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Crispr-Based Editing of Human Pluripotent Stem Cells for Disease Modeling

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Abstract

The CRISPR system, as an effective genome editing technology, has been extensively utilized for the construction of disease models in human pluripotent stem cells. Establishment of a gene mutant or knockout stem cell line typically relies on Cas nuclease-generated double-stranded DNA breaks and exogenous templates, which can produce uncontrollable editing byproducts and toxicity. The recently developed adenine base editors (ABE) have greatly facilitated related research by introducing A/T > G/C mutations in the coding regions or splitting sites (AG-GT) of genes, enabling mutant gene knock-in or knock-out without introducing DNA breaks. In this study, we edit the AG bases in exons anterior to achieve gene knockout via the ABE8e-SpRY, which recognizes most expanded protospacer adjacent motif to target the genome. Except for gene-knockout, ABE8e-SpRY can also efficiently establish disease-related A/T-to-G/C variation cell lines by targeting coding sequences. The method we generated is simple and time-saving, and it only takes two weeks to obtain the desired cell line. This protocol provides operating instructions step-by-step for constructing knockout and point mutation cell lines.

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Materials related to this paper is available from the corresponding author.

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Funding

We gratefully acknowledge funding support from the National Natural Science Foundation of China (82070258, 81970205), Shenzhen Fundamental Research Program (ZDSYS20200923172000001), China Postdoctoral Science Foundation (2022M720498).

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Authors and Affiliations

  1. Fuwai Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College/National Center for Cardiovascular Diseases, Beijing, 100037, China

    Yun Chang, Yongshuai Zhang & Shuhong Ma

  2. Fuwai Hospital Chinese Academy of Medical Sciences, Shenzhen, Shenzhen Key Laboratory of Cardiovascular Disease, State Key Laboratory of Cardiovascular Disease, Key Laboratory of Pluripotent Stem Cells in Cardiac Repair and Regeneration, Chinese Academy of Medical Sciences and Peking Union Medical College, Shenzhen, China

    Feng Lan & Shuhong Ma

  3. National Health Commission Key Laboratory of Cardiovascular Regenerative Medicine, Fuwai Central-China Hospital, Central-China Branch of National Center for Cardiovascular Diseases, Zhengzhou, China

    Feng Lan

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MSH and LF designed this study; CY and ZYS performed the majority of cell experiments and data analysis; CY and ZYS provided the manuscript preparation. All authors read and approved the final manuscript.

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Correspondence to Yongshuai Zhang.

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Chang, Y., Lan, F., Zhang, Y. et al. Crispr-Based Editing of Human Pluripotent Stem Cells for Disease Modeling. Stem Cell Rev and Rep (2024). https://doi.org/10.1007/s12015-024-10713-7

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