利用CRISPR/Cas9系统编辑拟南芥2个双链结合蛋白

Mutanted Double DRBs Based on CRSPR/Cas9 Technology in Arabidopsis thaliana

CRSPR/Cas9系统可对植物的内源基因进行有效定点编辑,为获得基因缺失突变体提供了新的方向。本研究在双链结合蛋白(ds RNA-binding protein,DRB)DRB3和DRB5两个基因外显子的保守序列设计2个靶点,并构建与t RNA串联的双靶点CRISPR/Cas9表达载体。通过一次转化,获得了DRB3或DRB5单独被编辑或同时被编辑的拟南芥突变体dcl2drb4转基因T_1代植株,为研究DRB3、DRB5是否参与DCL4介导的抗病毒RNA沉默通路奠定基础。

The CRSPR/Cas9 system can directly edit endogenous genomic loci in plant, and provide a new opportunity to obtain knockout mutants. In this study, two guide RNAs were designed based on the conserve sequence of the exon between DRB3 and DRB5 gene in Arabidopsis thaliana. To achieve efficient editing capability of the CRISPR/Cas9 system we developed a construct with double small guide RNA（g RNA） which tandemly arrayed with t RNA. The sequencing results showed the DRB3 or DRB5 gene in Arabidopsis dcl2 drb4 double mutant was successfully edited individuallly or altogether in T1 generation. The result would pave the way on research whether the DRB3 or DRB5 gene involved in DCL4-initiated antiviral RNA silencing pathway which is independent of DRB4 protein.