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Nature. 2015 Nov 12;527(7577):192-7. doi: 10.1038/nature15521. Epub 2015 Sep 16.

BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis.

Author information

1
Division of Hematology/Oncology, Boston Children's Hospital, Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Stem Cell Institute, Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA.
2
Department of Biostatistics and Computational Biology, Dana-Farber Cancer Institute and Harvard School of Public Health, Boston, Massachusetts 02115, USA.
3
Broad Institute of MIT and Harvard, McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences and Department of Biological Engineering, MIT, Cambridge, Massachusetts 02142, USA.
4
Cell Engineering Division, RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan.
5
Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan.
6
Howard Hughes Medical Institute, Boston, Massachusetts 02115, USA.
7
Division of Hematology, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.

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.

PMID:
26375006
PMCID:
PMC4644101
DOI:
10.1038/nature15521
[Indexed for MEDLINE]
Free PMC Article

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