CRISPR/Cas9编辑花生FAD2基因研究

张旺, 冼俊霖, 孙超, 王春明, 石丽, 于为常

Preliminary study of genome editing of peanut FAD2 genes by CRISPR/Cas9

ZHANG Wang, XIAN Jun-Lin, SUN Chao, WANG Chun-Ming, SHI Li, YU Wei-Chang

图1 基因组编辑载体的构建 合成含有gRNA1和gRNA2序列的引物1和引物2, 以pCBC-DT1T2载体为模版合成sgRNA片段, 并通过引物1和引物2上的Bsa I酶切位点, 克隆到pSKE401载体, 获得含2个靶点的基因组编辑载体FAD2T1T2。LB、RB分别为农杆菌T-DNA的左右边界; U6-26p、U6-29p为拟南芥U6基因启动子; U6-29T、U6-26T为转录终止子; 35Sp-Cas9-NosT、35Sp-NptII-polyA分别为Cas9和NptII基因表达框。

Fig. 1 Construction of genome editing vector Primer 1 and primer 2 containing gRNA1 and gRNA2 sequences, are used to synthesize sgRNA by PCR with pCBC-DT1T2 as template. The synthesized sgRNA was cloned into pSKE401 plasmid by Bsa I restriction enzyme sites to obtain FAD2T1T1 genome editing construct. LB, RB denote T-DNA left and right border sequences; U6-26p, U6-29p are promoters while U6-29T and U6-26T are terminators of Arabidopsis U6 gene; 35Sp-Cas9-NosT, 35Sp-NptII-polyA are gene expression cassettes of Cas9 and NptII genes.