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

Elife. 2022 Feb 11:11:e65421. doi: 10.7554/eLife.65421.


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 -124 bp of HBG promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (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.

Keywords: CRISPR/Cas9; HPFH mutations; base editing; beta hemoglobinopathies; beta-hemoglobinopathies; beta-thalassemia; cd34+ hspcs; chromosomes; crispr/cas9; fetal hemoglobin; gene expression; genetics; genomics; globin regulation; hbgpromoter; hpfh mutations; large deletions; sickle cell disease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenine / metabolism
  • Anemia, Sickle Cell / genetics*
  • CRISPR-Cas Systems*
  • Cell Line
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Cytosine / metabolism
  • Fetal Hemoglobin / genetics*
  • Gene Editing / methods*
  • Hematopoietic Stem Cells / metabolism
  • Humans
  • Point Mutation
  • Promoter Regions, Genetic
  • beta-Globins / genetics
  • beta-Thalassemia / genetics
  • gamma-Globins / genetics


  • beta-Globins
  • gamma-Globins
  • Cytosine
  • Fetal Hemoglobin
  • Adenine

Associated data

  • GEO/GSE192801
  • Dryad/10.5061/dryad.bzkh1897h

Grants and funding

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.