CRISPR/Cas9-Mediated Knockout of the White Gene in Agasicles hygrophila
Abstract
1. Introduction
2. Results
2.1. Phylogenetic Identification of the A. hygrophila White (AhW) Gene
2.2. Creation of an AhW Knockout White-Eyed Strain
2.3. Mutations in the Two sgRNA Target Sites in AhW gDNA from the AhW-KO Strain
2.4. Inheritance of Cas9-Introduced White Eye Mutations
2.5. Time Course of AhW Expression and Blackening of Eyes
3. Discussion
4. Materials and Methods
4.1. Insects
4.2. Sequence Amplification and Analysis
4.3. sgRNA Design and Synthesis
4.4. Embryonic Microinjection
4.5. Mutagenesis Analysis
4.6. Reverse Transcription Quantitative PCR (RT-qPCR)
4.7. Eye Pigment Observation
4.8. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene | Total Score | Query Covery | E Value | Identity |
---|---|---|---|---|
Dm-white | 694 | 100% | 0 | 49.35% |
Dm-scarlet | 399 | 85% | 5 × 10−130 | 36.12% |
Dm-brown | 250 | 86% | 2 × 10−73 | 27.26% |
Generations | Parents | Total Adult Progeny | Female Progeny | Male Progeny | Females with Black Eyes | Females with White Eyes | Males with Black Eyes | Males with White Eyes | Black/White Ratio for All Progeny | Black/White Ratio for Females | Black/White Ratio for Males |
---|---|---|---|---|---|---|---|---|---|---|---|
G1 hybrid | 1 ♀AhW × 1 ♂AhW-KO | 83 | 44 | 39 | 44 | 0 | 39 | 0 | 1.0:0 | 1.0:0 | 1.0:0 |
G1′ hybrid | 1 ♀AhW-KO × 1 ♂AhW | 72 | 38 | 34 | 38 | 0 | 0 | 34 | 1.1:1 | 1:0 | 0:1.0 |
Self-inbred G2 | 44 ♀G1 × 39 ♂ G1 | 486 | 232 | 254 | 232 | 0 | 130 | 124 | 2.92:1 | 1:0 | 1.05:1.0 |
Self-inbred G2′ | 38 ♀G1′ × 34 ♂ G1′ | 433 | 226 | 207 | 115 | 111 | 108 | 99 | 1.06:1.0 | 1.04:1.0 | 1.09:1.0 |
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Fu, L.; Li, P.; Rui, Z.; Sun, J.; Yang, J.; Wang, Y.; Jia, D.; Hu, J.; Li, X.; Ma, R. CRISPR/Cas9-Mediated Knockout of the White Gene in Agasicles hygrophila. Int. J. Mol. Sci. 2025, 26, 4586. https://doi.org/10.3390/ijms26104586
Fu L, Li P, Rui Z, Sun J, Yang J, Wang Y, Jia D, Hu J, Li X, Ma R. CRISPR/Cas9-Mediated Knockout of the White Gene in Agasicles hygrophila. International Journal of Molecular Sciences. 2025; 26(10):4586. https://doi.org/10.3390/ijms26104586
Chicago/Turabian StyleFu, Li, Penghui Li, Zhiyi Rui, Jiang Sun, Jun Yang, Yuanxin Wang, Dong Jia, Jun Hu, Xianchun Li, and Ruiyan Ma. 2025. "CRISPR/Cas9-Mediated Knockout of the White Gene in Agasicles hygrophila" International Journal of Molecular Sciences 26, no. 10: 4586. https://doi.org/10.3390/ijms26104586
APA StyleFu, L., Li, P., Rui, Z., Sun, J., Yang, J., Wang, Y., Jia, D., Hu, J., Li, X., & Ma, R. (2025). CRISPR/Cas9-Mediated Knockout of the White Gene in Agasicles hygrophila. International Journal of Molecular Sciences, 26(10), 4586. https://doi.org/10.3390/ijms26104586