International Journal of Stem Cells

Korean Society for Stem Cell Research (한국줄기세포학회)
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Transition Substitution of Desired Bases in Human Pluripotent Stem Cells with Base Editors: A Step-by-Step Guide

  • Ju-Chan Park (College of Pharmacy, Seoul National University) ;
  • Keun-Tae Kim (College of Pharmacy, Seoul National University) ;
  • Hyeon-Ki Jang (Division of Chemical Engineering and Bioengineering, College of Art Culture and Engineering, Kangwon National University) ;
  • Hyuk-Jin Cha (College of Pharmacy, Seoul National University)
  • Received : 2022.10.10
  • Accepted : 2022.11.12
  • Published : 2023.05.30
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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.

Keywords

Acknowledgement

This research was supported by grants from the National Research Foundation of Korea (NRF) (NRF-2022R1C1C2011617 to H.-K.J) and by Korean Fund for Regenerative Medicine funded by Ministry of Science and ICT, Ministry of Health and Welfare (RS-2022-00070316 to H.-J.C), Republic of Korea, and Seoul National University Research Grant.

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