TY  - JA
AU  - Yongping Zhao
AU  - Congsheng Zhang
AU  - Wenwen Liu
AU  - Wei Gao
AU  - Changlin Liu
AU  - Gaoyuan Song
AU  - Wen-Xue Li
AU  - Long Mao
AU  - Beijiu Chen
AU  - Yunbi Xu
AU  - Xinhai Li
AU  - Chuanxiao Xie
TI  - An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design
PY  - 2016///
CY  - London
PB  - Nature Publishing Group
KW  - Genes
KW  - AGROVOC
KW  - Plants
KW  - Genetic engineering
KW  - Molecular genetics
N1  - Peer review; Open Access; MCRP; FP2; FP3
N2  - 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
UR  - http://hdl.handle.net/10883/18311
T2  - Nature Scientific reports
DO  - https://doi.org/10.1038/srep23890
ER  -