eLife (Feb 2022)
Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin
- Nithin Sam Ravi,
- Beeke Wienert,
- Stacia K Wyman,
- Henry William Bell,
- Anila George,
- Gokulnath Mahalingam,
- Jonathan T Vu,
- Kirti Prasad,
- Bhanu Prasad Bandlamudi,
- Nivedhitha Devaraju,
- Vignesh Rajendiran,
- Nazar Syedbasha,
- Aswin Anand Pai,
- Yukio Nakamura,
- Ryo Kurita,
- Muthuraman Narayanasamy,
- Poonkuzhali Balasubramanian,
- Saravanabhavan Thangavel,
- Srujan Marepally,
- Shaji R Velayudhan,
- Alok Srivastava,
- Mark A DeWitt,
- Merlin Crossley,
- Jacob E Corn,
- Kumarasamypet M Mohankumar
Affiliations
- Nithin Sam Ravi
- ORCiD
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, India; Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, India
- Beeke Wienert
- Innovative Genomics Institute, University of California, Berkeley, Berkeley, United States; Institute of Data Science and Biotechnology, Gladstone Institutes, San Francisco, United States; School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia
- Stacia K Wyman
- Innovative Genomics Institute, University of California, Berkeley, Berkeley, United States
- Henry William Bell
- ORCiD
- School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia
- Anila George
- ORCiD
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, India; Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, India
- Gokulnath Mahalingam
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, India
- Jonathan T Vu
- ORCiD
- Innovative Genomics Institute, University of California, Berkeley, Berkeley, United States
- Kirti Prasad
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, India; Manipal Academy of Higher Education, Karnataka, India
- Bhanu Prasad Bandlamudi
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, India
- Nivedhitha Devaraju
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, India; Manipal Academy of Higher Education, Karnataka, India
- Vignesh Rajendiran
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, India; Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, India
- Nazar Syedbasha
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, India
- Aswin Anand Pai
- Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, India; Department of Haematology, Christian Medical College & Hospital, Vellore, India
- Yukio Nakamura
- Cell Engineering Division, RIKEN BioResource Center, Ibaraki, Japan
- Ryo Kurita
- Research and Development Department, Central Blood Institute Blood Service Headquarters, Japanese Red Cross Society, Japan, Tokyo, Japan
- Muthuraman Narayanasamy
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, India; Department of Biochemistry, Christian Medical College, Vellore, India
- Poonkuzhali Balasubramanian
- Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, India; Department of Haematology, Christian Medical College & Hospital, Vellore, India
- Saravanabhavan Thangavel
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, India
- Srujan Marepally
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, India
- Shaji R Velayudhan
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, India; Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, India; Department of Haematology, Christian Medical College & Hospital, Vellore, India
- Alok Srivastava
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, India; Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, India; Department of Haematology, Christian Medical College & Hospital, Vellore, India
- Mark A DeWitt
- Innovative Genomics Institute, University of California, Berkeley, Berkeley, United States; Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, United States
- Merlin Crossley
- School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia
- Jacob E Corn
- ORCiD
- Innovative Genomics Institute, University of California, Berkeley, Berkeley, United States; Institute of Molecular Health Sciences, Department of Biology, Zurich, Switzerland
- Kumarasamypet M Mohankumar
- ORCiD
- Centre for Stem Cell Research (a Unit of inStem, Bengaluru), Christian Medical College Campus, Vellore, India; Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram, India
- DOI
- https://doi.org/10.7554/eLife.65421
- Journal volume & issue
-
Vol. 11
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 –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