An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells. Issue 5 (23rd January 2017)

• Record Type: Journal Article
• Authors: Guo, Jianying; Ma, Dacheng; Huang, Rujin; Ming, Jia; Ye, Min; Kee, Kehkooi; Xie, Zhen; Na, Jie
• Is Part Of: Protein & cell. Volume 8:Issue 5(2017)
• Page Start: 379
• Page End: 393
• Publication Date: 2017-01-23
• Subjects: CRISPR -- transcription activation -- human pluripotent stem cells -- NANOG -- pluripotency; Proteins -- Periodicals; Cells -- Periodicals; Cytology -- Periodicals; 572.6
• DOI: 10.1007/s13238-016-0360-8
• Languages: English
• ISSNs: 1674-800X

Abstract

Abstract: 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.