Conditionally Stabilized dCas9 Activator for Controlling Gene Expression in Human Cell Reprogramming and Differentiation.
CRISPR/Cas9 protein fused to transactivation domains can be used to control gene expression in human cells. In this study, we demonstrate that a dCas9 fusion with repeats of VP16 activator domains can efficiently activate human genes involved in pluripotency in various cell types. This activator in combination with guide RNAs targeted to the OCT4 promoter can be used to completely replace transgenic OCT4 in human cell reprogramming. Furthermore, we generated a chemically controllable dCas9 activator version by fusion with the dihydrofolate reductase (DHFR) destabilization domain. Finally, we show that the destabilized dCas9 activator can be used to control human pluripotent stem cell differentiation into endodermal lineages.
Research Programs Unit, Molecular Neurology, Biomedicum Stem Cell Center, University of Helsinki, Haartmaninkatu 8, 00290 Helsinki, Finland.
Diego
D
Balboa
Research Programs Unit, Molecular Neurology, Biomedicum Stem Cell Center, University of Helsinki, Haartmaninkatu 8, 00290 Helsinki, Finland.
Jere
J
Weltner
Research Programs Unit, Molecular Neurology, Biomedicum Stem Cell Center, University of Helsinki, Haartmaninkatu 8, 00290 Helsinki, Finland.
Solja
S
Eurola
Research Programs Unit, Molecular Neurology, Biomedicum Stem Cell Center, University of Helsinki, Haartmaninkatu 8, 00290 Helsinki, Finland.
Ras
R
Trokovic
Research Programs Unit, Molecular Neurology, Biomedicum Stem Cell Center, University of Helsinki, Haartmaninkatu 8, 00290 Helsinki, Finland; Clinical Genetics, HUSLAB, Helsinki University Central Hospital, Haartmaninkatu 4, 00290 Helsinki, Finland.
Kirmo
K
Wartiovaara
Research Programs Unit, Molecular Neurology, Biomedicum Stem Cell Center, University of Helsinki, Haartmaninkatu 8, 00290 Helsinki, Finland; Children's Hospital, University of Helsinki and Helsinki University Central Hospital, Stenbäckinkatu 11, 00290 Helsinki, Finland. Electronic address: timo.otonkoski@helsinki.fi.
Timo
T
Otonkoski
D000293:Adolescent; D000368:Aged; D064113:CRISPR-Cas Systems; D002454:Cell Differentiation; D065150:Cellular Reprogramming; D000067470:Cellular Reprogramming Techniques; D005260:Female; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D008297:Male