A p21 reporter iPSC line for evaluating CRISPR-Cas9 and vector-induced stress responses.

Autor:	Sun YD; State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, People's Republic of China.; Tianjin Institutes of Health Science, Tianjin 301600, People's Republic of China., Li GH; State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, People's Republic of China.; Tianjin Institutes of Health Science, Tianjin 301600, People's Republic of China., Zhang F; State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, People's Republic of China.; Tianjin Institutes of Health Science, Tianjin 301600, People's Republic of China., Cheng T; State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, People's Republic of China.; Tianjin Institutes of Health Science, Tianjin 301600, People's Republic of China., Zhang JP; State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, People's Republic of China.; Tianjin Institutes of Health Science, Tianjin 301600, People's Republic of China., Zhang XB; State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Diseases, Haihe Laboratory of Cell Ecosystem, Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin 300020, People's Republic of China.; Tianjin Institutes of Health Science, Tianjin 301600, People's Republic of China.
Jazyk:	angličtina
Zdroj:	Stem cells (Dayton, Ohio) [Stem Cells] 2024 Nov 05; Vol. 42 (11), pp. 992-1005.
DOI:	10.1093/stmcls/sxae056
Abstrakt:	CRISPR-Cas9 editing triggers activation of the TP53-p21 pathway, but the impacts of different editing components and delivery methods have not been fully explored. In this study, we introduce a p21-mNeonGreen reporter iPSC line to monitor TP53-p21 pathway activation. This reporter enables dynamic tracking of p21 expression via flow cytometry, revealing a strong correlation between p21 expression and indel frequencies, and highlighting its utility in guide RNA screening. Our findings show that p21 activation is significantly more pronounced with double-stranded oligodeoxynucleotides (ODNs) or adeno-associated viral vectors (AAVs) compared to their single-stranded counterparts. Lentiviral vectors (LVs) and integrase-defective lentiviral vectors induce notably lower p21 expression than AAVs, suggesting their suitability for gene therapy in sensitive cells such as hematopoietic stem cells or immune cells. Additionally, specific viral promoters like SFFV significantly amplify p21 activation, emphasizing the critical role of promoter selection in vector development. Thus, the p21-mNeonGreen reporter iPSC line is a valuable tool for assessing the potential adverse effects of gene editing methodologies and vectors. Highlights Established a p21-mNeonGreen reporter iPSC line to track activation of the TP53-p21 pathway. Found a direct correlation between p21-mNeonGreen expression and indel frequencies, aiding in gRNA screening. Showed that LVs are preferable over AAVs for certain cells due to lower p21 activation, with viral promoter choice impacting p21 response. (© The Author(s) 2024. Published by Oxford University Press.)
Databáze:	MEDLINE
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