Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin
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).
-
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
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.
Metrics
-
- 4,570
- Page views
-
- 569
- Downloads
-
- 24
- Citations
Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.
Download links
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Further reading
-
- Chromosomes and Gene Expression
- Genetics and Genomics
Endothelial cell subpopulations are characterized by unique gene expression profiles, epigenetic landscapes and functional properties.
-
- Cell Biology
- Chromosomes and Gene Expression
Histone H1 participates in chromatin condensation and regulates nuclear processes. Human somatic cells may contain up to seven histone H1 variants, although their functional heterogeneity is not fully understood. Here, we have profiled the differential nuclear distribution of the somatic H1 repertoire in human cells through imaging techniques including super-resolution microscopy. H1 variants exhibit characteristic distribution patterns in both interphase and mitosis. H1.2, H1.3, and H1.5 are universally enriched at the nuclear periphery in all cell lines analyzed and co-localize with compacted DNA. H1.0 shows a less pronounced peripheral localization, with apparent variability among different cell lines. On the other hand, H1.4 and H1X are distributed throughout the nucleus, being H1X universally enriched in high-GC regions and abundant in the nucleoli. Interestingly, H1.4 and H1.0 show a more peripheral distribution in cell lines lacking H1.3 and H1.5. The differential distribution patterns of H1 suggest specific functionalities in organizing lamina-associated domains or nucleolar activity, which is further supported by a distinct response of H1X or phosphorylated H1.4 to the inhibition of ribosomal DNA transcription. Moreover, H1 variants depletion affects chromatin structure in a variant-specific manner. Concretely, H1.2 knock-down, either alone or combined, triggers a global chromatin decompaction. Overall, imaging has allowed us to distinguish H1 variants distribution beyond the segregation in two groups denoted by previous ChIP-Seq determinations. Our results support H1 variants heterogeneity and suggest that variant-specific functionality can be shared between different cell types.