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Efficient gene correction of an aberrant splice site in ?-thalassaemia iPSCs by CRISPR/Cas9 and single-strand oligodeoxynucleotides.


ABSTRACT: ?-thalassaemia is a prevalent hereditary haematological disease caused by mutations in the human haemoglobin ? (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 ?-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 ?-Thalassaemia, and the expression of the HBB gene can be restored, which can be used for gene therapy in the future.

SUBMITTER: Xiong Z 

PROVIDER: S-EPMC6850948 | BioStudies | 2019-01-01

REPOSITORIES: biostudies

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