RT Journal Article SR Electronic T1 An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells JF bioRxiv FD Cold Spring Harbor Laboratory SP 106112 DO 10.1101/106112 A1 Jianying Guo A1 Dacheng Ma A1 Rujin Huang A1 Jia Ming A1 Min Ye A1 Kehkooi Kee A1 Zhen Xie A1 Jie Na YR 2017 UL http://biorxiv.org/content/early/2017/02/05/106112.abstract AB 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 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 did not only promote naïve pluripotent gene expression but also enhanced cell survival and clonogenicity, and it enabled integration of hESCs 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.