CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs

Mohammad A Mandegar; Nathaniel Huebsch; Ekaterina B Frolov; Edward Shin; Annie Truong; Michael P Olvera; Amanda H Chan; Yuichiro Miyaoka; Kristin Holmes; C Ian Spencer; Luke M Judge; David E Gordon; Tilde V Eskildsen; Jacqueline E Villalta; Max A Horlbeck; Luke A Gilbert; Nevan J Krogan; Søren P Sheikh; Jonathan S Weissman; Lei S Qi; Po-Lin So; Bruce R Conklin

doi:10.1016/j.stem.2016.01.022

CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs

Cell Stem Cell. 2016 Apr 7;18(4):541-53. doi: 10.1016/j.stem.2016.01.022. Epub 2016 Mar 10.

Authors

Mohammad A Mandegar¹, Nathaniel Huebsch², Ekaterina B Frolov³, Edward Shin³, Annie Truong³, Michael P Olvera³, Amanda H Chan³, Yuichiro Miyaoka³, Kristin Holmes³, C Ian Spencer³, Luke M Judge², David E Gordon⁴, Tilde V Eskildsen⁵, Jacqueline E Villalta⁶, Max A Horlbeck⁶, Luke A Gilbert⁶, Nevan J Krogan⁴, Søren P Sheikh⁵, Jonathan S Weissman⁶, Lei S Qi⁷, Po-Lin So³, Bruce R Conklin⁸

Affiliations

¹ Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA. Electronic address: mo.mandegar@gladstone.ucsf.edu.
² Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA; Department of Pediatrics, University of California, San Francisco, San Francisco, CA 94158, USA.
³ Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA.
⁴ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biosciences, QB3, University of California, San Francisco, San Francisco, CA 94158, USA; Gladstone Institute of Virology and Immunology, San Francisco, CA 94158, USA.
⁵ Department of Cardiovascular and Renal Research, University of Southern Denmark, 5000 Odense C, Denmark; Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, 5000 Odense C, Denmark.
⁶ Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biosciences, QB3, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA.
⁷ Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
⁸ Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA; Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biosciences, QB3, University of California, San Francisco, San Francisco, CA 94158, USA; Department of Medicine and Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address: bconklin@gladstone.ucsf.edu.

Abstract

Developing technologies for efficient and scalable disruption of gene expression will provide powerful tools for studying gene function, developmental pathways, and disease mechanisms. Here, we develop clustered regularly interspaced short palindromic repeat interference (CRISPRi) to repress gene expression in human induced pluripotent stem cells (iPSCs). CRISPRi, in which a doxycycline-inducible deactivated Cas9 is fused to a KRAB repression domain, can specifically and reversibly inhibit gene expression in iPSCs and iPSC-derived cardiac progenitors, cardiomyocytes, and T lymphocytes. This gene repression system is tunable and has the potential to silence single alleles. Compared with CRISPR nuclease (CRISPRn), CRISPRi gene repression is more efficient and homogenous across cell populations. The CRISPRi system in iPSCs provides a powerful platform to perform genome-scale screens in a wide range of iPSC-derived cell types, dissect developmental pathways, and model disease.

CRISPR Interference Efficiently Induces Specific and Reversible Gene Silencing in Human iPSCs

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