行比较，我们证明 HaloPROTAC3 具有更快的降解速率和相似的降解程度。 © 2020 作者。 基本方案 1：HaloTag 或 HiBiT-HaloTag 的 CRISPR/Cas9 插入 基本方案 2：内源 HaloTag 融合物的 HaloPROTAC3 降解
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