An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells

被引：40

作者：

Guo, Jianying ^{[1
]}

Ma, Dacheng ^{[2
,3
]}

Huang, Rujin ^{[1
]}

Ming, Jia ^{[1
]}

Ye, Min ^{[1
]}

Kee, Kehkooi ^{[1
]}

Xie, Zhen ^{[2
,3
]}

Na, Jie ^{[1
]}

机构：

[1] Tsinghua Univ, Ctr Stem Cell Biol, Sch Med, Dept Basic Med Sci, Beijing 100084, Peoples R China

[2] Tsinghua Univ, Ctr Synthet & Syst Biol, Dept Automat, TNLIST,MOE Key Lab Bioinformat, Beijing 100084, Peoples R China

[3] Tsinghua Univ, Ctr Synthet & Syst Biol, Dept Automat, TNLIST,Bioinformat Div, Beijing 100084, Peoples R China

来源：

PROTEIN & CELL | 2017年 / 8卷 / 05期

基金：

中国国家自然科学基金;

关键词：

CRISPR; transcription activation; human pluripotent stem cells; NANOG; pluripotency;

D O I：

10.1007/s13238-016-0360-8

中图分类号：

Q2 [细胞生物学];

学科分类号：

071009 ; 090102 ;

摘要：

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 na < ve 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.

引用

页码：379 / 393

页数：15

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