Adenine base editors induce off-target structure variations in mouse embryos and primary human T cells
Abstract Background The safety of CRISPR-based gene editing methods is of the utmost priority in clinical applications. Previous studies have reported that Cas9 cleavage induced frequent aneuploidy in primary human T cells, but whether cleavage-mediated editing of base editors would generate off-tar...
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BMC
2024-11-01
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| Series: | Genome Biology |
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| Online Access: | https://doi.org/10.1186/s13059-024-03434-0 |
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| author | Leilei Wu Shutan Jiang Meisong Shi Tanglong Yuan Yaqin Li Pinzheng Huang Yingqi Li Erwei Zuo Changyang Zhou Yidi Sun |
| author_facet | Leilei Wu Shutan Jiang Meisong Shi Tanglong Yuan Yaqin Li Pinzheng Huang Yingqi Li Erwei Zuo Changyang Zhou Yidi Sun |
| author_sort | Leilei Wu |
| collection | DOAJ |
| description | Abstract Background The safety of CRISPR-based gene editing methods is of the utmost priority in clinical applications. Previous studies have reported that Cas9 cleavage induced frequent aneuploidy in primary human T cells, but whether cleavage-mediated editing of base editors would generate off-target structure variations remains unknown. Here, we investigate the potential off-target structural variations associated with CRISPR/Cas9, ABE, and CBE editing in mouse embryos and primary human T cells by whole-genome sequencing and single-cell RNA-seq analyses. Results The results show that both Cas9 and ABE generate off-target structural variations (SVs) in mouse embryos, while CBE induces rare SVs. In addition, off-target large deletions are detected in 32.74% of primary human T cells transfected with Cas9 and 9.17% of cells transfected with ABE. Moreover, Cas9-induced aneuploid cells activate the P53 and apoptosis pathways, whereas ABE-associated aneuploid cells significantly upregulate cell cycle-related genes and are arrested in the G0 phase. A percentage of 16.59% and 4.29% aneuploid cells are still observable at 3 weeks post transfection of Cas9 or ABE. These off-target phenomena in ABE are universal as observed in other cell types such as B cells and Huh7. Furthermore, the off-target SVs are significantly reduced in cells treated with high-fidelity ABE (ABE-V106W). Conclusions This study shows both CRISPR/Cas9 and ABE induce off-target SVs in mouse embryos and primary human T cells, raising an urgent need for the development of high-fidelity gene editing tools. |
| format | Article |
| id | doaj-art-a173f9a0e0c44725899cd86c3aaab2fd |
| institution | Kabale University |
| issn | 1474-760X |
| language | English |
| publishDate | 2024-11-01 |
| publisher | BMC |
| record_format | Article |
| series | Genome Biology |
| spelling | doaj-art-a173f9a0e0c44725899cd86c3aaab2fd2024-11-17T12:31:04ZengBMCGenome Biology1474-760X2024-11-0125112210.1186/s13059-024-03434-0Adenine base editors induce off-target structure variations in mouse embryos and primary human T cellsLeilei Wu0Shutan Jiang1Meisong Shi2Tanglong Yuan3Yaqin Li4Pinzheng Huang5Yingqi Li6Erwei Zuo7Changyang Zhou8Yidi Sun9Institute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, University of Chinese Academy of Sciences, Chinese Academy of SciencesEpigenic Therapeutics, IncEpigenic Therapeutics, IncShenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Key Laboratory of Synthetic Biology, Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural SciencesEpigenic Therapeutics, IncEpigenic Therapeutics, IncEpigenic Therapeutics, IncShenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Key Laboratory of Synthetic Biology, Ministry of Agriculture and Rural Affairs, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural SciencesInstitute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, University of Chinese Academy of Sciences, Chinese Academy of SciencesInstitute of Neuroscience, CAS Center for Excellence in Brain Science and Intelligence Technology, University of Chinese Academy of Sciences, Chinese Academy of SciencesAbstract Background The safety of CRISPR-based gene editing methods is of the utmost priority in clinical applications. Previous studies have reported that Cas9 cleavage induced frequent aneuploidy in primary human T cells, but whether cleavage-mediated editing of base editors would generate off-target structure variations remains unknown. Here, we investigate the potential off-target structural variations associated with CRISPR/Cas9, ABE, and CBE editing in mouse embryos and primary human T cells by whole-genome sequencing and single-cell RNA-seq analyses. Results The results show that both Cas9 and ABE generate off-target structural variations (SVs) in mouse embryos, while CBE induces rare SVs. In addition, off-target large deletions are detected in 32.74% of primary human T cells transfected with Cas9 and 9.17% of cells transfected with ABE. Moreover, Cas9-induced aneuploid cells activate the P53 and apoptosis pathways, whereas ABE-associated aneuploid cells significantly upregulate cell cycle-related genes and are arrested in the G0 phase. A percentage of 16.59% and 4.29% aneuploid cells are still observable at 3 weeks post transfection of Cas9 or ABE. These off-target phenomena in ABE are universal as observed in other cell types such as B cells and Huh7. Furthermore, the off-target SVs are significantly reduced in cells treated with high-fidelity ABE (ABE-V106W). Conclusions This study shows both CRISPR/Cas9 and ABE induce off-target SVs in mouse embryos and primary human T cells, raising an urgent need for the development of high-fidelity gene editing tools.https://doi.org/10.1186/s13059-024-03434-0ABECas9Off-target structure variationLarge deletionT cell |
| spellingShingle | Leilei Wu Shutan Jiang Meisong Shi Tanglong Yuan Yaqin Li Pinzheng Huang Yingqi Li Erwei Zuo Changyang Zhou Yidi Sun Adenine base editors induce off-target structure variations in mouse embryos and primary human T cells Genome Biology ABE Cas9 Off-target structure variation Large deletion T cell |
| title | Adenine base editors induce off-target structure variations in mouse embryos and primary human T cells |
| title_full | Adenine base editors induce off-target structure variations in mouse embryos and primary human T cells |
| title_fullStr | Adenine base editors induce off-target structure variations in mouse embryos and primary human T cells |
| title_full_unstemmed | Adenine base editors induce off-target structure variations in mouse embryos and primary human T cells |
| title_short | Adenine base editors induce off-target structure variations in mouse embryos and primary human T cells |
| title_sort | adenine base editors induce off target structure variations in mouse embryos and primary human t cells |
| topic | ABE Cas9 Off-target structure variation Large deletion T cell |
| url | https://doi.org/10.1186/s13059-024-03434-0 |
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