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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...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society of Gene & Cell Therapy
2022
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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. |
format | Online Article Text |
id | pubmed-9375152 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | American Society of Gene & Cell Therapy |
record_format | MEDLINE/PubMed |
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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