BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • CRISPR-Associated Proteins / metabolism*
  • CRISPR-Cas Systems / genetics
  • Carrier Proteins / genetics*
  • Cells, Cultured
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • DNA-Binding Proteins
  • Enhancer Elements, Genetic / genetics*
  • Erythroblasts / metabolism
  • Fetal Hemoglobin / genetics
  • Genetic Engineering*
  • Genome / genetics
  • Humans
  • Mice
  • Molecular Sequence Data
  • Mutagenesis / genetics*
  • Nuclear Proteins / genetics*
  • Organ Specificity
  • RNA, Guide / genetics
  • Repressor Proteins
  • Reproducibility of Results
  • Species Specificity

Substances

  • BCL11A protein, human
  • Bcl11a protein, mouse
  • CRISPR-Associated Proteins
  • Carrier Proteins
  • DNA-Binding Proteins
  • Nuclear Proteins
  • RNA, Guide
  • Repressor Proteins
  • Fetal Hemoglobin