BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis
Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of hum...
| Main Authors: | , , , , , , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | en_US |
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Nature Publishing Group
2016
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| Online Access: | http://hdl.handle.net/1721.1/102584 https://orcid.org/0000-0003-2782-2509 |
| _version_ | 1811072737467170816 |
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| author | Canver, Matthew C. Smith, Elenoe C. Sher, Falak Pinello, Luca Shalem, Ophir Chen, Diane D. Schupp, Patrick G. Vinjamur, Divya S. Garcia, Sara P. Luc, Sidinh Kurita, Ryo Nakamura, Yukio Fujiwara, Yuko Maeda, Takahiro Yuan, Guo-Cheng Zhang, Feng Orkin, Stuart H. Bauer, Daniel E. Sanjana, Neville |
| author2 | Massachusetts Institute of Technology. Department of Biological Engineering |
| author_facet | Massachusetts Institute of Technology. Department of Biological Engineering Canver, Matthew C. Smith, Elenoe C. Sher, Falak Pinello, Luca Shalem, Ophir Chen, Diane D. Schupp, Patrick G. Vinjamur, Divya S. Garcia, Sara P. Luc, Sidinh Kurita, Ryo Nakamura, Yukio Fujiwara, Yuko Maeda, Takahiro Yuan, Guo-Cheng Zhang, Feng Orkin, Stuart H. Bauer, Daniel E. Sanjana, Neville |
| author_sort | Canver, Matthew C. |
| collection | MIT |
| description | Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements. |
| first_indexed | 2024-09-23T09:11:29Z |
| format | Article |
| id | mit-1721.1/102584 |
| institution | Massachusetts Institute of Technology |
| language | en_US |
| last_indexed | 2024-09-23T09:11:29Z |
| publishDate | 2016 |
| publisher | Nature Publishing Group |
| record_format | dspace |
| spelling | mit-1721.1/1025842022-09-26T11:01:04Z BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis Canver, Matthew C. Smith, Elenoe C. Sher, Falak Pinello, Luca Shalem, Ophir Chen, Diane D. Schupp, Patrick G. Vinjamur, Divya S. Garcia, Sara P. Luc, Sidinh Kurita, Ryo Nakamura, Yukio Fujiwara, Yuko Maeda, Takahiro Yuan, Guo-Cheng Zhang, Feng Orkin, Stuart H. Bauer, Daniel E. Sanjana, Neville 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 Sanjana, Neville Shalem, Ophir Zhang, Feng Enhancers, critical determinants of cellular identity, are commonly recognized by correlative chromatin marks and gain-of-function potential, although only loss-of-function studies can demonstrate their requirement in the native genomic context. Previously, we identified an erythroid enhancer of human BCL11A, subject to common genetic variation associated with the fetal haemoglobin level, the mouse orthologue of which is necessary for erythroid BCL11A expression. Here we develop pooled clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 guide RNA libraries to perform in situ saturating mutagenesis of the human and mouse enhancers. This approach reveals critical minimal features and discrete vulnerabilities of these enhancers. Despite conserved function of the composite enhancers, their architecture diverges. The crucial human sequences appear to be primate-specific. Through editing of primary human progenitors and mouse transgenesis, we validate the BCL11A erythroid enhancer as a target for fetal haemoglobin reinduction. The detailed enhancer map will inform therapeutic genome editing, and the screening approach described here is generally applicable to functional interrogation of non-coding genomic elements. Massachusetts Institute of Technology. Simons Center for the Social Brain National Human Genome Research Institute (U.S.) (Award K99-HG008171) Klarman Family Foundation (Fellowship) National Institute of Mental Health (U.S.) (K99-HG008171) National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (5R01-DK097768) National Science Foundation (U.S.) (Waterman Award) W. M. Keck Foundation McKnight Foundation Damon Runyon Cancer Research Foundation Kinship Foundation. Searle Scholars Program Merkin Foundation Vallee Foundation Simons Foundation 2016-05-22T23:55:16Z 2016-05-22T23:55:16Z 2015-09 2015-04 Article http://purl.org/eprint/type/JournalArticle 0028-0836 1476-4687 http://hdl.handle.net/1721.1/102584 Canver, Matthew C., Elenoe C. Smith, Falak Sher, Luca Pinello, Neville E. Sanjana, Ophir Shalem, Diane D. Chen, et al. “BCL11A Enhancer Dissection by Cas9-Mediated in Situ Saturating Mutagenesis.” Nature 527, no. 7577 (September 16, 2015): 192–197. https://orcid.org/0000-0003-2782-2509 en_US http://dx.doi.org/10.1038/nature15521 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 Nature Publishing Group PMC |
| spellingShingle | Canver, Matthew C. Smith, Elenoe C. Sher, Falak Pinello, Luca Shalem, Ophir Chen, Diane D. Schupp, Patrick G. Vinjamur, Divya S. Garcia, Sara P. Luc, Sidinh Kurita, Ryo Nakamura, Yukio Fujiwara, Yuko Maeda, Takahiro Yuan, Guo-Cheng Zhang, Feng Orkin, Stuart H. Bauer, Daniel E. Sanjana, Neville BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis |
| title | BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis |
| title_full | BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis |
| title_fullStr | BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis |
| title_full_unstemmed | BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis |
| title_short | BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis |
| title_sort | bcl11a enhancer dissection by cas9 mediated in situ saturating mutagenesis |
| url | http://hdl.handle.net/1721.1/102584 https://orcid.org/0000-0003-2782-2509 |
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