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An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design
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An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design
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Journal Article
An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design
2016
Overview
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.
