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

  1. Nithin Sam Ravi
  2. Beeke Wienert
  3. Stacia K Wyman
  4. Henry William Bell
  5. Anila George
  6. Gokulnath Mahalingam
  7. Jonathan T Vu
  8. Kirti Prasad
  9. Bhanu Prasad Bandlamudi
  10. Nivedhitha Devaraju
  11. Vignesh Rajendiran
  12. Nazar Syedbasha
  13. Aswin Anand Pai
  14. Yukio Nakamura
  15. Ryo Kurita
  16. Muthuraman Narayanasamy
  17. Poonkuzhali Balasubramanian
  18. Saravanabhavan Thangavel
  19. Srujan Marepally
  20. Shaji R Velayudhan
  21. Alok Srivastava
  22. Mark A DeWitt
  23. Merlin Crossley
  24. Jacob E Corn
  25. Kumarasamypet Murugesan Mohankumar  Is a corresponding author
  1. Christian Medical College, India
  2. Gladstone Institutes, United States
  3. University of California, Berkeley, United States
  4. University of New South Wales, Australia
  5. Christian Medical College & Hospital, India
  6. RIKEN BioResource Center, Japan
  7. Japanese Red Cross Society, Japan, Japan
  8. University of California, Los Angeles, United States
  9. 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).

The following data sets were generated

Article and author information

Author details

  1. Nithin Sam Ravi

    Centre for Stem Cell Research, Christian Medical College, Vellore, India
    Competing interests
    The authors declare that no competing interests exist.
  2. Beeke Wienert

    Institute of Data Science and Biotechnology, Gladstone Institutes, San Francisco, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Stacia K Wyman

    Innovative Genomics Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Henry William Bell

    School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4677-8208
  5. Anila George

    Centre for Stem Cell Research, Christian Medical College, Vellore, India
    Competing interests
    The authors declare that no competing interests exist.
  6. Gokulnath Mahalingam

    Centre for Stem Cell Research, Christian Medical College, Vellore, India
    Competing interests
    The authors declare that no competing interests exist.
  7. Jonathan T Vu

    Innovative Genomics Institute, University of California, Berkeley, Berkeley, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4950-7967
  8. Kirti Prasad

    Centre for Stem Cell Research, Christian Medical College, Vellore, India
    Competing interests
    The authors declare that no competing interests exist.
  9. Bhanu Prasad Bandlamudi

    Centre for Stem Cell Research, Christian Medical College, Vellore, India
    Competing interests
    The authors declare that no competing interests exist.
  10. Nivedhitha Devaraju

    Centre for Stem Cell Research, Christian Medical College, Vellore, India
    Competing interests
    The authors declare that no competing interests exist.
  11. Vignesh Rajendiran

    Centre for Stem Cell Research, Christian Medical College, Vellore, India
    Competing interests
    The authors declare that no competing interests exist.
  12. Nazar Syedbasha

    Centre for Stem Cell Research, Christian Medical College, Vellore, India
    Competing interests
    The authors declare that no competing interests exist.
  13. Aswin Anand Pai

    Department of Haematology, Christian Medical College & Hospital, Vellore, India
    Competing interests
    The authors declare that no competing interests exist.
  14. Yukio Nakamura

    Cell Engineering Division, RIKEN BioResource Center, Ibaraki, Japan
    Competing interests
    The authors declare that no competing interests exist.
  15. Ryo Kurita

    Research and Development Department, Central Blood Institute Blood Service Headquarters, Japanese Red Cross Society, Japan, Tokyo, Japan
    Competing interests
    The authors declare that no competing interests exist.
  16. Muthuraman Narayanasamy

    Centre for Stem Cell Research, Christian Medical College, Vellore, India
    Competing interests
    The authors declare that no competing interests exist.
  17. Poonkuzhali Balasubramanian

    Department of Haematology, Christian Medical College & Hospital, Vellore, India
    Competing interests
    The authors declare that no competing interests exist.
  18. Saravanabhavan Thangavel

    Centre for Stem Cell Research, Christian Medical College, Vellore, India
    Competing interests
    The authors declare that no competing interests exist.
  19. Srujan Marepally

    Centre for Stem Cell Research, Christian Medical College, Vellore, India
    Competing interests
    The authors declare that no competing interests exist.
  20. Shaji R Velayudhan

    Department of Haematology, Christian Medical College & Hospital, Vellore, India
    Competing interests
    The authors declare that no competing interests exist.
  21. Alok Srivastava

    Department of Haematology, Christian Medical College & Hospital, Vellore, India
    Competing interests
    The authors declare that no competing interests exist.
  22. Mark A DeWitt

    Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.
  23. Merlin Crossley

    School of Biotechnology and Biomolecular Sciences, University of New South Wales, Kensington, Australia
    Competing interests
    The authors declare that no competing interests exist.
  24. Jacob E Corn

    Department of Biology, ETH Zurich, Zurich, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7798-5309
  25. Kumarasamypet Murugesan Mohankumar

    Centre for Stem Cell Research, Christian Medical College, Vellore, India
    For correspondence
    mohankumarkm@cmcvellore.ac.in
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9407-1800

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

  1. 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

  1. Preprint posted: July 1, 2020 (view preprint)
  2. Received: December 3, 2020
  3. Accepted: February 11, 2022
  4. Accepted Manuscript published: February 11, 2022 (version 1)
  5. 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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  1. Nithin Sam Ravi
  2. Beeke Wienert
  3. Stacia K Wyman
  4. Henry William Bell
  5. Anila George
  6. Gokulnath Mahalingam
  7. Jonathan T Vu
  8. Kirti Prasad
  9. Bhanu Prasad Bandlamudi
  10. Nivedhitha Devaraju
  11. Vignesh Rajendiran
  12. Nazar Syedbasha
  13. Aswin Anand Pai
  14. Yukio Nakamura
  15. Ryo Kurita
  16. Muthuraman Narayanasamy
  17. Poonkuzhali Balasubramanian
  18. Saravanabhavan Thangavel
  19. Srujan Marepally
  20. Shaji R Velayudhan
  21. Alok Srivastava
  22. Mark A DeWitt
  23. Merlin Crossley
  24. Jacob E Corn
  25. Kumarasamypet Murugesan Mohankumar
(2022)
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

Share this article

https://doi.org/10.7554/eLife.65421

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