BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis

Matthew C. Canver, Elenoe C. Smith, Falak Sher, Luca Pinello, Neville E. Sanjana, Ophir Shalem, Diane D. Chen, Patrick G. Schupp, Divya S. Vinjamur, Sara P. Garcia, Sidinh Luc, Ryo Kurita, Yukio Nakamura, Yuko Fujiwara, Takahiro Maeda, Guo-Cheng Yuan, Feng Zhang (), Stuart H. Orkin () and Daniel E. Bauer ()
Additional contact information
Matthew C. Canver: Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School
Elenoe C. Smith: Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School
Falak Sher: Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School
Luca Pinello: Dana-Farber Cancer Institute and Harvard School of Public Health
Neville E. Sanjana: Broad Institute of MIT and Harvard, McGovern Institute for Brain Research, MIT
Ophir Shalem: Broad Institute of MIT and Harvard, McGovern Institute for Brain Research, MIT
Diane D. Chen: Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School
Patrick G. Schupp: Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School
Divya S. Vinjamur: Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School
Sara P. Garcia: Dana-Farber Cancer Institute and Harvard School of Public Health
Sidinh Luc: Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School
Ryo Kurita: RIKEN BioResource Center
Yukio Nakamura: RIKEN BioResource Center
Yuko Fujiwara: Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School
Takahiro Maeda: Brigham and Women’s Hospital, Harvard Medical School
Guo-Cheng Yuan: Dana-Farber Cancer Institute and Harvard School of Public Health
Feng Zhang: Broad Institute of MIT and Harvard, McGovern Institute for Brain Research, MIT
Stuart H. Orkin: Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School
Daniel E. Bauer: Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Harvard Medical School

Nature, 2015, vol. 527, issue 7577, 192-197

Abstract: 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.

Date: 2015
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DOI: 10.1038/nature15521

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