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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DOI：
10.1093/nar/gkab727
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发表时间：
2021-11-18
影响因子：
14.9
通讯作者：
Szczelkun MD
中科院分区：
文献类型：
--
作者：
Torres Montaguth OE;Cross SJ;Ingram KWA;Lee L;Diffin FM;Szczelkun MD
绘制DNA切割事件的精确位置图在确定内切酶的机制和功能方面起着关键作用。内孔是一种高通量的基于纳米孔的方法，它允许在体外绘制单分子DNA切割事件的时间分辨图谱。在核酸内切酶使环状DNA底物线性化后，连接一个抗性盒，记录切割事件的位置。构建单个切割事件文库，并进行滚环扩增以产生串联序列。这些序列被测序，并用于产生准确的共识序列。为了确定切割位点(S)，我们开发了切割位点调查器(CSI)。CSI识别连接到裂解底物中的盒式磁带的末端，并对dsDNA断裂的位置进行三角定位。我们首次使用II型限制性内切酶对内孔进行了基准检测。其次，我们分析了crRNA长度对CRISPR Cas12a切割模式的影响。最后，我们通过在其DNA底物中引入随机双链断裂的ISP型限制性内切酶LlaGI绘制了时间分辨的DNA切割图谱。
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 events in 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.
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影响因子：
14.9
作者：
Smith RM;Josephsen J;Szczelkun MD
通讯作者：
Szczelkun MD
影响因子：
14.9
作者：
Lei C;Li SY;Liu JK;Zheng X;Zhao GP;Wang J
通讯作者：
Wang J
影响因子：
64.8
作者：
Anders, Carolin;Niewoehner, Ole;Duerst, Alessia;Jinek, Martin
通讯作者：
Jinek, Martin
影响因子：
46.9
作者：
Tsai, Shengdar Q.;Zheng, Zongli;Nguyen, Nhu T.;Liebers, Matthew;Topkar, Ved V.;Thapar, Vishal;Wyvekens, Nicolas;Khayter, Cyd;Iafrate, A. John;Le, Long P.;Aryee, Martin J.;Joung, J. Keith
通讯作者：
Joung, J. Keith
影响因子：
48
作者：
Karst, Soren M.;Ziels, Ryan M.;Albertsen, Mads
通讯作者：
Albertsen, Mads