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An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design

Autor: Yongping Zhao, /
Autor: Congsheng Zhang
Autor: Wenwen Liu
Autor: Wei Gao
Autor: Changlin Liu
Autor: Gaoyuan, Song
Autor: Wen-Xue Li, /
Autor: Long Mao, /
Autor: Cheng, Beijiu
Autor: Yunbi Xu
Autor: Xinhai Li, /
Autor: Chuanxiao Xie, /
Año: 2016
URI: http://hdl.handle.net/10883/18311
Descripción: Genes
Descripción: Plants
Resumen: 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.
Lenguaje: English
Editor: Nature Publishing Group
Copyright: 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.
Tipo: Article
Lugar: London
No de Revista: art. 23890
Revista: Scientific Reports
Volumen de la Revista: 6
DOI: 10.1038/srep23890


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