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Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity
Shuai Jin, Hongyuan Fei, Zixu Zhu, Yingfeng Luo, Jinxing Liu, Shenghan Gao, Feng Zhang, Yu-Hang Chen, Yanpeng Wang, Caixia Gao
Molecular Cell
Abstract
Cytosine base editors (CBEs) generate C-to-T nucleotide substitutions in genomic target sites without inducing double-strand breaks. However, CBEs such as BE3 can cause genome-wide off-target changes via sgRNA-independent DNA deamination. By leveraging the orthogonal R-loops generated by SaCas9 nickase to mimic actively transcribed genomic loci that are more susceptible to cytidine deaminase, we set up a high-throughput assay for assessing sgRNA-independent off-target effects of CBEs in rice protoplasts. The reliability of this assay was confirmed by the whole-genome sequencing (WGS) of 10 base editors in regenerated rice plants. The R-loop assay was used to screen a series of rationally designed A3Bctd-BE3 variants for improved specificity. We obtained 2 efficient CBE variants, A3Bctd-VHM-BE3 and A3Bctd-KKR-BE3, and the WGS analysis revealed that these new CBEs eliminated sgRNA-independent DNA off-target edits in rice plants. Moreover, these 2 base editor variants were more precise at their target sites by producing fewer multiple C edits.
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论文编号: |
DOI:10.1016/j.molcel.2020.07.005 |
论文题目: |
Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity |
英文论文题目: |
Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity |
第一作者: |
Shuai Jin, Hongyuan Fei, Zixu Zhu, Yingfeng Luo, Jinxing Liu, Shenghan Gao, Feng Zhang, Yu-Hang Chen, Yanpeng Wang, Caixia Gao |
英文第一作者: |
Shuai Jin, Hongyuan Fei, Zixu Zhu, Yingfeng Luo, Jinxing Liu, Shenghan Gao, Feng Zhang, Yu-Hang Chen, Yanpeng Wang, Caixia Gao |
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2020-07-28 |
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摘要: |
Cytosine base editors (CBEs) generate C-to-T nucleotide substitutions in genomic target sites without inducing double-strand breaks. However, CBEs such as BE3 can cause genome-wide off-target changes via sgRNA-independent DNA deamination. By leveraging the orthogonal R-loops generated by SaCas9 nickase to mimic actively transcribed genomic loci that are more susceptible to cytidine deaminase, we set up a high-throughput assay for assessing sgRNA-independent off-target effects of CBEs in rice protoplasts. The reliability of this assay was confirmed by the whole-genome sequencing (WGS) of 10 base editors in regenerated rice plants. The R-loop assay was used to screen a series of rationally designed A3Bctd-BE3 variants for improved specificity. We obtained 2 efficient CBE variants, A3Bctd-VHM-BE3 and A3Bctd-KKR-BE3, and the WGS analysis revealed that these new CBEs eliminated sgRNA-independent DNA off-target edits in rice plants. Moreover, these 2 base editor variants were more precise at their target sites by producing fewer multiple C edits. |
英文摘要: |
Cytosine base editors (CBEs) generate C-to-T nucleotide substitutions in genomic target sites without inducing double-strand breaks. However, CBEs such as BE3 can cause genome-wide off-target changes via sgRNA-independent DNA deamination. By leveraging the orthogonal R-loops generated by SaCas9 nickase to mimic actively transcribed genomic loci that are more susceptible to cytidine deaminase, we set up a high-throughput assay for assessing sgRNA-independent off-target effects of CBEs in rice protoplasts. The reliability of this assay was confirmed by the whole-genome sequencing (WGS) of 10 base editors in regenerated rice plants. The R-loop assay was used to screen a series of rationally designed A3Bctd-BE3 variants for improved specificity. We obtained 2 efficient CBE variants, A3Bctd-VHM-BE3 and A3Bctd-KKR-BE3, and the WGS analysis revealed that these new CBEs eliminated sgRNA-independent DNA off-target edits in rice plants. Moreover, these 2 base editor variants were more precise at their target sites by producing fewer multiple C edits. |
刊物名称: |
Molecular Cell |
英文刊物名称: |
Molecular Cell |
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其它备注: |
Shuai Jin, Hongyuan Fei, Zixu Zhu, Yingfeng Luo, Jinxing Liu, Shenghan Gao, Feng Zhang, Yu-Hang Chen, Yanpeng Wang, Caixia Gao. Rationally Designed APOBEC3B Cytosine Base Editors with Improved Specificity. Molecular Cell. DOI:10.1016/j.molcel.2020.07.005 |
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