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

Nature
Authors
Keywords
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.

Year of Publication
2015
Journal
Nature
Volume
527
Issue
7577
Pages
192-7
Date Published
2015 Nov 12
ISSN
1476-4687
URL
DOI
10.1038/nature15521
PubMed ID
26375006
PubMed Central ID
PMC4644101
Links
Grant list
R01 HL119099 / HL / NHLBI NIH HHS / United States
K08 DK093705 / DK / NIDDK NIH HHS / United States
R01 HL032259 / HL / NHLBI NIH HHS / United States
R01 MH110049 / MH / NIMH NIH HHS / United States
K99 HG008171 / HG / NHGRI NIH HHS / United States
R01HL119099 / HL / NHLBI NIH HHS / United States
5DP1-MH100706 / DP / NCCDPHP CDC HHS / United States
P30 DK049216 / DK / NIDDK NIH HHS / United States
K99 HG008399 / HG / NHGRI NIH HHS / United States
P01HL032262 / HL / NHLBI NIH HHS / United States
P01 HL032262 / HL / NHLBI NIH HHS / United States
K99-HG008171 / HG / NHGRI NIH HHS / United States
R01 HG005085 / HG / NHGRI NIH HHS / United States
Howard Hughes Medical Institute / United States
DP1 MH100706 / MH / NIMH NIH HHS / United States
R01 A1084905 / PHS HHS / United States
R01HG005085 / HG / NHGRI NIH HHS / United States
R01 DK097768 / DK / NIDDK NIH HHS / United States
5R01-DK097768 / DK / NIDDK NIH HHS / United States
K08DK093705 / DK / NIDDK NIH HHS / United States
F30DK103359-01A1 / DK / NIDDK NIH HHS / United States
P30DK049216 / DK / NIDDK NIH HHS / United States
K99HG008399 / HG / NHGRI NIH HHS / United States
F30 DK103359 / DK / NIDDK NIH HHS / United States