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Engineering an adenine base editor in human embryonic stem cells with minimal DNA and RNA off-target activities

Genome editing in pluripotent stem cells (PSCs) using CRISPR technology holds great promise for therapeutic applications. Yet, it has been reported that Cas9-mediated cleavage could cause large deletions or rearrangements of DNA, and the selection of edited PSCs could acquire p53 mutations. Adenine...

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Detalles Bibliográficos
Autores principales: Zhang, Zhenwu, Tao, Wanyu, Huang, Shisheng, Sun, Wenjun, Wang, Yue, Jiang, Wen, Huang, Xingxu, Lin, Chao-Po
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society of Gene & Cell Therapy 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9375152/
https://www.ncbi.nlm.nih.gov/pubmed/35991312
http://dx.doi.org/10.1016/j.omtn.2022.07.026
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author Zhang, Zhenwu
Tao, Wanyu
Huang, Shisheng
Sun, Wenjun
Wang, Yue
Jiang, Wen
Huang, Xingxu
Lin, Chao-Po
author_facet Zhang, Zhenwu
Tao, Wanyu
Huang, Shisheng
Sun, Wenjun
Wang, Yue
Jiang, Wen
Huang, Xingxu
Lin, Chao-Po
author_sort Zhang, Zhenwu
collection PubMed
description Genome editing in pluripotent stem cells (PSCs) using CRISPR technology holds great promise for therapeutic applications. Yet, it has been reported that Cas9-mediated cleavage could cause large deletions or rearrangements of DNA, and the selection of edited PSCs could acquire p53 mutations. Adenine base editors (ABEs) do not introduce DNA double-strand breaks and thus have been proposed as alternatives to circumvent those problems, but their off-target effects still limit their applications. Here, we tested different combinations of off-target reduction methods to further diminish off-target effects of ABEs without compromising their on-target editing efficiencies. We subsequently chose the best editor, CE-8e-dV, which contains V106W substitution, R153 deletion, and Cas-embedding strategy, to establish a single-cell-derived human embryonic stem cell (hESC) line expressing tetracycline-inducible CE-8e-dV. By performing RNA and whole-genome sequencing, we demonstrated that the expression of CE-8e-dV did not produce nearly any DNA or RNA off-target effects in hESCs. Our results provide stringent proof of the safety of ABEs in PSCs and suggest that CE-8e-dV could be suitable for related therapeutic strategies, such as generation of engineered stem cells in vitro and gene therapy in vivo.
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spelling pubmed-93751522022-08-18 Engineering an adenine base editor in human embryonic stem cells with minimal DNA and RNA off-target activities Zhang, Zhenwu Tao, Wanyu Huang, Shisheng Sun, Wenjun Wang, Yue Jiang, Wen Huang, Xingxu Lin, Chao-Po Mol Ther Nucleic Acids Original Article Genome editing in pluripotent stem cells (PSCs) using CRISPR technology holds great promise for therapeutic applications. Yet, it has been reported that Cas9-mediated cleavage could cause large deletions or rearrangements of DNA, and the selection of edited PSCs could acquire p53 mutations. Adenine base editors (ABEs) do not introduce DNA double-strand breaks and thus have been proposed as alternatives to circumvent those problems, but their off-target effects still limit their applications. Here, we tested different combinations of off-target reduction methods to further diminish off-target effects of ABEs without compromising their on-target editing efficiencies. We subsequently chose the best editor, CE-8e-dV, which contains V106W substitution, R153 deletion, and Cas-embedding strategy, to establish a single-cell-derived human embryonic stem cell (hESC) line expressing tetracycline-inducible CE-8e-dV. By performing RNA and whole-genome sequencing, we demonstrated that the expression of CE-8e-dV did not produce nearly any DNA or RNA off-target effects in hESCs. Our results provide stringent proof of the safety of ABEs in PSCs and suggest that CE-8e-dV could be suitable for related therapeutic strategies, such as generation of engineered stem cells in vitro and gene therapy in vivo. American Society of Gene & Cell Therapy 2022-08-01 /pmc/articles/PMC9375152/ /pubmed/35991312 http://dx.doi.org/10.1016/j.omtn.2022.07.026 Text en © 2022 The Author(s) https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Original Article
Zhang, Zhenwu
Tao, Wanyu
Huang, Shisheng
Sun, Wenjun
Wang, Yue
Jiang, Wen
Huang, Xingxu
Lin, Chao-Po
Engineering an adenine base editor in human embryonic stem cells with minimal DNA and RNA off-target activities
title Engineering an adenine base editor in human embryonic stem cells with minimal DNA and RNA off-target activities
title_full Engineering an adenine base editor in human embryonic stem cells with minimal DNA and RNA off-target activities
title_fullStr Engineering an adenine base editor in human embryonic stem cells with minimal DNA and RNA off-target activities
title_full_unstemmed Engineering an adenine base editor in human embryonic stem cells with minimal DNA and RNA off-target activities
title_short Engineering an adenine base editor in human embryonic stem cells with minimal DNA and RNA off-target activities
title_sort engineering an adenine base editor in human embryonic stem cells with minimal dna and rna off-target activities
topic Original Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9375152/
https://www.ncbi.nlm.nih.gov/pubmed/35991312
http://dx.doi.org/10.1016/j.omtn.2022.07.026
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