Chapter 14
Correction of Hemoglobin E/Beta-Thalassemia
Patient-Derived iPSCs Using CRISPR/Cas9
Methichit Wattanapanitch
Abstract
HbE/β-thalassemia is one of the most common thalassemic syndromes in Southeast Asia and Thailand.
Patients have mutations in β hemoglobin (HBB) gene resulting in decreased and/or abnormal production
of β hemoglobin. Here, we describe a protocol for CRISPR/Cas9-mediated gene correction of the mutated
hemoglobin E from one allele of the HBB gene by homology-directed repair (HDR) in HbE/β-thalassemia
patient-derived induced pluripotent stem cells (iPSCs) using a CRISPR/Cas9 plasmid-based transfection
method and a single-stranded DNA oligonucleotide (ssODN) repair template harboring the correct
nucleotides. Our strategy allows the seamless HbE gene correction with the editing efficiency (HDR) up
to 3%, as confirmed by Sanger sequencing. This protocol provides a simple one-step genetic correction of
HbE mutation in the patient-derived iPSCs. Further differentiation of the corrected iPSCs into hematopoietic stem/progenitor cells will provide an alternative renewable source of cells for the application in
autologous transplantation in the future.
Key words Beta-thalassemia, Hemoglobinopathy, Genetic correction, Induced pluripotent stem cells,
CRISPR/Cas9, Homology-directed repair (HDR), Clonal isolation
1 Introduction
Thalassemia is an inherited autosomal recessive disease characterized by decreased production or structural alteration of the α or β
hemoglobin chain leading to improper oxygen transport and
hemolytic anemia [1, 2]. The complexity of thalassemia syndromes
is a result of interactions between different combinations of globin
gene defects. Several different combinations of abnormal genes can
result in over 60 thalassemic syndromes.
One of the most common syndromes in Thailand and Southeast Asia is HbE/β-thalassemia, which has heterogeneous clinical
manifestations from very mild and asymptomatic anemia to very
severe disorder requiring transfusions. Patients with HbE/β-thalassemia can have various mutations in one allele (β
0 or β
+ ) producing no or decreased β-globin chain, and a missense mutation at
Kumaran Narayanan (ed.), Bio-Carrier Vectors: Methods and Protocols, Methods in Molecular Biology, vol. 2211,
https://doi.org/10.1007/978-1-0716-0943-9_14, © Springer Science+Business Media, LLC, part of Springer Nature 2021
193
Correction of Hemoglobin E/Beta-Thalassemia
Patient-Derived iPSCs Using CRISPR/Cas9
Methichit Wattanapanitch
Abstract
HbE/β-thalassemia is one of the most common thalassemic syndromes in Southeast Asia and Thailand.
Patients have mutations in β hemoglobin (HBB) gene resulting in decreased and/or abnormal production
of β hemoglobin. Here, we describe a protocol for CRISPR/Cas9-mediated gene correction of the mutated
hemoglobin E from one allele of the HBB gene by homology-directed repair (HDR) in HbE/β-thalassemia
patient-derived induced pluripotent stem cells (iPSCs) using a CRISPR/Cas9 plasmid-based transfection
method and a single-stranded DNA oligonucleotide (ssODN) repair template harboring the correct
nucleotides. Our strategy allows the seamless HbE gene correction with the editing efficiency (HDR) up
to 3%, as confirmed by Sanger sequencing. This protocol provides a simple one-step genetic correction of
HbE mutation in the patient-derived iPSCs. Further differentiation of the corrected iPSCs into hematopoietic stem/progenitor cells will provide an alternative renewable source of cells for the application in
autologous transplantation in the future.
Key words Beta-thalassemia, Hemoglobinopathy, Genetic correction, Induced pluripotent stem cells,
CRISPR/Cas9, Homology-directed repair (HDR), Clonal isolation
1 Introduction
Thalassemia is an inherited autosomal recessive disease characterized by decreased production or structural alteration of the α or β
hemoglobin chain leading to improper oxygen transport and
hemolytic anemia [1, 2]. The complexity of thalassemia syndromes
is a result of interactions between different combinations of globin
gene defects. Several different combinations of abnormal genes can
result in over 60 thalassemic syndromes.
One of the most common syndromes in Thailand and Southeast Asia is HbE/β-thalassemia, which has heterogeneous clinical
manifestations from very mild and asymptomatic anemia to very
severe disorder requiring transfusions. Patients with HbE/β-thalassemia can have various mutations in one allele (β
0 or β
+ ) producing no or decreased β-globin chain, and a missense mutation at
Kumaran Narayanan (ed.), Bio-Carrier Vectors: Methods and Protocols, Methods in Molecular Biology, vol. 2211,
https://doi.org/10.1007/978-1-0716-0943-9_14, © Springer Science+Business Media, LLC, part of Springer Nature 2021
193