Exploring C-to-G and A-to-Y Base Editing in Rice by Using New Vector Tools
CRISPR/Cas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) can efficiently mediate C-to-T/G-to-A and A-to-G/T-to-C substitutions, respectively; however, achieving base transversions (C-to-G/C-to-A and A-to-T/A-to-C) is challenging and has been rarely studied in plants. Here, we c...
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| Format: | Article |
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MDPI AG
2022-07-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/23/14/7990 |
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| author | Dongchang Zeng Zhiye Zheng Yuxin Liu Taoli Liu Tie Li Jianhong Liu Qiyu Luo Yang Xue Shengting Li Nan Chai Suize Yu Xianrong Xie Yao-Guang Liu Qinlong Zhu |
| author_facet | Dongchang Zeng Zhiye Zheng Yuxin Liu Taoli Liu Tie Li Jianhong Liu Qiyu Luo Yang Xue Shengting Li Nan Chai Suize Yu Xianrong Xie Yao-Guang Liu Qinlong Zhu |
| author_sort | Dongchang Zeng |
| collection | DOAJ |
| description | CRISPR/Cas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) can efficiently mediate C-to-T/G-to-A and A-to-G/T-to-C substitutions, respectively; however, achieving base transversions (C-to-G/C-to-A and A-to-T/A-to-C) is challenging and has been rarely studied in plants. Here, we constructed new plant C-to-G base editors (CGBEs) and new A-to-Y (T/C) base editors and explored their base editing characteristics in rice. First, we fused the highly active cytidine deaminase evoFENRY and the PAM-relaxed Cas9-nickase variant Cas9n-NG with rice and human uracil DNA N-glycosylase (rUNG and hUNG), respectively, to construct CGBE-rUNG and CGBE-hUNG vector tools. The analysis of five NG-PAM target sites showed that these CGBEs achieved C-to-G conversions with monoallelic editing efficiencies of up to 27.3% in T<sub>0</sub> rice, with major byproducts being insertion/deletion mutations. Moreover, for the A-to-Y (C or T) editing test, we fused the highly active adenosine deaminase TadA8e and the Cas9-nickase variant SpGn (with NG-PAM) with <i>Escherichia coli</i> endonuclease V (EndoV) and human alkyladenine DNA glycosylase (hAAG), respectively, to generate ABE8e-EndoV and ABE8e-hAAG vectors. An assessment of five NG-PAM target sites showed that these two vectors could efficiently produce A-to-G substitutions in a narrow editing window; however, no A-to-Y editing was detected. Interestingly, the ABE8e-EndoV also generated precise small fragment deletions in the editing window from the 5′-deaminated A base to the SpGn cleavage site, suggesting its potential value in producing predictable small-fragment deletion mutations. Overall, we objectively evaluated the editing performance of CGBEs in rice, explored the possibility of A-to-Y editing, and developed a new ABE8e-EndoV tool, thus providing a valuable reference for improving and enriching base editing tools in plants. |
| first_indexed | 2024-03-09T03:20:25Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-09T03:20:25Z |
| publishDate | 2022-07-01 |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-8098e2736b0840ea9afefc4a2936dfb02023-12-03T15:10:41ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-07-012314799010.3390/ijms23147990Exploring C-to-G and A-to-Y Base Editing in Rice by Using New Vector ToolsDongchang Zeng0Zhiye Zheng1Yuxin Liu2Taoli Liu3Tie Li4Jianhong Liu5Qiyu Luo6Yang Xue7Shengting Li8Nan Chai9Suize Yu10Xianrong Xie11Yao-Guang Liu12Qinlong Zhu13State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Sciences, South China Agricultural University, Guangzhou 510642, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Sciences, South China Agricultural University, Guangzhou 510642, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Sciences, South China Agricultural University, Guangzhou 510642, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Sciences, South China Agricultural University, Guangzhou 510642, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Sciences, South China Agricultural University, Guangzhou 