Efficient gene correction of an aberrant splice site in β‐thalassaemia iPSCs by CRISPR/Cas9 and single‐strand oligodeoxynucleotides

β‐thalassaemia is a prevalent hereditary haematological disease caused by mutations in the human haemoglobin β (HBB) gene. Among them, the HBB IVS2‐654 (C > T) mutation, which is in the intron, creates an aberrant splicing site. Bone marrow transplantation for curing β‐thalassaemia is limited due...

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Bibliographic Details
Published in:Journal of cellular and molecular medicine 2019-12, Vol.23 (12), p.8046-8057
Main Authors: Xiong, Zeyu, Xie, Yingjun, Yang, Yi, Xue, Yanting, Wang, Ding, Lin, Shouheng, Chen, Diyu, Lu, Dian, He, Lina, Song, Bing, Yang, Yinghong, Sun, Xiaofang
Format: Article
Language:English
Subjects:
beta-Globins - genetics
beta-Globins - metabolism
beta-Thalassemia - genetics
beta-Thalassemia - metabolism
Blood diseases
Bone marrow
Bone marrow transplantation
Cell cycle
CRISPR
CRISPR-Cas Systems
CRISPR/Cas9
Data analysis
Deoxyribonucleic acid
Design
DNA
DNA, Single-Stranded - genetics
gene correction
Gene Editing - methods
Gene therapy
Genetic disorders
Genetic modification
Genetic Therapy - methods
Genome editing
Genomes
HBB gene
Hematology
Hematopoiesis
Hemoglobin
Humans
induced pluripotent stem cells
Induced Pluripotent Stem Cells - chemistry
Induced Pluripotent Stem Cells - metabolism
Mutation
Oligodeoxyribonucleotides - genetics
Oligonucleotides
Original
Pluripotency
Polymerase chain reaction
RNA Cleavage - genetics
RNA Splice Sites
RNA Splicing - genetics
Science
secondary cleavage
single‐stranded oligodeoxynucleotide
Splicing
Stem cells
Thalassemia
Transplantation
Whole Exome Sequencing
β‐Thalassaemia
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Summary:β‐thalassaemia is a prevalent hereditary haematological disease caused by mutations in the human haemoglobin β (HBB) gene. Among them, the HBB IVS2‐654 (C > T) mutation, which is in the intron, creates an aberrant splicing site. Bone marrow transplantation for curing β‐thalassaemia is limited due to the lack of matched donors. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9), as a widely used tool for gene editing, is able to target specific sequence and create double‐strand break (DSB), which can be combined with the single‐stranded oligodeoxynucleotide (ssODN) to correct mutations. In this study, according to two different strategies, the HBB IVS2‐654 mutation was seamlessly corrected in iPSCs by CRISPR/Cas9 system and ssODN. To reduce the occurrence of secondary cleavage, a more efficient strategy was adopted. The corrected iPSCs kept pluripotency and genome stability. Moreover, they could differentiate normally. Through CRISPR/Cas9 system and ssODN, our study provides improved strategies for gene correction of β‐Thalassaemia, and the expression of the HBB gene can be restored, which can be used for gene therapy in the future.
ISSN:1582-1838
1582-4934
DOI:10.1111/jcmm.14669