Repositorium
An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design
264
Journal Article / 2016
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
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.
Techniques
ID | Corresponding Author Country |
Plant Species | GE Technique Sequence Identifier |
Trait Type of Alteration |
Progress in Research Key Topic |
---|---|---|---|---|---|
637 |
Xie, Chuanxiao China |
Arabidopsis thaliana |
CRISPR/Cas9 mir169a |
Improved drought resistance SDN1 |
Basic research Basic research |
638 |
Xie, Chuanxiao China |
Arabidopsis thaliana |
CRISPR/Cas9 mir827a |
Improved drought resistance SDN1 |
Basic research Basic research |
639 |
Xie, Chuanxiao China |
Arabidopsis thaliana |
CRISPR/Cas9 TFL1 |
No information SDN3 |
Basic research Basic research |