CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide
CRISPR-Mediated Tagging of Endogenous Proteins with a Luminescent Peptide
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DOI：
10.1021/acschembio.7b00549
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发表时间：
2018-02-01
影响因子：
4
通讯作者：
Wood, Keith V.
中科院分区：
文献类型：
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作者：
Schwinn, Marie K.;Machleidt, Thomas;Wood, Keith V.
细胞内信号传导途径由蛋白质丰度的变化和翻译后修饰介导。研究合成化合物诱导的信号传导机制和效应的常用方法是通过重组报告基因的过表达。使用CRISPR/Cas9进行基因组编辑提供了一种通过将报告基因直接附加到内源基因上来更好地保护天然生物学的方法。用于此目的的最佳报告子将是小的以可忽略地影响细胞内过程，容易地以最小的实验努力与内源基因连接，并且足够灵敏以检测低表达蛋白。HiBiT是一种1.3 kDa肽（11个氨基酸），能够通过与NanoLuc（LgBiT）衍生的18 kDa亚基的高亲和力互补（KD = 700 pM）产生明亮和定量发光。使用CRISPR/Cas9，我们证明HiBiT可以快速有效地整合到基因组中，作为内源性蛋白质的报告标签。在不需要克隆分离编辑的细胞的情况下，我们能够量化响应于各种刺激的缺氧诱导因子IA（HIF 1 α）及其几个下游转录靶标的丰度的变化。结合荧光抗体，我们进一步使用HiBiT将HIF 1 α水平与介导其降解的羟脯氨酸修饰直接相关。这些结果证明了用小的发光肽有效标记内源性蛋白质的能力，允许灵敏地定量其调节表达和共价修饰中的响应动力学。
Intracellular signaling pathways are mediated by changes in protein abundance and post-translational modifications. A common approach for investigating signaling mechanisms and the effects induced by synthetic compounds is through overexpression of recombinant reporter genes. Genome editing with CRISPR/Cas9 offers a means to better preserve native biology by appending reporters directly onto the endogenous genes. An optimal reporter for this purpose would be small to negligibly influence intracellular processes, be readily linked to the endogenous genes with minimal experimental effort, and be sensitive enough to detect low expressing proteins. HiBiT is a 1.3 kDa peptide (11 amino acids) capable of producing bright and quantitative luminescence through high affinity complementation (K-D = 700 pM) with an 18 kDa subunit derived from NanoLuc (LgBiT). Using CRISPR/Cas9, we demonstrate that HiBiT can be rapidly and efficiently integrated into the genome to serve as a reporter tag for endogenous proteins. Without requiring clonal isolation of the edited cells, we were able to quantify changes in abundance of the hypoxia inducible factor IA (HIF1 alpha) and several of its downstream transcriptional targets in response to various stimuli. In combination with fluorescent antibodies, we further used HiBiT to directly correlate HIF1 alpha levels with the hydroxyproline modification that mediates its degradation. These results demonstrate the ability to efficiently tag endogenous proteins with a small luminescent peptide, allowing sensitive quantitation of the response dynamics in their regulated expression and covalent modifications.