Optimization and Application of CRISPR/Cas9 Genome Editing in a Cosmopolitan Pest, Diamondback Moth
Abstract
:1. Introduction
2. Results
2.1. High Transfection Efficiency of the P. xylostella Cell Lines
2.2. Identification of a Strong Endogenous Promoters
2.3. Characterization of the 217–2 Promoter
2.4. CRISPR/Cas9-Mediated Knockout of Endogenous and Exogenous Gene in P. xylostella Cell Lines
2.5. Generation of a Transgenic Cas9 P. xylostella Cell Lines
3. Discussion
4. Materials and Methods
4.1. The Subculture and Transfection of the P. xylostella Cell Lines
4.1.1. Cell Subculture
4.1.2. Cell Transfection
4.2. Calculation of Transfection Efficiency of the P. xylostella Cell Lines
4.3. Construction of Phylogenetic Tree and Characterization of Promoters
4.4. Acquisition of Potential Promoter Regions
4.5. Construction of Plasmids
4.5.1. Construction of luc2 (Photinus pyralis) Luciferase Expression Plasmids
4.5.2. Construction of pB-Cas9-Neo Plasmid
4.5.3. Construction of pU6-sgRNA Plasmid and pU6-shRNA Plasmid
4.6. Dual-Luciferase Reporter Assay Experiment
4.7. RT-qPCR Detection
4.8. DAPI Staining Assay
4.9. T7 Endonuclease I Digestion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Zhang, Z.; Xiong, L.; Xie, C.; Shen, L.; Chen, X.; Ye, M.; Sun, L.; Yang, X.; Yao, S.; Yue, Z.; et al. Optimization and Application of CRISPR/Cas9 Genome Editing in a Cosmopolitan Pest, Diamondback Moth. Int. J. Mol. Sci. 2022, 23, 13042. https://doi.org/10.3390/ijms232113042
Zhang Z, Xiong L, Xie C, Shen L, Chen X, Ye M, Sun L, Yang X, Yao S, Yue Z, et al. Optimization and Application of CRISPR/Cas9 Genome Editing in a Cosmopolitan Pest, Diamondback Moth. International Journal of Molecular Sciences. 2022; 23(21):13042. https://doi.org/10.3390/ijms232113042
Chicago/Turabian StyleZhang, Zhen, Lei Xiong, Chao Xie, Lingling Shen, Xuanhao Chen, Min Ye, Linyang Sun, Xiaozhen Yang, Shuyuan Yao, Zhen Yue, and et al. 2022. "Optimization and Application of CRISPR/Cas9 Genome Editing in a Cosmopolitan Pest, Diamondback Moth" International Journal of Molecular Sciences 23, no. 21: 13042. https://doi.org/10.3390/ijms232113042