CRISPR/Cas9基因编辑技术在小菜蛾细胞系中的优化与应用

Abstract

The CRISPR/Cas9 system is an efficient tool for reverse genetics validation, and the application of this system in the cell lines provides a new perspective on target gene analysis for the development of biotechnology tools. However, in the cell lines of diamondback moth,Plutella xylostella, the integrity of the CRISPR/Cas9 system and the utilization of this cell lines still need to be improved to ensure the application of the system. Here, we stabilize the transfection efficiency of theP. xylostellacell lines at different passages at about 60% by trying different transfection reagents and adjusting the transfection method. For Cas9 expression in the CRIPSPR/Cas9 system, we identified a strong endogenous promoter: the 217–2 promoter. The dual-luciferase and EGFP reporter assay demonstrated that it has a driving efficiency close to that of the IE1 promoter. We constructed pB-Cas9-Neo plasmid and pU6-sgRNA plasmid for CRISPR/Cas9 system and subsequent cell screening. The feasibility of the CRISPR/Cas9 system inP. xylostellacell lines was verified by knocking out endogenous and exogenous genes. Finally, we generated a transgenic Cas9 cell line ofP. xylostellathat would benefit future exploitation, such as knock-in and multi-threaded editing. Our works provides the validity of the CRISPR/Cas9 system in theP. xylostellacell lines and lays the foundation for further genetic and molecular studies on insects, particularly favoring gene function analysis.Keywords:cell lines;Plutella xylostella;endogenous promoter;plasmid delivery