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, Daniel E. Bauer

Research output: Contribution to journalArticlepeer-review

614 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)192-197
Number of pages6
Issue number7577
Publication statusPublished - Nov 12 2015
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • General


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