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...

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Main Authors: Canver, Matthew C. (Author), Smith, Elenoe C. (Author), Sher, Falak (Author), Pinello, Luca (Author), Shalem, Ophir (Contributor), Chen, Diane D. (Author), Schupp, Patrick G. (Author), Vinjamur, Divya S. (Author), Garcia, Sara P. (Author), Luc, Sidinh (Author), Kurita, Ryo (Author), Nakamura, Yukio (Author), Fujiwara, Yuko (Author), Maeda, Takahiro (Author), Yuan, Guo-Cheng (Author), Zhang, Feng (Contributor), Orkin, Stuart H. (Author), Bauer, Daniel E. (Author), Sanjana, Neville (Contributor)
Other Authors: Massachusetts Institute of Technology. Department of Biological Engineering (Contributor), Massachusetts Institute of Technology. Department of Brain and Cognitive Sciences (Contributor), McGovern Institute for Brain Research at MIT (Contributor)
Format: Article
Language:English
Published: Nature Publishing Group, 2016-05-22T23:55:16Z.
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Summary: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