Flow cytometric assays for interrogating LAGLIDADG homing endonuclease DNA-binding and cleavage properties.
Flow cytometric assays for interrogating LAGLIDADG homing endonuclease DNA-binding and cleavage properties.
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DOI：
10.1007/978-1-62703-293-3_4
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发表时间：
2013-01-01
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影响因子：
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通讯作者：
Jarjour, Jordan
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文献类型：
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作者：
Baxter, Sarah K;Lambert, Abigail R;Jarjour, Jordan
一种快速、简单和可扩展的方法来评估位点特异性核酸酶的性质，对于理解它们在基因组工程或群体水平基因驱动应用中的细胞内行为至关重要。在这里，我们描述了一个分析平台，使高通量，半定量询问的DNA结合和催化性能的LAGLIDADG归巢核酸内切酶（LHE）。使用该平台，天然或工程化的LHE在酿酒酵母的表面上表达，其中它们可以使用流式细胞术针对合成的DNA靶序列进行快速评估。
A fast, easy, and scalable method to assess the properties of site-specific nucleases is crucial to -understanding their in cellulo behavior in genome engineering or population-level gene drive applications. Here we describe an analytical platform that enables high-throughput, semiquantitative interrogation of the DNA-binding and catalytic properties of LAGLIDADG homing endonucleases (LHEs). Using this platform, natural or engineered LHEs are expressed on the surface of Saccharomyces cerevisiae yeast where they can be rapidly evaluated against synthetic DNA target sequences using flow cytometry.