An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design
Precision DNA/gene replacement is a promising genome-editing tool that is highly desirable for molecular engineering and breeding by design. Although the CRISPR/Cas9 system works well as a tool for gene knockout in plants, gene replacement has rarely been reported. Towards this end, we first designed a combinatory dual-sgRNA/Cas9 vector (construct #1) that successfully deleted miRNA gene regions (MIR169a and MIR827a). The deletions were confirmed by PCR and subsequent sequencing, yielding deletion efficiencies of 20% and 24% on MIR169a and MIR827a loci, respectively. We designed a second structure (construct #2) that contains sites homologous to Arabidopsis TERMINAL FLOWER 1 (TFL1) for homology-directed repair (HDR) with regions corresponding to the two sgRNAs on the modified construct #1. The two constructs were co-transformed into Arabidopsis plants to provide both targeted deletion and donor repair for targeted gene replacement by HDR. Four of 500 stably transformed T0 transgenic plants (0.8%) contained replaced fragments. The presence of the expected recombination sites was further confirmed by sequencing. Therefore, we successfully established a gene deletion/replacement system in stably transformed plants that can potentially be utilized to introduce genes of interest for targeted crop improvement.
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Nature Publishing Group
2016
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Subjects: | AGRICULTURAL SCIENCES AND BIOTECHNOLOGY, GENES, PLANTS, DNA SEQUENCE, CHROMOSOME MANIPULATION, |
Online Access: | http://hdl.handle.net/10883/18311 |
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dig-cimmyt-10883-183112023-11-14T21:21:51Z An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design Yongping Zhao Congsheng Zhang Wenwen Liu Wei Gao Changlin Liu Gaoyuan, Song Wen-Xue Li Long Mao Cheng, Beijiu Yunbi Xu Xinhai Li Chuanxiao Xie AGRICULTURAL SCIENCES AND BIOTECHNOLOGY GENES PLANTS DNA SEQUENCE CHROMOSOME MANIPULATION Precision DNA/gene replacement is a promising genome-editing tool that is highly desirable for molecular engineering and breeding by design. Although the CRISPR/Cas9 system works well as a tool for gene knockout in plants, gene replacement has rarely been reported. Towards this end, we first designed a combinatory dual-sgRNA/Cas9 vector (construct #1) that successfully deleted miRNA gene regions (MIR169a and MIR827a). The deletions were confirmed by PCR and subsequent sequencing, yielding deletion efficiencies of 20% and 24% on MIR169a and MIR827a loci, respectively. We designed a second structure (construct #2) that contains sites homologous to Arabidopsis TERMINAL FLOWER 1 (TFL1) for homology-directed repair (HDR) with regions corresponding to the two sgRNAs on the modified construct #1. The two constructs were co-transformed into Arabidopsis plants to provide both targeted deletion and donor repair for targeted gene replacement by HDR. Four of 500 stably transformed T0 transgenic plants (0.8%) contained replaced fragments. The presence of the expected recombination sites was further confirmed by sequencing. Therefore, we successfully established a gene deletion/replacement system in stably transformed plants that can potentially be utilized to introduce genes of interest for targeted crop improvement. 2017-05-09T19:11:24Z 2017-05-09T19:11:24Z 2016 Article http://hdl.handle.net/10883/18311 10.1038/srep23890 English https://www.nature.com/articles/srep23890#Sec23 CIMMYT manages Intellectual Assets as International Public Goods. The user is free to download, print, store and share this work. In case you want to translate or create any other derivative work and share or distribute such translation/derivative work, please contact CIMMYT-Knowledge-Center@cgiar.org indicating the work you want to use and the kind of use you intend; CIMMYT will contact you with the suitable license for that purpose. Open Access PDF London Nature Publishing Group 6 Scientific Reports 23890 |
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AGRICULTURAL SCIENCES AND BIOTECHNOLOGY GENES PLANTS DNA SEQUENCE CHROMOSOME MANIPULATION AGRICULTURAL SCIENCES AND BIOTECHNOLOGY GENES PLANTS DNA SEQUENCE CHROMOSOME MANIPULATION |
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AGRICULTURAL SCIENCES AND BIOTECHNOLOGY GENES PLANTS DNA SEQUENCE CHROMOSOME MANIPULATION AGRICULTURAL SCIENCES AND BIOTECHNOLOGY GENES PLANTS DNA SEQUENCE CHROMOSOME MANIPULATION Yongping Zhao Congsheng Zhang Wenwen Liu Wei Gao Changlin Liu Gaoyuan, Song Wen-Xue Li Long Mao Cheng, Beijiu Yunbi Xu Xinhai Li Chuanxiao Xie An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design |
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Precision DNA/gene replacement is a promising genome-editing tool that is highly desirable for molecular engineering and breeding by design. Although the CRISPR/Cas9 system works well as a tool for gene knockout in plants, gene replacement has rarely been reported. Towards this end, we first designed a combinatory dual-sgRNA/Cas9 vector (construct #1) that successfully deleted miRNA gene regions (MIR169a and MIR827a). The deletions were confirmed by PCR and subsequent sequencing, yielding deletion efficiencies of 20% and 24% on MIR169a and MIR827a loci, respectively. We designed a second structure (construct #2) that contains sites homologous to Arabidopsis TERMINAL FLOWER 1 (TFL1) for homology-directed repair (HDR) with regions corresponding to the two sgRNAs on the modified construct #1. The two constructs were co-transformed into Arabidopsis plants to provide both targeted deletion and donor repair for targeted gene replacement by HDR. Four of 500 stably transformed T0 transgenic plants (0.8%) contained replaced fragments. The presence of the expected recombination sites was further confirmed by sequencing. Therefore, we successfully established a gene deletion/replacement system in stably transformed plants that can potentially be utilized to introduce genes of interest for targeted crop improvement. |
format |
Article |
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AGRICULTURAL SCIENCES AND BIOTECHNOLOGY GENES PLANTS DNA SEQUENCE CHROMOSOME MANIPULATION |
author |
Yongping Zhao Congsheng Zhang Wenwen Liu Wei Gao Changlin Liu Gaoyuan, Song Wen-Xue Li Long Mao Cheng, Beijiu Yunbi Xu Xinhai Li Chuanxiao Xie |
author_facet |
Yongping Zhao Congsheng Zhang Wenwen Liu Wei Gao Changlin Liu Gaoyuan, Song Wen-Xue Li Long Mao Cheng, Beijiu Yunbi Xu Xinhai Li Chuanxiao Xie |
author_sort |
Yongping Zhao |
title |
An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design |
title_short |
An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design |
title_full |
An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design |
title_fullStr |
An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design |
title_full_unstemmed |
An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design |
title_sort |
alternative strategy for targeted gene replacement in plants using a dual-sgrna/cas9 design |
publisher |
Nature Publishing Group |
publishDate |
2016 |
url |
http://hdl.handle.net/10883/18311 |
work_keys_str_mv |
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