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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Veröffentlicht in:	Nature (London) Jg. 527; H. 7577; S. 192 - 197
Hauptverfasser:	Canver, Matthew C., Smith, Elenoe C., Sher, Falak, Pinello, Luca, Sanjana, Neville E., Shalem, Ophir, Chen, Diane D., Schupp, Patrick G., Vinjamur, Divya S., Garcia, Sara P., Luc, Sidinh, Kurita, Ryo, Nakamura, Yukio, Fujiwara, Yuko, Maeda, Takahiro, Yuan, Guo-Cheng, Zhang, Feng, Orkin, Stuart H., Bauer, Daniel E.
Format:	Journal Article
Sprache:	Englisch
Veröffentlicht:	London Nature Publishing Group UK 12.11.2015 Nature Publishing Group
Schlagworte:	13/100 13/109 13/31 38/22 38/70 38/77 38/88 45/23 49/47 631/1647/1511 631/208/200 631/337/572/2102 64/60 692/308/2056 Animals Base Sequence Carrier Proteins - genetics Cells, Cultured Clustered Regularly Interspaced Short Palindromic Repeats - genetics CRISPR-Associated Proteins - metabolism CRISPR-Cas Systems - genetics DNA methylation DNA-Binding Proteins Enhancer Elements, Genetic - genetics Erythroblasts - metabolism Fetal Hemoglobin - genetics Gene expression Genetic diversity Genetic Engineering Genome - genetics Genomes Genomics Humanities and Social Sciences Humans Methods Mice Molecular Sequence Data multidisciplinary Mutagenesis Mutagenesis - genetics Nuclear Proteins - genetics Organ Specificity Physiological aspects Repressor Proteins Reproducibility of Results RNA, Guide, CRISPR-Cas Systems - genetics Science Species Specificity Studies
ISSN:	0028-0836, 1476-4687, 1476-4687
Online-Zugang:	Volltext
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Zusammenfassung:	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. A CRISPR-Cas9 approach is used to perform saturating mutagenesis of the human and mouse BCL11A enhancers, producing a map that reveals critical regions and specific vulnerabilities; BCL11A enhancer disruption is validated by CRISPR-Cas9 as a therapeutic strategy for inducing fetal haemoglobin by applying it in both mice and primary human erythroblast cells. BCL11A enhancer disruption analysed BCL11A is a transcriptional repressor that inhibits expression of fetal globin genes in adults, and is a potential therapeutic target for the treatment of β-globinopathies such as β-thalassemia and sickle cell disease. The enhancer of BCL11A is subject to common genetic variation associated with fetal hemoglobin level. Here, Daniel Bauer and colleagues use a CRISPR–Cas9 approach to perform saturation mutagenesis of the human and mouse BCL11A enhancers, producing a map that reveals critical regions and specific vulnerabilities. They validate BCL11A enhancer disruption by CRISPR–Cas9 as a therapeutic strategy for inducing fetal haemoglobin by applying it in both mice and primary human erythroblast cells.
Bibliographie:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 14 content type line 23 These authors contributed equally to this work. These authors jointly supervised this work.
ISSN:	0028-0836 1476-4687 1476-4687
DOI:	10.1038/nature15521