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

Author: Yongping Zhao, /
Author: Congsheng Zhang
Author: Wenwen Liu
Author: Wei Gao
Author: Changlin Liu
Author: Gaoyuan, Song
Author: Wen-Xue Li, /
Author: Long Mao, /
Author: Cheng, Beijiu
Author: Yunbi Xu
Author: Xinhai Li, /
Author: Chuanxiao Xie, /
Year: 2016
Descriptors: Genes
Descriptors: Plants
Abstract: 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.
Language: English
Publisher: Nature Publishing Group
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Type: Article
Place: London
Journal issue: art. 23890
Journal: Scientific Reports
Journal volume: 6
DOI: 10.1038/srep23890

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  • Maize
    Maize breeding, phytopathology, entomology, physiology, quality, and biotech

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