Optimization and Application of CRISPR/Cas9 Genome Editing in a Cosmopolitan Pest, Diamondback Moth

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 the P. xylostella cell 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 in P. xylostella cell lines was verified by knocking out endogenous and exogenous genes. Finally, we generated a transgenic Cas9 cell line of P. xylostella that would benefit future exploitation, such as knock-in and multi-threaded editing. Our works provides the validity of the CRISPR/Cas9 system in the P. xylostella cell lines and lays the foundation for further genetic and molecular studies on insects, particularly favoring gene function analysis.

New Insights into the Plutella xylostella Detoxifying Enzymes: Sequence Evolution, Structural Similarity, Functional Diversity, and Application Prospects of Glucosinolate Sulfatases

“…Recent developments include the successful establishment of multithreaded editing tools in cells. A transgenic insect line has been used to test gene drive efficiency in P. xylostella , confirming the availability of the U6 promoter for transcribing fluorescent/phenotypic marker genes − and potential germline-specific promoters for driving Cas9. , However, the feasibility of using U6 Pol III promoters and germline-active Pol II promoters ( Pxvasa , PxnanosP , and Pxmeiw68 ) has been confirmed in the corresponding transgenic lines. The gene drive rate was extremely minimal in hybrid offspring based on various cross-combinations. , Although Pxvasa and PxnanosP drove higher rates of Cas9 activity than Pxmeiw68 , both promoters also displayed unexpected leaky somatic expression of Cas9 and a low homing rate.…”