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 HBG proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the -123 region. Base editing at -123 and -124bp of HBG promoter induced HbF to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human 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 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.

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CRISPR/Cas9 system, a bacterial adaptive immune system developed into a genome editing technology, has emerged as a powerful tool revolutionising genome engineering in all branches of biological science including agriculture, research and medicine. Rapid evolution of CRISPR/Cas9 system from the generation of double strand breaks to more advanced applications on gene regulation has made the wide-spread use of this technology possible. Medical science has benefited greatly from CRISPR/Cas9; being both a versatile and economical tool, it has brought gene therapy closer to reality. In this review, the development of CRISPR/Cas9 system, variants thereof and its application in different walks of medical science- research, diagnostics and therapy, will be discussed.