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 designe...

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Published in:Scientific reports 2016-04, Vol.6 (1), p.23890-23890, Article 23890
Main Authors: Zhao, Yongping, Zhang, Congsheng, Liu, Wenwen, Gao, Wei, Liu, Changlin, Song, Gaoyuan, Li, Wen-Xue, Mao, Long, Chen, Beijiu, Xu, Yunbi, Li, Xinhai, Xie, Chuanxiao
Format: Article
Language:English
Subjects:
Arabidopsis - genetics
Arabidopsis Proteins - genetics
CRISPR
CRISPR-Cas Systems
Crop improvement
Design
Gene Deletion
Gene Editing - methods
Gene Knockout Techniques
Genes, Plant
Genetic Vectors - genetics
Genome editing
Genomes
Homology
MicroRNAs - genetics
miRNA
Mutation
Plant breeding
Plants, Genetically Modified
Polymerase chain reaction
Recombination
RNA, Plant - genetics
Transformation, Genetic
Transgenic plants
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Summary: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.
ISSN:2045-2322
2045-2322
DOI:10.1038/srep23890