A Split-Cre system designed to detect simultaneous expression of two genes based on SpyTag/SpyCatcher conjugation and Split-GFP dimerization.
A Split-Cre system designed to detect simultaneous expression of two genes based on SpyTag/SpyCatcher conjugation and Split-GFP dimerization.
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Split-Cre 系统旨在基于 SpyTag/SpyCatcher 接合和 Split-GFP 二聚化检测两个基因的同时表达
DOI：
10.1016/j.jbc.2021.101119
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发表时间：
2021-10
期刊：
影响因子：
--
通讯作者：
Ma J
中科院分区：
文献类型：
--
作者：
Wei X;Zhang J;Cui J;Xu W;Zhou X;Ma J
Split-Cre系统是一种强有力的遗传操作工具，可用于在体内时空控制基因表达。然而，在Split-Cre系统中重构的NCre/CCre重组酶的低活性限制了其作为一对感兴趣的基因的同时表达的指示剂的应用。在这里，我们描述了两种方法，用于提高活性的Split-Cre系统后Cre重建的基础上自缔合分裂GFP（Split-GFP）和SpyTag/SpyCatcher共轭。首先，我们通过构建NCre和CCre分别与GFP的N-末端和C-末端亚基的融合蛋白来创建Split-GFP-Cre系统。通过GFP介导的两种融合蛋白的二聚化重建Cre导致重组酶活性接近活细胞中全长Cre的活性。其次，为了在低水平的Split-Cre表达下进一步增加重组酶活性，通过将SpyTag和SpyCatcher的序列并入Split-GFP-Cre系统的组分中来建立Split-Spy-GCre系统。如预期的，SpyTag和SpyCatcher区段的共价缀合改善了Split-GFP二聚化，以进一步增加活细胞中的Cre重组酶活性。这种双分裂系统（Split-Cre和Split-GFP）的增加的效率和稳健性最小化了由于差的NCre/CCre重组酶活性而导致的不完全的双基因特异性KO或低标记效率的问题。因此，这种Split-Spy-GCre系统允许对细胞亚群进行更精确的基因操作，这将提供对免疫系统等复杂组织中基因和细胞功能的高级分析。
The Split-Cre system is a powerful tool for genetic manipulation and can be used to spatiotemporally control gene expression in vivo. However, the low activity of the reconstituted NCre/CCre recombinase in the Split-Cre system limits its application as an indicator of the simultaneous expression of a pair of genes of interest. Here, we describe two approaches for improving the activity of the Split-Cre system after Cre reconstitution based on self-associating split GFP (Split-GFP) and SpyTag/SpyCatcher conjugation. First, we created the Split-GFP-Cre system by constructing fusion proteins of NCre and CCre with the N-terminal and C-terminal subunits of GFP, respectively. Reconstitution of Cre by GFP-mediated dimerization of the two fusion proteins resulted in recombinase activity approaching that of full-length Cre in living cells. Second, to further increase recombinase activity at low levels of Split-Cre expression, the Split-Spy-GCre system was established by incorporating the sequences for SpyTag and SpyCatcher into the components of the Split-GFP-Cre system. As anticipated, covalent conjugation of the SpyTag and SpyCatcher segments improved Split-GFP dimerization to further increase Cre recombinase activity in living cells. The increased efficiency and robustness of this dual-split system (Split-Cre and Split-GFP) minimize the problems of incomplete double gene-specific KO or low labeling efficiency due to poor NCre/CCre recombinase activity. Thus, this Split-Spy-GCre system allows more precise gene manipulation of cell subpopulations, which will provide advanced analysis of genes and cell functions in complex tissue such as the immune system.
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