Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation
Author(s)
Gilbert, Luke A.; Horlbeck, Max A.; Adamson, Britt; Villalta, Jacqueline E.; Chen, Yuwen; Whitehead, Evan H.; Guimaraes, Carla; Panning, Barbara; Bassik, Michael C.; Qi, Lei S.; Kampmann, Martin; Weissman, Jonathan S.; Ploegh, Hidde; ... Show more Show less
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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.
Date issued
2014-10Department
Massachusetts Institute of Technology. Department of Biology; Whitehead Institute for Biomedical ResearchJournal
Cell
Publisher
Elsevier
Citation
Gilbert, Luke A. et al. “Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation.” Cell 159.3 (2014): 647–661.
Version: Author's final manuscript
ISSN
0092-8674
1097-4172