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

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.

Col0 wild type; 8,12,31,32,33,34,35,36,39,42: the 10 lines in which the heterozygous targeted mutation was identified. Supplementary File 6. The raw sequencing peak of the reverse complementary sequence harboring re-joining junction site after DNA donor had been target deleted in plant.

Supplementary File 7, 8 and 9.
Three more biological replicates of the sequencing evidence for harboring re-joining junction site after DNA donor had been target deleted in plants. are expected to be joined together after precision repair of both DSB lesions induced by the two sgRNAs.
Supplementary file 1. The desired sequence detail of partial AtTFL1 was replaced with eGFP.

Captions:
 Sequence in red letters is the replaced sequence harboring the expression cassette of eGFP.  Sequence with light blue shadowed is the sequence of the homologs arms.  All primer regions were underlined in lowercase letters.  At the middle junction sites.  The captions were inserted between the lines of the sequence.  The junction sites 1-4 were indicated. The different color letters in word "junction" indicate the real junction site located in the sequence. For example,"Junction 1" indicates that the junction sites were located within"ATCGGTTAGC"showing different shadow colors. "GT"was the very junction site.  Title ： An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design Authors： Zhao Y., Zhang C., Liu W., Gao W., Liu C., Song G., Li W-X, Mao L., Chen B., Xu Y., Li X., Xie C.
Supplementary File 11. The list of primers used in this study.

Primer orientation
Sequence (