Simple promotion of Cas9 and Cas12a expression improves gene targeting via an all-in-one strategy
Gene targeting (GT) is a promising tool for precise manipulation of genome sequences, however, GT in seed plants remains a challenging task. The simple and direct way to improve the efficiency of GT via homology-directed repair (HDR) is to increase the frequency of double-strand breaks (DSBs) at tar...
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2024-03-01
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| Series: | Frontiers in Plant Science |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fpls.2024.1360925/full |
| _version_ | 1797263647931629568 |
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| author | Yiqiu Cheng Yiqiu Cheng Lei Zhang Lei Zhang Jing Li Jing Li Xiaofei Dang Jian-Kang Zhu Jian-Kang Zhu Hiroaki Shimada Daisuke Miki |
| author_facet | Yiqiu Cheng Yiqiu Cheng Lei Zhang Lei Zhang Jing Li Jing Li Xiaofei Dang Jian-Kang Zhu Jian-Kang Zhu Hiroaki Shimada Daisuke Miki |
| author_sort | Yiqiu Cheng |
| collection | DOAJ |
| description | Gene targeting (GT) is a promising tool for precise manipulation of genome sequences, however, GT in seed plants remains a challenging task. The simple and direct way to improve the efficiency of GT via homology-directed repair (HDR) is to increase the frequency of double-strand breaks (DSBs) at target sites in plants. Here we report an all-in-one approach of GT in Arabidopsis by combining a transcriptional and a translational enhancer for the Cas expression. We find that facilitating the expression of Cas9 and Cas12a variant by using enhancers can improve DSB and subsequent knock-in efficiency in the Arabidopsis genome. These results indicate that simply increasing Cas protein expression at specific timings - egg cells and early embryos - can improve the establishment of heritable GTs. This simple approach allows for routine genome engineering in plants. |
| first_indexed | 2024-04-25T00:16:20Z |
| format | Article |
| id | doaj.art-255362da47864c3cad066af07b1a37f1 |
| institution | Directory Open Access Journal |
| issn | 1664-462X |
| language | English |
| last_indexed | 2024-04-25T00:16:20Z |
| publishDate | 2024-03-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Plant Science |
| spelling | doaj.art-255362da47864c3cad066af07b1a37f12024-03-13T04:35:53ZengFrontiers Media S.A.Frontiers in Plant Science1664-462X2024-03-011510.3389/fpls.2024.13609251360925Simple promotion of Cas9 and Cas12a expression improves gene targeting via an all-in-one strategyYiqiu Cheng0Yiqiu Cheng1Lei Zhang2Lei Zhang3Jing Li4Jing Li5Xiaofei Dang6Jian-Kang Zhu7Jian-Kang Zhu8Hiroaki Shimada9Daisuke Miki10Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, ChinaUniversity of Chinese Academy of Sciences, Beijing, ChinaShanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, ChinaUniversity of Chinese Academy of Sciences, Beijing, ChinaShanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, ChinaUniversity of Chinese Academy of Sciences, Beijing, ChinaShanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, ChinaInstitute of Advanced Biotechnology and School of Life Sciences, Southern University of Science and Technology, Shenzhen, ChinaCenter for Advanced Bioindustry Technologies, Chinese Academy of Agricultural Sciences, Beijing, ChinaDepartment of Biological Science and Technology, Tokyo University of Science, Tokyo, JapanShanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai, ChinaGene targeting (GT) is a promising tool for precise manipulation of genome sequences, however, GT in seed plants remains a challenging task. The simple and direct way to improve the efficiency of GT via homology-directed repair (HDR) is to increase the frequency of double-strand breaks (DSBs) at target sites in plants. Here we report an all-in-one approach of GT in Arabidopsis by combining a transcriptional and a translational enhancer for the Cas expression. We find that facilitating the expression of Cas9 and Cas12a variant by using enhancers can improve DSB and subsequent knock-in efficiency in the Arabidopsis genome. These results indicate that simply increasing Cas protein expression at specific timings - egg cells and early embryos - can improve the establishment of heritable GTs. This simple approach allows for routine genome engineering in plants.https://www.frontiersin.org/articles/10.3389/fpls.2024.1360925/fullgenome engineeringCRISPR/Cas9CRISPR/ttCas12aRPS5aall-in-onegene targeting (GT) |
| spellingShingle | Yiqiu Cheng Yiqiu Cheng Lei Zhang Lei Zhang Jing Li Jing Li Xiaofei Dang Jian-Kang Zhu Jian-Kang Zhu Hiroaki Shimada Daisuke Miki Simple promotion of Cas9 and Cas12a expression improves gene targeting via an all-in-one strategy Frontiers in Plant Science genome engineering CRISPR/Cas9 CRISPR/ttCas12a RPS5a all-in-one gene targeting (GT) |
| title | Simple promotion of Cas9 and Cas12a expression improves gene targeting via an all-in-one strategy |
| title_full | Simple promotion of Cas9 and Cas12a expression improves gene targeting via an all-in-one strategy |
| title_fullStr | Simple promotion of Cas9 and Cas12a expression improves gene targeting via an all-in-one strategy |
| title_full_unstemmed | Simple promotion of Cas9 and Cas12a expression improves gene targeting via an all-in-one strategy |
| title_short | Simple promotion of Cas9 and Cas12a expression improves gene targeting via an all-in-one strategy |
| title_sort | simple promotion of cas9 and cas12a expression improves gene targeting via an all in one strategy |
| topic | genome engineering CRISPR/Cas9 CRISPR/ttCas12a RPS5a all-in-one gene targeting (GT) |
| url | https://www.frontiersin.org/articles/10.3389/fpls.2024.1360925/full |
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