ENDO-Pore: High-throughput linked-end mapping of single DNA cleavage events using nanopore sequencing
ENDO-Pore: High-throughput linked-end mapping of single DNA cleavage events using nanopore sequencing
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ENDO-Pore：使用纳米孔测序对单个 DNA 切割事件进行高通量连接末端图谱
DOI：
10.1101/2021.07.02.450912
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发表时间：
2021
期刊：
影响因子：
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通讯作者：
Torres Montaguth O
中科院分区：
文献类型：
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作者：
Torres Montaguth O
绘制 DNA 切割事件的精确位置对于确定核酸内切酶的机制和功能起着关键作用。 ENDO-Pore 是一种基于纳米孔的高通量方法，可在体外对单分子 DNA 切割事件进行时间分辨绘图。通过核酸内切酶对环状 DNA 底物进行线性化后，连接一个抗性盒，记录切割事件的位置。构建单切割事件文库并进行滚环扩增以生成多联体。这些被测序并用于产生准确的共有序列。为了识别切割位点，我们开发了 CSI（切割位点调查器）。 CSI 识别连接到切割底物的盒末端并三角测量 dsDNA 断裂的位置。我们首先使用 II 型限制性内切酶对 ENDO-Pore 进行基准测试。其次，我们分析了crRNA长度对CRISPR Cas12a切割模式的影响。最后，我们绘制了 ISP 型限制性内切核酸酶 LlaGI 的时间分辨 DNA 切割图谱，该酶将随机双链断裂引入其 DNA 底物中。
Mapping the precise position of DNA cleavage events plays a key role in determining the mechanism and function of endonucleases. ENDO-Pore is a high-throughput nanopore-based method that allows the time resolved mapping single molecule DNA cleavage eventsin vitro. Following linearisation of a circular DNA substrate by the endonuclease, a resistance cassette is ligated recording the position of the cleavage event. A library of single cleavage events is constructed and subjected to rolling circle amplification to generate concatemers. These are sequenced and used to produce accurate consensus sequences. To identify the cleavage site(s), we developed CSI (Cleavage Site Investigator). CSI recognizes the ends of the cassette ligated into the cleaved substrate and triangulates the position of the dsDNA break. We firstly benchmarked ENDO-Pore using Type II restriction endonucleases. Secondly, we analysed the effect of crRNA length on the cleavage pattern of CRISPR Cas12a. Finally, we mapped the time-resolved DNA cleavage by the Type ISP restriction endonuclease LlaGI that introduces random double-strand breaks into its DNA substrates.