510642, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Sciences, South China Agricultural University, Guangzhou 510642, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Sciences, South China Agricultural University, Guangzhou 510642, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Sciences, South China Agricultural University, Guangzhou 510642, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Sciences, South China Agricultural University, Guangzhou 510642, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Sciences, South China Agricultural University, Guangzhou 510642, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Sciences, South China Agricultural University, Guangzhou 510642, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Sciences, South China Agricultural University, Guangzhou 510642, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Sciences, South China Agricultural University, Guangzhou 510642, ChinaState Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Sciences, South China Agricultural University, Guangzhou 510642, ChinaCRISPR/Cas9-based cytosine base editors (CBEs) and adenine base editors (ABEs) can efficiently mediate C-to-T/G-to-A and A-to-G/T-to-C substitutions, respectively; however, achieving base transversions (C-to-G/C-to-A and A-to-T/A-to-C) is challenging and has been rarely studied in plants. Here, we constructed new plant C-to-G base editors (CGBEs) and new A-to-Y (T/C) base editors and explored their base editing characteristics in rice. First, we fused the highly active cytidine deaminase evoFENRY and the PAM-relaxed Cas9-nickase variant Cas9n-NG with rice and human uracil DNA N-glycosylase (rUNG and hUNG), respectively, to construct CGBE-rUNG and CGBE-hUNG vector tools. The analysis of five NG-PAM target sites showed that these CGBEs achieved C-to-G conversions with monoallelic editing efficiencies of up to 27.3% in T<sub>0</sub> rice, with major byproducts being insertion/deletion mutations. Moreover, for the A-to-Y (C or T) editing test, we fused the highly active adenosine deaminase TadA8e and the Cas9-nickase variant SpGn (with NG-PAM) with <i>Escherichia coli</i> endonuclease V (EndoV) and human alkyladenine DNA glycosylase (hAAG), respectively, to generate ABE8e-EndoV and ABE8e-hAAG vectors. An assessment of five NG-PAM target sites showed that these two vectors could efficiently produce A-to-G substitutions in a narrow editing window; however, no A-to-Y editing was detected. Interestingly, the ABE8e-EndoV also generated precise small fragment deletions in the editing window from the 5′-deaminated A base to the SpGn cleavage site, suggesting its potential value in producing predictable small-fragment deletion mutations. Overall, we objectively evaluated the editing performance of CGBEs in rice, explored the possibility of A-to-Y editing, and developed a new ABE8e-EndoV tool, thus providing a valuable reference for improving and enriching base editing tools in plants.https://www.mdpi.com/1422-0067/23/14/7990CGBEABE8e-EndoVCBEABECRISPR/Cas9rice |
| spellingShingle | Dongchang Zeng Zhiye Zheng Yuxin Liu Taoli Liu Tie Li Jianhong Liu Qiyu Luo Yang Xue Shengting Li Nan Chai Suize Yu Xianrong Xie Yao-Guang Liu Qinlong Zhu Exploring C-to-G and A-to-Y Base Editing in Rice by Using New Vector Tools International Journal of Molecular Sciences CGBE ABE8e-EndoV CBE ABE CRISPR/Cas9 rice |
| title | Exploring C-to-G and A-to-Y Base Editing in Rice by Using New Vector Tools |
| title_full | Exploring C-to-G and A-to-Y Base Editing in Rice by Using New Vector Tools |
| title_fullStr | Exploring C-to-G and A-to-Y Base Editing in Rice by Using New Vector Tools |
| title_full_unstemmed | Exploring C-to-G and A-to-Y Base Editing in Rice by Using New Vector Tools |
| title_short | Exploring C-to-G and A-to-Y Base Editing in Rice by Using New Vector Tools |
| title_sort | exploring c to g and a to y base editing in rice by using new vector tools |
| topic | CGBE ABE8e-EndoV CBE ABE CRISPR/Cas9 rice |
| url | https://www.mdpi.com/1422-0067/23/14/7990 |
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