END-seq: An Unbiased, High-Resolution, and Genome-Wide Approach to Map DNA Double-Strand Breaks and Resection in Human Cells
END-seq: An Unbiased, High-Resolution, and Genome-Wide Approach to Map DNA Double-Strand Breaks and Resection in Human Cells
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END-seq：一种无偏见、高分辨率、全基因组的方法来绘制人类细胞中 DNA 双链断裂和切除的图谱
DOI：
10.1007/978-1-0716-0644-5_2
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发表时间：
2021
影响因子：
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通讯作者：
Canela A.
中科院分区：
文献类型：
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作者：
Wong N;John S;Nussenzweig A;Canela A.
DNA双链断裂（DSB）是DNA损伤中毒性最强的一种形式，可在生理或病理条件下发生。如果不修复，这些DSB可能导致基因组不稳定，这是肿瘤发生和其他病理学的主要驱动因素。因此，定位DSB和理解断裂形成和修复过程的动力学对于解剖潜在机制和靶向治疗的发展具有极大的意义。在这里，我们描述了END-seq，一种高灵敏度的下一代测序技术，用于以无偏的方式在整个基因组中以核苷酸分辨率定量绘制DNA双链断裂（DSB）。END-seq基于测序衔接子与DSB末端的直接连接，并提供有关DSB处DNA加工（末端切除）的信息，这是选择修复途径的关键决定因素。不存在细胞固定和使用琼脂糖包埋细胞以及使用核酸外切酶钝化DSB末端是关键的进步，这些进步有助于该技术相对于先前建立的方法提高灵敏度和稳健性。总的来说，END-seq为DSB作图提供了重大的技术进步，也有助于为复杂的生物学过程提供信息，包括基因组组织，复制叉崩溃和染色体脆性，RAG重组酶和基因编辑核酸酶的脱靶鉴定，以及DSB位点的DNA末端切除。
DNA double-strand breaks (DSBs) represent the most toxic form of DNA damage and can arise in either physiological or pathological conditions. If left unrepaired, these DSBs can lead to genome instability which serves as a major driver to tumorigenesis and other pathologies. Consequently, localizing DSBs and understanding the dynamics of break formation and the repair process are of great interest for dissecting underlying mechanisms and in the development of targeted therapies. Here, we describe END-seq, a highly sensitive next-generation sequencing technique for quantitatively mapping DNA double-strand breaks (DSB) at nucleotide resolution across the genome in an unbiased manner. END-seq is based on the direct ligation of a sequencing adapter to the ends of DSBs and provides information about DNA processing (end resection) at DSBs, a critical determinant in the selection of repair pathways. The absence of cell fixation and the use of agarose for embedding cells and exonucleases for blunting the ends of DSBs are key advances that contribute to the technique’s increased sensitivity and robustness over previously established methods. Overall, END-seq has provided a major technical advance for mapping DSBs and has also helped inform the biology of complex biological processes including genome organization, replication fork collapse and chromosome fragility, off-target identification of RAG recombinase and gene-editing nucleases, and DNA end resection at sites of DSBs.