Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin

Naturally occurring point mutations in the HBG promoter switch hemoglobin
synthesis from defective adult beta-globin to fetal gamma-globin in sickle
cell patients with hereditary persistence of fetal hemoglobin (HPFH) and
ameliorate the clinical severity. Inspired by this natural phenomenon, we
tiled the highly homologous HBG1 and HBG2 proximal promoters using adenine
and cytosine base editors that avoid the generation of large deletions.
Among the novel regulatory region identified, base editing at -123 region
induced HbF to a higher level than disruption of a well-known BCL11A
binding site in erythroblasts derived from healthy donor CD34+
HSPC. We further demonstrated in vitro that the introduction of
-123T>C and -124T>C HPFH-like mutations drives gamma-globin
expression by creating a de novo binding site for the master erythroid
regulator KLF1. Overall, our findings shed light on so far unknown
regulatory elements within the HBG promoter and identified
additional targets for therapeutic upregulation of fetal
hemoglobin.