An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design
Author(s) -
Yongping Zhao,
Congsheng Zhang,
Wénwén Liú,
Wei Gao,
Changlin Liu,
Gaoyuan Song,
Wenxue Li,
Long Mao,
Beijiu Chen,
Yunbi Xu,
Xinhai Li,
Chuanxiao Xie
Publication year - 2016
Publication title -
scientific reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.24
H-Index - 213
ISSN - 2045-2322
DOI - 10.1038/srep23890
Subject(s) - cas9 , genome editing , gene , crispr , genome engineering , biology , subgenomic mrna , computational biology , arabidopsis , genetics , guide rna , homologous recombination , gene targeting , transgene , mutant
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.
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