Zhao Yongping, Zhang Congsheng, Liu Wenwen, Gao Wei, Liu Changlin, Song Gaoyuan, Li Wen-Xue, Mao Long, Chen Beijiu, Xu Yunbi, Li Xinhai, Xie Chuanxiao
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.
Sci Rep. 2016 Apr 1;6:23890. doi: 10.1038/srep23890.
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.
精确DNA/基因替换是一种很有前景的基因组编辑工具，对于分子工程和设计育种来说是非常理想的。尽管CRISPR/Cas9系统作为植物基因敲除工具效果良好，但基因替换却鲜有报道。为此，我们首先设计了一种组合式双sgRNA/Cas9载体（构建体#1），该载体成功删除了miRNA基因区域（MIR169a和MIR827a）。通过PCR和后续测序确认了缺失情况，MIR169a和MIR827a位点的缺失效率分别为20%和24%。我们设计了第二种结构（构建体#2），其包含与拟南芥TERMINAL FLOWER 1（TFL1）同源的位点，用于同源定向修复（HDR），该区域与修饰后的构建体#1上的两个sgRNA相对应。将这两种构建体共转化到拟南芥植株中，通过HDR为靶向基因替换提供靶向缺失和供体修复。500株稳定转化的T0转基因植株中有4株（0.8%）含有替换片段。通过测序进一步确认了预期重组位点的存在。因此，我们成功地在稳定转化的植株中建立了一个基因缺失/替换系统，该系统有可能用于引入感兴趣的基因以进行靶向作物改良。
