CRISPR-Cas9-mediated functional dissection of 3'-UTRs.
CRISPR-Cas9-mediated functional dissection of 3'-UTRs.
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DOI：
10.1093/nar/gkx675
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发表时间：
2017-10-13
影响因子：
14.9
通讯作者：
Erle DJ
中科院分区：
文献类型：
--
作者：
Zhao W;Siegel D;Biton A;Tonqueze OL;Zaitlen N;Ahituv N;Erle DJ
报道基因分析表明3′非翻译区（3′-UTR）通过控制mRNA的稳定性和翻译来调控基因的表达。由于异源报告基因检测的内在局限性，我们试图开发一种基因编辑方法来研究3′-UTR在其天然环境中的调控活性。我们最初使用双CRISPR（成簇的，规则间隔的，短回文重复序列）-Cas9靶向来删除对应于9个趋化因子3′-UTR的DNA区域，这些区域在报告基因测定中使mRNA不稳定。靶向6个趋化因子3′-UTR增加了趋化因子mRNA水平。然而，靶向CXCL 1、CXCL 6和CXCL 8 3′-UTR出乎意料地导致mRNA显著降低。代谢标记分析表明，靶向这三个3′-UTR增加了mRNA的稳定性，正如报告基因分析所预测的那样，同时也显著降低了转录，证明了3′-UTR序列在转录调控中的意想不到的作用。我们进一步表明，CRISPR-Cas9靶向特异性3 '-UTR元件可用于调节基因表达和在天然环境中高度平行定位活性3'-UTR元件。我们的工作证明了3′-UTR序列在基因表达调控中的双重性和复杂性，为调控基因表达和3′-UTR在天然环境中的功能注释提供了有用的方法。
Many studies using reporter assays have demonstrated that 3′ untranslated regions (3′-UTRs) regulate gene expression by controlling mRNA stability and translation. Due to intrinsic limitations of heterologous reporter assays, we sought to develop a gene editing approach to investigate the regulatory activity of 3′-UTRs in their native context. We initially used dual-CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 targeting to delete DNA regions corresponding to nine chemokine 3′-UTRs that destabilized mRNA in a reporter assay. Targeting six chemokine 3′-UTRs increased chemokine mRNA levels as expected. However, targeting CXCL1, CXCL6 and CXCL8 3′-UTRs unexpectedly led to substantial mRNA decreases. Metabolic labeling assays showed that targeting these three 3′-UTRs increased mRNA stability, as predicted by the reporter assay, while also markedly decreasing transcription, demonstrating an unexpected role for 3′-UTR sequences in transcriptional regulation. We further show that CRISPR–Cas9 targeting of specific 3′-UTR elements can be used for modulating gene expression and for highly parallel localization of active 3′-UTR elements in the native context. Our work demonstrates the duality and complexity of 3′-UTR sequences in regulation of gene expression and provides a useful approach for modulating gene expression and for functional annotation of 3′-UTRs in the native context.
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影响因子：
3.7
作者：
Kramer M;Sponholz C;Slaba M;Wissuwa B;Claus RA;Menzel U;Huse K;Platzer M;Bauer M
通讯作者：
Bauer M
影响因子：
48
作者：
Diao Y;Fang R;Li B;Meng Z;Yu J;Qiu Y;Lin KC;Huang H;Liu T;Marina RJ;Jung I;Shen Y;Guan KL;Ren B
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影响因子：
7
作者：
Inoue, Fumitaka;Kircher, Martin;Shendure, Jay
通讯作者：
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影响因子：
46.9
作者：
通讯作者：
--
影响因子：
11.2
作者：
Brennan SE;Kuwano Y;Alkharouf N;Blackshear PJ;Gorospe M;Wilson GM
通讯作者：
Wilson GM