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Dryad

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

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Dec 12, 2022 version files 1.41 GB

Abstract

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.