title: Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation.
abstract: While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%-99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Luke A
authors.initials: LA
authors.lastname: Gilbert
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Max A
authors.initials: MA
authors.lastname: Horlbeck
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Britt
authors.initials: B
authors.lastname: Adamson
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Jacqueline E
authors.initials: JE
authors.lastname: Villalta
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Yuwen
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research, San Francisco, CA 94158, USA; Center for Systems and Synthetic Biology, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Evan H
authors.initials: EH
authors.lastname: Whitehead
authors.affiliation: Department of Biology, Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
authors.forename: Carla
authors.initials: C
authors.lastname: Guimaraes
authors.affiliation: Department of Biochemistry and Biophysics University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Barbara
authors.initials: B
authors.lastname: Panning
authors.affiliation: Department of Biology, Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
authors.forename: Hidde L
authors.initials: HL
authors.lastname: Ploegh
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Bassik
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research, San Francisco, CA 94158, USA; Center for Systems and Synthetic Biology, University of California, San Francisco, San Francisco, CA 94158, USA.
authors.forename: Lei S
authors.initials: LS
authors.lastname: Qi
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address: martin.kampmann@ucsf.edu.
authors.forename: Martin
authors.initials: M
authors.lastname: Kampmann
authors.affiliation: Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA 94158, USA; California Institute for Quantitative Biomedical Research, San Francisco, CA 94158, USA; Center for RNA Systems Biology, University of California, San Francisco, San Francisco, CA 94158, USA. Electronic address: jonathan.weissman@ucsf.edu.
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Weissman
mesh_terms: D064113:CRISPR-Cas Systems; D002460:Cell Line; D002772:Cholera Toxin / Q000378:metabolism; D004167:Diphtheria Toxin / Q000378:metabolism; D005821:Genetic Techniques; D015894:Genome, Human; D006801:Humans; D014158:Transcription, Genetic
chemical_list: D004167:Diphtheria Toxin; D002772:Cholera Toxin