title: An alternative strategy for targeted gene replacement in plants using a dual-sgRNA/Cas9 design.
abstract: 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.
authors.affiliation: Institute of Crop Science, Chinese Academy of Agricultural Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing, 100081 China.
authors.forename: Yongping
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Institute of Crop Science, Chinese Academy of Agricultural Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing, 100081 China.|Anhui Agricultural University, Hefei, Anhui Province, 230036 China.
authors.forename: Congsheng
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Institute of Crop Science, Chinese Academy of Agricultural Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing, 100081 China.
authors.forename: Wenwen
authors.initials: W
authors.lastname: Liu
authors.affiliation: Institute of Crop Science, Chinese Academy of Agricultural Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing, 100081 China.
authors.forename: Wei
authors.initials: W
authors.lastname: Gao
authors.affiliation: Institute of Crop Science, Chinese Academy of Agricultural Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing, 100081 China.
authors.forename: Changlin
authors.initials: C
authors.lastname: Liu
authors.affiliation: Institute of Crop Science, Chinese Academy of Agricultural Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing, 100081 China.
authors.forename: Gaoyuan
authors.initials: G
authors.lastname: Song
authors.affiliation: Institute of Crop Science, Chinese Academy of Agricultural Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing, 100081 China.
authors.forename: Wen-Xue
authors.initials: WX
authors.lastname: Li
authors.affiliation: Institute of Crop Science, Chinese Academy of Agricultural Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing, 100081 China.
authors.forename: Long
authors.initials: L
authors.lastname: Mao
authors.affiliation: Anhui Agricultural University, Hefei, Anhui Province, 230036 China.
authors.forename: Beijiu
authors.initials: B
authors.lastname: Chen
authors.affiliation: Institute of Crop Science, Chinese Academy of Agricultural Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing, 100081 China.
authors.forename: Yunbi
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Institute of Crop Science, Chinese Academy of Agricultural Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing, 100081 China.
authors.forename: Xinhai
authors.initials: X
authors.lastname: Li
authors.affiliation: Institute of Crop Science, Chinese Academy of Agricultural Sciences, National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing, 100081 China.
authors.forename: Chuanxiao
authors.initials: C
authors.lastname: Xie
mesh_terms: D017360:Arabidopsis / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics; D064113:CRISPR-Cas Systems; D017353:Gene Deletion; D000072669:Gene Editing / Q000379:methods*; D055786:Gene Knockout Techniques; D017343:Genes, Plant; D005822:Genetic Vectors / Q000235:genetics*; D035683:MicroRNAs / Q000235:genetics; D009154:Mutation; D030821:Plants, Genetically Modified; D018749:RNA, Plant / Q000235:genetics; D014170:Transformation, Genetic
chemical_list: D029681:Arabidopsis Proteins; C000591832:MIRN169 microRNA, Arabidopsis; C559523:MIRN827 microRNA, Arabidopsis; D035683:MicroRNAs; D018749:RNA, Plant; C102864:TFL1 protein, Arabidopsis