Analysis of chromatin accessbility in erythroid cells following CRISPR editing or ASO perturbation of BCL11A enhancer
CRISPR editing of an intronic enhancer within the BCL11A gene reactivates fetal hemoglobin (HbF) in adult erythroid cells, serving as a gene-based therapy for hemoglobinopathies. However, the mechanisms underlying the remarkable efficacy of CRISPR-mediated enhancer ablation remain poorly understood. We describe an evolutionarily conserved, enhancer-dependent chromatin architecture, the enhancer-CTCF rosette, essential for chromatin insulation and the developmental expression of BCL11A in hematopoiesis. CRISPR-mediated disruption of lineage-specific BCL11A enhancers impairs enhancer RNA transcription and NIPBL-cohesin loading, leading to destabilized chromatin configuration, impaired chromatin insulation, and epigenetic silencing of BCL11A. Antisense oligonucleotide-mediated depletion of enhancer RNA silences BCL11A by disrupting chromatin insulation in adult erythroid cells. Overall design: ATAC-seq analysis was performed on wild-type and sg1617-edited HUDEP-2 cells, as well as control AAVS-edited CD34+ HSPCs and Cas9-sg1617 RNP-edited CD34+ HSPCs following erythroid differentiation. Additionally, ATAC-seq was conducted on HUDEP-2 cells treated with either control ASO or E+58 eRNA ASO.