Transition Substitution of Desired Bases in Human Pluripotent Stem Cells with Base Editors: A Step-by-Step Guide.

IF 2.5 4区 医学 Q3 CELL & TISSUE ENGINEERING International journal of stem cells Pub Date : 2023-05-30 DOI:10.15283/ijsc22171
Ju-Chan Park, Keun-Tae Kim, Hyeon-Ki Jang, Hyuk-Jin Cha
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引用次数: 1

Abstract

The recent advances in human pluripotent stem cells (hPSCs) enable to precisely edit the desired bases in hPSCs to be used for the establishment of isogenic disease models and autologous ex vivo cell therapy. The knock-in approach based on the homologous directed repair with Cas9 endonuclease, causing DNA double-strand breaks (DSBs), produces not only insertion and deletion (indel) mutations but also deleterious large deletions. On the contrary, due to the lack of Cas9 endonuclease activity, base editors (BEs) such as adenine base editor (ABE) and cytosine base editor (CBE) allow precise base substitution by conjugated deaminase activity, free from DSB formation. Despite the limitation of BEs in transition substitution, precise base editing by BEs with no massive off-targets is suggested to be a prospective alternative in hPSCs for clinical applications. Considering the unique cellular characteristics of hPSCs, a few points should be considered. Herein, we describe an updated and optimized protocol for base editing in hPSCs. We also describe an improved methodology for CBE-based C to T substitutions, which are generally lower than A to G substitutions in hPSCs.

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人类多能干细胞中所需碱基的过渡替代与碱基编辑器:一步一步的指南。
人类多能干细胞(human pluripotent stem cells, hPSCs)的最新进展使其能够精确编辑所需的碱基,用于建立等基因疾病模型和自体离体细胞治疗。基于Cas9内切酶同源定向修复的敲入方法,引起DNA双链断裂(DSBs),不仅产生插入和删除(indel)突变,而且产生有害的大缺失。相反,由于缺乏Cas9内切酶活性,碱基编辑器(BEs),如腺嘌呤碱基编辑器(ABE)和胞嘧啶碱基编辑器(CBE)可以通过偶联脱氨酶活性进行精确的碱基替换,而不会形成DSB。尽管BEs在过渡替代方面存在局限性,但通过BEs进行精确的碱基编辑而没有大规模脱靶被认为是临床应用于人造血干细胞的一种有前景的替代方法。考虑到造血干细胞独特的细胞特性,有几点需要考虑。在此,我们描述了一种更新和优化的hPSCs碱基编辑方案。我们还描述了一种改进的基于cbe的C到T替换方法,该方法通常低于hPSCs中A到G的替换。
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来源期刊
International journal of stem cells
International journal of stem cells Biochemistry, Genetics and Molecular Biology-Cell Biology
CiteScore
5.10
自引率
4.30%
发文量
38
期刊介绍: International Journal of Stem Cells (Int J Stem Cells), a peer-reviewed open access journal, principally aims to provide a forum for investigators in the field of stem cell biology to present their research findings and share their visions and opinions. Int J Stem Cells covers all aspects of stem cell biology including basic, clinical and translational research on genetics, biochemistry, and physiology of various types of stem cells including embryonic, adult and induced stem cells. Reports on epigenetics, genomics, proteomics, metabolomics of stem cells are welcome as well. Int J Stem Cells also publishes review articles, technical reports and treatise on ethical issues.
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