Detection and Quantification of HDR and NHEJ Induced by Genome Editing at Endogenous Gene Loci Using Droplet Digital PCR.
Detection and Quantification of HDR and NHEJ Induced by Genome Editing at Endogenous Gene Loci Using Droplet Digital PCR.
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DOI：
10.1007/978-1-4939-7778-9_20
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发表时间：
2018
期刊：
影响因子：
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通讯作者：
Conklin BR
中科院分区：
文献类型：
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作者：
Miyaoka Y;Mayerl SJ;Chan AH;Conklin BR
基因组编辑在实验生物学和潜在的临床应用方面大有可为。为了成功地利用基因组编辑，敏感地检测和量化其结果是至关重要的：同源定向修复(HDR)和非同源末端连接(NHEJ)。这在内源基因座上一直是困难的，取而代之的是经常使用人工报告系统来完成。在这里，我们描述了一种基于滴状数字聚合酶链式反应(DdPCR)的方法，可以同时检测内源基因座上的HDR和NHEJ。这种高度敏感和定量的方法可能有助于更好地理解基因组编辑背后的DNA修复机制，并通过允许对许多基因组编辑条件进行有效和系统的测试来改进基因组编辑技术。
Genome editing holds great promise for experimental biology and potential clinical use. To successfully utilize genome editing, it is critical to sensitively detect and quantify its outcomes: homology-directed repair (HDR) and nonhomologous end joining (NHEJ). This has been difficult at endogenous gene loci and instead is frequently done using artificial reporter systems. Here, we describe a droplet digital PCR (ddPCR)-based method to simultaneously measure HDR and NHEJ at endogenous gene loci. This highly sensitive and quantitative method may significantly contribute to a better understanding of DNA repair mechanisms underlying genome editing and to the improvement of genome editing technology by allowing for efficient and systematic testing of many genome editing conditions in parallel.