Park Ju-Chan, Kim Keun-Tae, Jang Hyeon-Ki, Cha Hyuk-Jin
College of Pharmacy, Seoul National University, Seoul, Korea.
Division of Chemical Engineering and Bioengineering, College of Art Culture and Engineering, Kangwon National University, Chuncheon, Korea.
Int J Stem Cells. 2023 May 30;16(2):234-243. doi: 10.15283/ijsc22171. Epub 2023 Feb 28.
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.
人类多能干细胞（hPSCs）的最新进展使得能够在hPSCs中精确编辑所需碱基，用于建立同基因疾病模型和自体体外细胞治疗。基于Cas9核酸内切酶同源定向修复的敲入方法会导致DNA双链断裂（DSB），不仅会产生插入和缺失（indel）突变，还会产生有害的大片段缺失。相反，由于缺乏Cas9核酸内切酶活性，腺嘌呤碱基编辑器（ABE）和胞嘧啶碱基编辑器（CBE）等碱基编辑器可通过共轭脱氨酶活性实现精确的碱基替换，不会形成DSB。尽管碱基编辑器在转换替换方面存在局限性，但在hPSCs中进行无大量脱靶效应的精确碱基编辑被认为是临床应用的一种有前景的替代方法。考虑到hPSCs独特的细胞特性，有几点需要考虑。在此，我们描述了一种用于hPSCs碱基编辑的更新和优化方案。我们还描述了一种改进的基于CBE的C到T替换方法，这种替换在hPSCs中通常低于A到G替换。
