Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin
- Christian Medical College, India
- Gladstone Institutes, United States
- University of California, Berkeley, United States
- University of New South Wales, Australia
- Christian Medical College & Hospital, India
- RIKEN BioResource Center, Japan
- Japanese Red Cross Society, Japan, Japan
- University of California, Los Angeles, United States
- ETH Zurich, Switzerland
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 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.
Data availability
The transcriptome data have been deposited in GEO under accession code GSE192801All the raw data from this study have been deposited in Dyrad (doi:10.5061/dryad.bzkh1897h).
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Data from: Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobinDryad Digital Repository, doi:10.5061/dryad.bzkh1897h.
Article and author information
Author details
Henry William Bell
Kumarasamypet Murugesan Mohankumar
Funding
Department of Biotechnology, Ministry of Science and Technology, India (BT/PR17316/MED/31/326/2015)
- Kumarasamypet Murugesan Mohankumar
National Health and Medical Research Council (National Health and Medical Research Council (NHMRC))
- Henry William Bell
National Health and Medical Research Council (Grant)
- Merlin Crossley
Science and Engineering Research Board (EMR/2017/004363)
- Kumarasamypet Murugesan Mohankumar
Indo-US Science and Technology Forum (Indo-U.S. GETin Fellowship_2018_066)
- Kumarasamypet Murugesan Mohankumar
Department of Biotechnology, Ministry of Science and Technology, India (BT/PR38392/GET/119/301/2020)
- Kumarasamypet Murugesan Mohankumar
Council of Scientific and Industrial Research, India (Senior Research Fellow)
- Nithin Sam Ravi
Council of Scientific and Industrial Research, India (Senior Research Fellow)
- Anila George
Department of Biotechnology, Ministry of Science and Technology, India (Senior Research Fellow)
- Vignesh Rajendiran
National Health and Medical Research Council (Early Career Research Fellowship)
- Beeke Wienert
Department of Biotechnology, Ministry of Science and Technology, India (BT/PR25841/GET/119/162/2017)
- Srujan Marepally
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Stephen C Ekker, Mayo Clinic, United States
Ethics
Human subjects: The left-over peripheral blood mononuclear cells (PBMNC) were obtained from a healthy donor after infusion according to the clinical protocols approved by the Intuitional Review Boards of Christian Medical College, Vellore.IRB Min. No. 12309 (OTHER) dated 30. 10.2019
Version history
- Preprint posted: July 1, 2020 (view preprint)
- Received: December 3, 2020
- Accepted: February 11, 2022
- Accepted Manuscript published: February 11, 2022 (version 1)
- Version of Record published: February 23, 2022 (version 2)
Copyright
© 2022, Ravi et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin
eLife 11:e65421.
https://doi.org/10.7554/eLife.65421
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