title: Optimized inducible shRNA and CRISPR/Cas9 platforms for in vitro studies of human development using hPSCs.
abstract: Inducible loss of gene function experiments are necessary to uncover mechanisms underlying development, physiology and disease. However, current methods are complex, lack robustness and do not work in multiple cell types. Here we address these limitations by developing single-step optimized inducible gene knockdown or knockout (sOPTiKD or sOPTiKO) platforms. These are based on genetic engineering of human genomic safe harbors combined with an improved tetracycline-inducible system and CRISPR/Cas9 technology. We exemplify the efficacy of these methods in human pluripotent stem cells (hPSCs), and show that generation of sOPTiKD/KO hPSCs is simple, rapid and allows tightly controlled individual or multiplexed gene knockdown or knockout in hPSCs and in a wide variety of differentiated cells. Finally, we illustrate the general applicability of this approach by investigating the function of transcription factors (OCT4 and T), cell cycle regulators (cyclin D family members) and epigenetic modifiers (DPY30). Overall, sOPTiKD and sOPTiKO provide a unique opportunity for functional analyses in multiple cell types relevant for the study of human development.
authors.affiliation: Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, CB2 0SZ, UK abertero@uw.edu lv225@cam.ac.uk.|Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Bertero
authors.affiliation: Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, CB2 0SZ, UK.|Department of Clinical Neuroscience, University of Cambridge, Cambridge, CB2 0QQ, UK.
authors.forename: Matthias
authors.initials: M
authors.lastname: Pawlowski
authors.affiliation: Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, CB2 0SZ, UK.|Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK.
authors.forename: Daniel
authors.initials: D
authors.lastname: Ortmann
authors.affiliation: Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, CB2 0SZ, UK.|Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Snijders
authors.affiliation: Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, CB2 0SZ, UK.|Division of Cardiovascular Medicine, University of Cambridge, Cambridge, UK.
authors.forename: Loukia
authors.initials: L
authors.lastname: Yiangou
authors.affiliation: Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, CB2 0SZ, UK.|Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK.
authors.forename: Miguel
authors.initials: M
authors.lastname: Cardoso de Brito
authors.affiliation: Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, CB2 0SZ, UK.|Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Brown
authors.affiliation: Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, CB2 0SZ, UK.|Division of Cardiovascular Medicine, University of Cambridge, Cambridge, UK.
authors.forename: William G
authors.initials: WG
authors.lastname: Bernard
authors.affiliation: Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, CB2 0SZ, UK.|Division of Cardiovascular Medicine, University of Cambridge, Cambridge, UK.
authors.forename: James D
authors.initials: JD
authors.lastname: Cooper
authors.affiliation: Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, CB2 0SZ, UK.|Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK.
authors.forename: Elisa
authors.initials: E
authors.lastname: Giacomelli
authors.affiliation: Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, CB2 0SZ, UK.|Division of Cardiovascular Medicine, University of Cambridge, Cambridge, UK.
authors.forename: Laure
authors.initials: L
authors.lastname: Gambardella
authors.affiliation: Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, CB2 0SZ, UK.|Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK.
authors.forename: Nicholas R F
authors.initials: NR
authors.lastname: Hannan
authors.affiliation: Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, CB2 0SZ, UK.|Division of Cardiovascular Medicine, University of Cambridge, Cambridge, UK.
authors.forename: Dharini
authors.initials: D
authors.lastname: Iyer
authors.affiliation: Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, CB2 0SZ, UK.|Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK.
authors.forename: Fotios
authors.initials: F
authors.lastname: Sampaziotis
authors.affiliation: Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, CB2 0SZ, UK.|Division of Cardiovascular Medicine, University of Cambridge, Cambridge, UK.
authors.forename: Felipe
authors.initials: F
authors.lastname: Serrano
authors.affiliation: Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, CB2 0SZ, UK.|Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK.
authors.forename: Mariëlle C F
authors.initials: MC
authors.lastname: Zonneveld
authors.affiliation: Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, CB2 0SZ, UK.|Division of Cardiovascular Medicine, University of Cambridge, Cambridge, UK.
authors.forename: Sanjay
authors.initials: S
authors.lastname: Sinha
authors.affiliation: Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, CB2 0SZ, UK.|Department of Clinical Neuroscience, University of Cambridge, Cambridge, CB2 0QQ, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Kotter
authors.affiliation: Wellcome Trust-MRC Stem Cell Institute, Anne McLaren Laboratory, University of Cambridge, Cambridge, CB2 0SZ, UK abertero@uw.edu lv225@cam.ac.uk.|Department of Surgery, University of Cambridge, Cambridge, CB2 0QQ, UK.|Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, UK.
authors.forename: Ludovic
authors.identifier: 0000-0002-3848-2602
authors.initials: L
authors.lastname: Vallier
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D056741:Cyclin D / Q000235:genetics*; D053595:Embryonic Stem Cells / Q000166:cytology; D005326:Fetal Proteins / Q000235:genetics*; D055786:Gene Knockout Techniques; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology; D009687:Nuclear Proteins / Q000235:genetics*; D050814:Octamer Transcription Factor-3 / Q000235:genetics*; D020825:T-Box Domain Proteins / Q000235:genetics*; D014157:Transcription Factors; D000098908:Brachyury Protein
chemical_list: D056741:Cyclin D; D005326:Fetal Proteins; D009687:Nuclear Proteins; D050814:Octamer Transcription Factor-3; D020825:T-Box Domain Proteins; D014157:Transcription Factors; D000098908:Brachyury Protein; C507432:DPY30 protein, human; C494173:POU5F1 protein, human
keywords: CRISPR/Cas9; DPY30; Human pluripotent stem cells; OCT4; POU5F1; T, brachyury; shRNA