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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.

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