Extended Data Fig. 12: MCC profiles at the main α-globin enhancer show the specific loss of contacts when an NF-E2 site is deleted.
a, Genome editing was used to make a small 2–4-bp deletion, determined by Illumina sequencing, in an NF-E2 consensus motif in the main enhancer at the α-globin locus (termed the R2 enhancer). b, ChIP–qPCR showing loss of NF-E2 binding at the R2 enhancer compared with enrichment at the adjacent R1 enhancer. Wild type, n = 6; NF-E2 edited, n = 8; data mean ± s.d., two-tailed Mann–Whitney U-test. Note that complete abrogation of NF-E2 binding would not be expected because there are two NF-E2-binding sites in the enhancer, which are separated by 26 bp. c, Deletion of the NF-E2-binding site at the R2 enhancer results in a significant reduction in expression of the α-globin genes in erythroid cells from normal donors that have undergone genome editing at the R2 NF-E2 site (editing efficiencies in excess of 90% with Cas9 ribonuclear protein, data not shown). n = 7, data are mean ± s.d., two-tailed Mann–Whitney U-test. d, e, MCC profiles of the promotors of HBA1 and HBA2 in human HUDEP-2 cells (d), showing that the interactions with the main enhancer (R2) reduce very specifically (e) at the site of an engineered 2–4-bp deletion at the binding site of NF-E2. f, In addition, the MCC footprint is specifically altered at the NF-E2-binding site. g, Quantification of read depth at all other hypersensitive sites at the α-globin locus showing that the only statistically significant change is at the site of the deletion. Data from the edited clone aligned to a modified hg19 genome with the deletion; n = 6, data are mean ± s.d., two-tailed Mann–Whitney U-test.
