Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex
Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered...
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| Format: | Article |
| Language: | en_US |
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2016
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| Online Access: | http://hdl.handle.net/1721.1/102589 https://orcid.org/0000-0003-2782-2509 https://orcid.org/0000-0001-7915-1685 https://orcid.org/0000-0001-6656-5002 https://orcid.org/0000-0002-7979-3220 |
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| author | Konermann, Silvana Brigham, Mark D. Trevino, Alexandro E. Joung, Julia Barcena, Clea Hsu, Patrick D. Habib, Naomi Gootenberg, Jonathan S. Nishimasu, Hiroshi Nureki, Osamu Zhang, Feng Abudayyeh, Omar Osama |
| author2 | Harvard University--MIT Division of Health Sciences and Technology |
| author_facet | Harvard University--MIT Division of Health Sciences and Technology Konermann, Silvana Brigham, Mark D. Trevino, Alexandro E. Joung, Julia Barcena, Clea Hsu, Patrick D. Habib, Naomi Gootenberg, Jonathan S. Nishimasu, Hiroshi Nureki, Osamu Zhang, Feng Abudayyeh, Omar Osama |
| author_sort | Konermann, Silvana |
| collection | MIT |
| description | Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology. |
| first_indexed | 2024-09-23T15:05:11Z |
| format | Article |
| id | mit-1721.1/102589 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T15:05:11Z |
| publishDate | 2016 |
| record_format | dspace |
| spelling | mit-1721.1/1025892022-09-29T12:36:25Z Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex Konermann, Silvana Brigham, Mark D. Trevino, Alexandro E. Joung, Julia Barcena, Clea Hsu, Patrick D. Habib, Naomi Gootenberg, Jonathan S. Nishimasu, Hiroshi Nureki, Osamu Zhang, Feng Abudayyeh, Omar Osama Harvard University--MIT Division of Health Sciences and Technology Massachusetts Institute of Technology. Department of Biological Engineering Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences McGovern Institute for Brain Research at MIT Konermann, Silvana Brigham, Mark D. Trevino, Alexandro E. Abudayyeh, Omar Osama Barcena, Clea Hsu, Patrick D. Gootenberg, Jonathan S. Zhang, Feng Joung, Julia Systematic interrogation of gene function requires the ability to perturb gene expression in a robust and generalizable manner. Here we describe structure-guided engineering of a CRISPR-Cas9 complex to mediate efficient transcriptional activation at endogenous genomic loci. We used these engineered Cas9 activation complexes to investigate single-guide RNA (sgRNA) targeting rules for effective transcriptional activation, to demonstrate multiplexed activation of ten genes simultaneously, and to upregulate long intergenic non-coding RNA (lincRNA) transcripts. We also synthesized a library consisting of 70,290 guides targeting all human RefSeq coding isoforms to screen for genes that, upon activation, confer resistance to a BRAF inhibitor. The top hits included genes previously shown to be able to confer resistance, and novel candidates were validated using individual sgRNA and complementary DNA overexpression. A gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and patient-derived samples. These results collectively demonstrate the potential of Cas9-based activators as a powerful genetic perturbation technology. National Science Foundation (U.S.) (Graduate Research Fellowship) United States. Dept. of Energy (Computational Science Graduate Fellowship) National Institute of Mental Health (U.S.) (DP1-MH100706) National Institute of Neurological Disorders and Stroke (U.S.) (R01-NS07312401) National Science Foundation (U.S.) W. M. Keck Foundation Kinship Foundation. Searle Scholars Program Klingenstein Foundation Vallee Foundation Simons Foundation 2016-05-23T00:51:14Z 2016-05-23T00:51:14Z 2016-05-22 Article http://purl.org/eprint/type/JournalArticle 0028-0836 1476-4687 http://hdl.handle.net/1721.1/102589 Konermann, Silvana, Mark D. Brigham, Alexandro E. Trevino, Julia Joung, Omar O. Abudayyeh, Clea Barcena, Patrick D. Hsu, et al. “Genome-Scale Transcriptional Activation by an Engineered CRISPR-Cas9 Complex.” Nature 517, no. 7536 (December 10, 2014): 583–88. https://orcid.org/0000-0003-2782-2509 https://orcid.org/0000-0001-7915-1685 https://orcid.org/0000-0001-6656-5002 https://orcid.org/0000-0002-7979-3220 en_US http://dx.doi.org/10.1038/nature14136 Nature Article is made available in accordance with the publisher's policy and may be subject to US copyright law. Please refer to the publisher's site for terms of use. application/pdf PMC |
| spellingShingle | Konermann, Silvana Brigham, Mark D. Trevino, Alexandro E. Joung, Julia Barcena, Clea Hsu, Patrick D. Habib, Naomi Gootenberg, Jonathan S. Nishimasu, Hiroshi Nureki, Osamu Zhang, Feng Abudayyeh, Omar Osama Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex |
| title | Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex |
| title_full | Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex |
| title_fullStr | Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex |
| title_full_unstemmed | Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex |
| title_short | Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex |
| title_sort | genome scale transcriptional activation by an engineered crispr cas9 complex |
| url | http://hdl.handle.net/1721.1/102589 https://orcid.org/0000-0003-2782-2509 https://orcid.org/0000-0001-7915-1685 https://orcid.org/0000-0001-6656-5002 https://orcid.org/0000-0002-7979-3220 |
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