Promega Corporation , Madison, Wisconsin 53711, United States.
Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah , Salt Lake City, Utah 84112, United States.
ACS Chem Biol. 2018 Feb 16;13(2):467-474. doi: 10.1021/acschembio.7b00549. Epub 2017 Sep 21.
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 = 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 1A (HIF1α) and several of its downstream transcriptional targets in response to various stimuli. In combination with fluorescent antibodies, we further used HiBiT to directly correlate HIF1α 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.
细胞内信号通路是通过蛋白质丰度和翻译后修饰的变化来介导的。研究信号机制和合成化合物诱导的作用的一种常见方法是通过重组报告基因的过表达。CRISPR/Cas9 基因组编辑提供了一种通过直接在内源性基因上附加报告基因来更好地保留天然生物学的方法。为此目的，一个理想的报告基因应该是体积小到对细胞内过程的影响可以忽略不计，能够以最小的实验工作量与内源性基因轻易连接，并且足够敏感以检测低表达的蛋白质。HiBiT 是一个 1.3 kDa 的肽（11 个氨基酸），通过与源自 NanoLuc（LgBiT）的 18 kDa 亚基的高亲和力互补（K = 700 pM），能够产生明亮和定量的发光。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基因标签。我们不需要对编辑细胞进行克隆分离，就能够定量检测缺氧诱导因子 1A（HIF1α）及其几个下游转录靶标对各种刺激的丰度变化。与荧光抗体结合使用，我们进一步使用 HiBiT 将 HIF1α 水平与介导其降解的羟脯氨酸修饰直接相关联。这些结果表明，能够有效地用小的发光肽标记内源性蛋白质，从而能够灵敏地定量其受调控表达和共价修饰的反应动力学。
