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Question

To evaluate the requirement for human BCL11A enhancer
sequences, we used HUDEP-2 cells, an immortalized human CD341
haematopoietic stem and progenitor cell (HSPC)-derived erythroid
precursor cell line that expresses BCL11A and predominantly b- rather
than c-globin34. We used the CRISPR-Cas9 nuclease system to generate
clones of HUDEP-2 cells with deletion of the 12-kb BCL11A composite
enhancer by introduction of a pair of chimaeric single guide RNAs
(sgRNAs). Enhancer deletion resulted in near-complete loss of
BCL11A expression and induction of c-globin and HbF protein to
similar levels as cells with BCL11A knockout (Fig. 1a–c), consistent
with the possibility that these sequences could serve as targets for
therapeutic genome editing for HbF reinduction for the b-haemoglobinopathies35. Although targeted deletions by paired double strand
breaks (DSBs) may be achieved by genome editing, competing genomic
outcomes include local insertion/deletion (indel) production at each

Answer 1

Deletion of human BCL11A enhancer sequences in HUDEP-2 cells resulted in loss of BCL11A expression and induction of c-globin and HbF protein, suggesting therapeutic potential for HbF reinduction in b-haemoglobinopathies through genome editing.