BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis

By Matthew C. Canver, Elenoe C Smith, F. Sher, Luca Pinello, Neville E. Sanjana, Ophir Shalem, Diane D. Chen, Patrick G. Schupp, Divya S. Vinjamur, Sara P. Garcia and 9 more. Nature, 2015

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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.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/f9fbbe9ea958020ec4cdef7ffee208cba2698b02

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Citations in the corpus

Activity-by-Contact model of enhancer-promoter regulation from thousands of CRISPR perturbations (2019, methodology): “The ABC model, CRISPRi-FlowFISH, and other approaches to map enhancer function 9,13,36–39 provide a means to test this principle and to further refine our understanding of noncoding regulatory elements by mapping and modeling promoter-promoter regulation, functions of CTCF sites, and combinatorial…”
Gene therapy comes of age (2018, positive): “NHEJ-mediated disruption of the erythroid-specific enhancer element responsible for expression of the BCL11A gene results in high-level HbF in animal models and in human sickle cell erythroid cells in vitro (122).”