利用 CRISPR/Cas9 系统编辑拟南芥 2 个双链结合蛋白

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（dsRNA-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 tRNA 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 T1 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。

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

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 (gRNA) which tandemly arrayed with tRNA. The sequencing results showed the DRB3 or DRB5 gene in Arabidopsis dcl2drb4 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.