Efficient gene correction of an aberrant splice site in beta-thalassaemia iPSCs by CRISPR/Cas9 and single-strand oligodeoxynucleotides
Efficient gene correction of an aberrant splice site in beta-thalassaemia iPSCs by CRISPR/Cas9 and single-strand oligodeoxynucleotides
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通过 CRISPR/Cas9 和单链寡脱氧核苷酸对β地中海贫血 iPSC 中的异常剪接位点进行有效的基因校正
DOI：
10.1111/jcmm.14669
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发表时间：
2019
影响因子：
5.3
通讯作者：
Xiaofang Sun
中科院分区：
文献类型：
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作者：
Zeyu Xiong;Yingjun Xie;Yi Yang;Yanting Xue;Ding Wang;Shouheng Lin;Diyu Chen;Dian Lu;Lina He;Bing Song;Yinghong Yang;Xiaofang Sun
β地中海贫血是一种流行的遗传性血液病，由人类血红蛋白β（HBB）基因突变引起。其中，内含子中的HBB IVS 2 - 654（C > T）突变会产生异常剪接位点。由于缺乏匹配的供体，用于治疗β地中海贫血的骨髓移植受到限制。成簇的规则间隔短回文重复序列（CRISPR）/CRISPR相关蛋白9（Cas9）作为一种广泛使用的基因编辑工具，能够靶向特定序列并产生双链断裂（DSB），其可以与单链寡核苷酸（ssODN）组合以纠正突变。在这项研究中，根据两种不同的策略，通过CRISPR/Cas9系统和ssODN在iPSCs中无缝纠正HBB IVS 2 - 654突变。为了减少二次切割的发生，采用了更有效的策略。校正的iPSC保持多能性和基因组稳定性。而且还能正常分化。通过CRISPR/Cas9系统和ssODN，我们的研究为β地中海贫血的基因校正提供了改进的策略，HBB基因的表达可以恢复，这可以用于未来的基因治疗。
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.