Targeting non-coding RNAs with the CRISPR/Cas9 system in human cell lines.
Targeting non-coding RNAs with the CRISPR/Cas9 system in human cell lines.
复制标题
DOI：
10.1093/nar/gku1198
复制
发表时间：
2015-02-18
影响因子：
14.9
通讯作者：
Mo YY
中科院分区：
文献类型：
--
作者：
Ho TT;Zhou N;Huang J;Koirala P;Xu M;Fung R;Wu F;Mo YY
CRISPR/Cas最近被证明是多种生物体中强大的基因组编辑工具。然而，这些研究主要集中在蛋白质编码基因。本研究旨在确定该技术是否可以应用于非编码基因。敲除非编码基因的挑战之一是由标准CRISPR/Cas系统产生的小缺失或插入可能不一定导致给定非编码基因的功能丧失，因为缺乏开放阅读框，特别是在多倍性人细胞系中。为了克服这一挑战，我们采用了一种选择系统，允许标记基因通过同源重组（HR）整合到基因组中。此外，我们构建了一个双向导RNA载体，可以在指定的位点同时进行两次切割，从而可以删除一个大片段。通过这些方法，我们能够在各种人类细胞系中成功地产生miR-21、miR-29 a、lncRNA-21 A、UCA 1和AK 023948的敲除。最后，我们表明HR介导的靶向效率可以通过抑制非同源末端连接途径进一步提高。总之，这些结果证明了在人类细胞系中通过CRISPR/Cas系统敲除非编码基因的可行性。
The CRISPR/Cas has been recently shown to be a powerful genome-editing tool in a variety of organisms. However, these studies are mainly focused on protein-coding genes. The present study aims to determine whether this technology can be applied to non-coding genes. One of the challenges for knockout of non-coding genes is that a small deletion or insertion generated by the standard CRISPR/Cas system may not necessarily lead to functional loss of a given non-coding gene because of lacking an open reading frame, especially in polyploidy human cell lines. To overcome this challenge, we adopt a selection system that allows for marker genes to integrate into the genome through homologous recombination (HR). Moreover, we construct a dual guide RNA vector that can make two cuts simultaneously at designated sites such that a large fragment can be deleted. With these approaches, we are able to successfully generate knockouts for miR-21, miR-29a, lncRNA-21A, UCA1 and AK023948 in various human cell lines. Finally, we show that the HR-mediated targeting efficiency can be further improved by suppression of the non-homologous end joining pathway. Together, these results demonstrate the feasibility of knockout for non-coding genes by the CRISPR/Cas system in human cell lines.
登录
查看更多内容
DOI：
10.1084/jem.20090831
发表时间：
2010-03-15
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Han YC;Park CY;Bhagat G;Zhang J;Wang Y;Fan JB;Liu M;Zou Y;Weissman IL;Gu H
通讯作者：
Gu H
影响因子：
4.8
作者：
Muylaert, Isabella;Elias, Per
通讯作者：
Elias, Per
新的小核RNA基因样转录单元作为调节转录的来源。
影响因子：
4.5
作者：
Pagano A;Castelnuovo M;Tortelli F;Ferrari R;Dieci G;Cancedda R
通讯作者：
Cancedda R
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
46.9
作者：
Cho, Seung Woo;Kim, Sojung;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo