Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks.
Novel fluorescent genome editing reporters for monitoring DNA repair pathway utilization at endonuclease-induced breaks.
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DOI：
10.1093/nar/gkt872
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发表时间：
2014-01
影响因子：
14.9
通讯作者：
Certo MT
中科院分区：
文献类型：
--
作者：
Kuhar R;Gwiazda KS;Humbert O;Mandt T;Pangallo J;Brault M;Khan I;Maizels N;Rawlings DJ;Scharenberg AM;Certo MT
通过设计核酸内切酶在特定位点产生DNA断裂，可以用来编辑基因组。然而，DNA断裂可能涉及几种相互竞争的修复途径之一，从而导致不同类型的基因组改变。因此，了解引入靶向DNA断裂后不同修复途径的贡献对于进一步提高核酸酶诱导的基因组修饰的安全性和效率至关重要。为了深入了解不同DNA修复途径在将核酸酶诱导的DNA断裂分解为基因组编辑结果中的作用，我们之前开发了一种基于荧光的报告系统，称为红绿灯报告系统，该系统提供了基因靶向和基因破坏的读出目标DNA双链断裂下游。在这里，我们描述了两个相关但新颖的报告器，扩展了这项技术：一个允许监测报告位点的转录活性，因此可以应用于询问活性和抑制位点的断裂分辨率；第二种除了基因靶向和基因破坏外，还能读出单链退火。应用这些报告者来评估几种常见基因编辑环境中修复途径的使用，证实了染色质状态和末端切除的起始对核酸酶诱导断裂的解决的重要性。
The creation of a DNA break at a specific locus by a designer endonuclease can be harnessed to edit a genome. However, DNA breaks may engage one of several competing repair pathways that lead to distinct types of genomic alterations. Therefore, understanding the contribution of different repair pathways following the introduction of a targeted DNA break is essential to further advance the safety and efficiency of nuclease-induced genome modification. To gain insight into the role of different DNA repair pathways in resolving nuclease-induced DNA breaks into genome editing outcomes, we previously developed a fluorescent-based reporter system, designated the Traffic Light Reporter, which provides a readout of gene targeting and gene disruption downstream of a targeted DNA double-strand break. Here we describe two related but novel reporters that extend this technology: one that allows monitoring of the transcriptional activity at the reporter locus, and thus can be applied to interrogate break resolution at active and repressed loci; and a second that reads out single-strand annealing in addition to gene targeting and gene disruption. Application of these reporters to assess repair pathway usage in several common gene editing contexts confirms the importance that chromatin status and initiation of end resection have on the resolution of nuclease-induced breaks.
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影响因子：
4.8
作者：
Bobis-Wozowicz, Sylwia;Osiak, Anna;Cathomen, Toni
通讯作者：
Cathomen, Toni
影响因子：
30.8
作者：
John S;Sabo PJ;Thurman RE;Sung MH;Biddie SC;Johnson TA;Hager GL;Stamatoyannopoulos JA
通讯作者：
Stamatoyannopoulos JA
影响因子：
46.9
作者：
Cho, Seung Woo;Kim, Sojung;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo
DOI：
10.1016/j.bbrc.2010.09.131
发表时间：
2010-11-12
影响因子：
3.1
作者：
Hsu, Chia-Chen;Li, Hsin-Pai;Hsiao, Shu-Huei
通讯作者：
Hsiao, Shu-Huei
影响因子：
46.9
作者：
Miller, Jeffrey C.;Tan, Siyuan;Rebar, Edward J.
通讯作者：
Rebar, Edward J.