Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation
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...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | en_US |
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Elsevier
2017
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| Online Access: | http://hdl.handle.net/1721.1/108008 https://orcid.org/0000-0002-1090-6071 |
| _version_ | 1811081896012021760 |
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| author | 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 |
| author2 | Massachusetts Institute of Technology. Department of Biology |
| author_facet | Massachusetts Institute of Technology. Department of Biology 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 |
| author_sort | Gilbert, Luke A. |
| collection | MIT |
| description | 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. |
| first_indexed | 2024-09-23T11:54:08Z |
| format | Article |
| id | mit-1721.1/108008 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T11:54:08Z |
| publishDate | 2017 |
| publisher | Elsevier |
| record_format | dspace |
| spelling | mit-1721.1/1080082022-09-27T22:43:32Z Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation 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 Massachusetts Institute of Technology. Department of Biology Whitehead Institute for Biomedical Research Ploegh, Hidde 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. 2017-04-10T15:27:17Z 2017-04-10T15:27:17Z 2014-10 2014-09 Article http://purl.org/eprint/type/JournalArticle 0092-8674 1097-4172 http://hdl.handle.net/1721.1/108008 Gilbert, Luke A. et al. “Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation.” Cell 159.3 (2014): 647–661. https://orcid.org/0000-0002-1090-6071 en_US http://dx.doi.org/10.1016/j.cell.2014.09.029 Cell Creative Commons Attribution-NonCommercial-NoDerivs License http://creativecommons.org/licenses/by-nc-nd/4.0/ application/pdf Elsevier PMC |
| spellingShingle | 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 Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation |
| title | Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation |
| title_full | Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation |
| title_fullStr | Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation |
| title_full_unstemmed | Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation |
| title_short | Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation |
| title_sort | genome scale crispr mediated control of gene repression and activation |
| url | http://hdl.handle.net/1721.1/108008 https://orcid.org/0000-0002-1090-6071 |
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