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

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 i...

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Bibliographic Details
Published in:Protein & cell 2017-05, Vol.8 (5), p.379-393
Main Authors: Guo, Jianying, Ma, Dacheng, Huang, Rujin, Ming, Jia, Ye, Min, Kee, Kehkooi, Xie, Zhen, Na, Jie
Format: Article
Language:English
Subjects:
Animals
Biochemistry
Biomedical and Life Sciences
Blastocysts
Cell Biology
Cell Line
Cell survival
Clustered Regularly Interspaced Short Palindromic Repeats
CRISPR
Developmental Biology
Doxycycline
Doxycycline - pharmacology
Gene expression
Gene Expression Regulation - drug effects
gRNA
Human Embryonic Stem Cells - metabolism
Human Genetics
human pluripotent stem cells
Humans
Life Sciences
Mice
NANOG
Nanog Homeobox Protein - biosynthesis
Nanog Homeobox Protein - genetics
Pluripotency
Pluripotent Stem Cells - metabolism
Protein Science
Research Article
Stem cell transplantation
Stem Cells
transcription activation
Transfection
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Summary: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 dCasg-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 pro- moter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naive pluripotsnt 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.
ISSN:1674-800X
1674-8018
DOI:10.1007/s13238-016-0360-8