BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis
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Zhang_BCL11A enhancer.pdf
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Author(s)
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
Date Issued
September 2015
Journal
Nature
Publisher
Nature Publishing Group
Citation
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.
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Author's final manuscript
Abstract
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.
MIT Department
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
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DOI of Published Version
https://doi.org/10.1038/nature15521
