An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells.
An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells.
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DOI：
10.1007/s13238-016-0360-8
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发表时间：
2017-05
期刊：
影响因子：
21.1
通讯作者：
Na J
中科院分区：
文献类型：
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作者：
Guo J;Ma D;Huang R;Ming J;Ye M;Kee K;Xie Z;Na J
人多能干细胞（Human pluripotent stem cells, hPSCs）是研究人类早期发育、模拟人类疾病和开发细胞替代疗法的重要系统。然而，人类造血干细胞的遗传操作具有挑战性，迫切需要一种以可控方式同时激活多个基因组位点的方法。在这里，我们构建了一个CRISPR-ON系统来有效上调人造血干细胞中的内源性基因。将多西环素（Dox）诱导的dCas9-VP64-p65-Rta （dCas9-VPR）转录激活子和Tet反转录激活子（rtTA）表达盒敲入AAVS1位点的两个等位基因中，生成iVPR hESC细胞系。我们发现dCas9-VPR水平可以通过添加和退出Dox精确和可逆地控制。通过转染靶向NANOG启动子的多重gRNA质粒并诱导Dox，我们能够从其内源位点控制NANOG基因的表达。有趣的是，NANOG水平的升高促进了naïve多能基因的表达，提高了细胞存活率和克隆原性，并使hESCs能够与体外小鼠囊胚的内细胞团（ICM）结合。因此，iVPR细胞为基因功能研究和高通量筛选提供了方便的平台。本文的在线版本（doi:10.1007/s13238-016-0360-8）包含补充材料，仅供授权用户使用。
Human pluripotent stem cells (hPSCs) are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR-ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline (Dox) inducible dCas9-VP64-p65-Rta (dCas9-VPR) transcription activator and a reverse Tet transactivator (rtTA) expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG promoter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naïve pluripotent gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass (ICM) of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs. The online version of this article (doi:10.1007/s13238-016-0360-8) contains supplementary material, which is available to authorized users.