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.
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
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
-
- 5,713
- views
-
- 695
- downloads
-
- 45
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
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
RNA interference (RNAi) is a conserved pathway that utilizes Argonaute proteins and their associated small RNAs to exert gene regulatory function on complementary transcripts. While the majority of germline-expressed RNAi proteins reside in perinuclear germ granules, it is unknown whether and how RNAi pathways are spatially organized in other cell types. Here, we find that the small RNA biogenesis machinery is spatially and temporally organized during Caenorhabditis elegans embryogenesis. Specifically, the RNAi factor, SIMR-1, forms visible concentrates during mid-embryogenesis that contain an RNA-dependent RNA polymerase, a poly-UG polymerase, and the unloaded nuclear Argonaute protein, NRDE-3. Curiously, coincident with the appearance of the SIMR granules, the small RNAs bound to NRDE-3 switch from predominantly CSR-class 22G-RNAs to ERGO-dependent 22G-RNAs. NRDE-3 binds ERGO-dependent 22G-RNAs in the somatic cells of larvae and adults to silence ERGO-target genes; here we further demonstrate that NRDE-3-bound, CSR-class 22G-RNAs repress transcription in oocytes. Thus, our study defines two separable roles for NRDE-3, targeting germline-expressed genes during oogenesis to promote global transcriptional repression, and switching during embryogenesis to repress recently duplicated genes and retrotransposons in somatic cells, highlighting the plasticity of Argonaute proteins and the need for more precise temporal characterization of Argonaute-small RNA interactions.
-
- Chromosomes and Gene Expression
- Genetics and Genomics
A new method for mapping torsion provides insights into the ways that the genome responds to the torsion generated by RNA polymerase II.