Genome editing of a hybridoma cell line via the CRISPR/Cas9 system: A new approach for constitutive high-level expression of heterologous proteins in eukaryotic system
Genome editing of a hybridoma cell line via the CRISPR/Cas9 system: A new approach for constitutive high-level expression of heterologous proteins in eukaryotic system
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DOI：
10.1016/j.vetimm.2021.110286
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发表时间：
2021-06-23
影响因子：
1.8
通讯作者：
Rosati, Sergio
中科院分区：
文献类型：
--
作者：
Scialli, Nicoletta Schibeci Natoli;Colitti, Barbara;Rosati, Sergio
CRISPR/Cas9 系统的强大功能彻底改变了生物学许多领域的基因组编辑。近年来，这些应用呈指数级增长，包括与蛋白质表达技术相关的应用。 CRISPR/Cas9系统避免了目的基因的随机整合，并且由于这一特性可用于获得稳定的细胞系以高产表达重组蛋白。在这里，我们提出了一种使用 CRISPR/Cas9 系统编辑杂交瘤细胞系以组成型表达感兴趣的蛋白质的方法。首先，通过对方法的优化，我们用绿色荧光蛋白（GFP）基因替换了部分免疫球蛋白轻链，在杂交瘤基因组中获得了精确的敲入。我们通过荧光分析确认了 GFP 的表达和分泌到培养基中，并通过 RNA 测序确认了正确的基因组编辑。然后，使用相同的方法，我们将编码具有诊断意义的蛋白质（牛疱疹病毒 1 糖蛋白 E）的基因纳入供体 DNA 中。我们获得了能够将与 GFP 融合的 gE 蛋白分泌到培养基中的稳定克隆。该结果通过 ELISA 和 Western Blot 分析得到证实。这项研究证实了该细胞系通过在哺乳动物系统中稳定的基因表达来生产具有诊断意义的蛋白质的适用性。这些实验将使该技术从概念验证发展到传染病诊断领域更具体的应用。
The power of the CRISPR/Cas9 system has revolutionized genome editing in many fields of biology. These applications have expanded exponentially over recent years, including those regarding protein expression technologies. The CRISPR/Cas9 system avoids random integration of the gene of interest and due to this characteristic can be exploited to obtain a stable cell line for the high-yield expression of recombinant proteins. Here we propose a method to edit a hybridoma cell line for the constitutive expression of proteins of interest using the CRISPR/Cas9 system. First, with the scope of optimizing the method, we replaced part of the light chain of immunoglobulin with the Green Fluorescent Protein (GFP) gene, obtaining a precise knock-in in the hybridoma genome. We confirmed the expression and secretion of GFP into the culture medium via fluorimetric analysis, as well as correct genome editing by RNA sequencing. Then, using the same approach, we included the gene encoding a protein of diagnostic interest, the Bovine Herpesvirus 1 glycoprotein E, in the donor DNA. We obtained a stable clone able to secrete gE protein in fusion with GFP into the culture medium. This result was confirmed by ELISA and Western Blot analysis. This study confirms the suitability of this cell line for the production of proteins of diagnostic interest by stable gene expression in a mammalian system. These experiments will enable the technique to be developed from its proof of concept to more specific applications in the field of infectious disease diagnostics.