Conditional expression of Cas9 and dCas9 in Lucilia cuprina reveals dCas9-associated lethality

Kriete, A.; Basika, T.; Novas, R.; Belikoff, E. J.; Scott, M. J.

doi:10.1101/2025.08.12.669956

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bioRxiv · 10.1101/2025.08.12.669956

Conditional expression of Cas9 and dCas9 in Lucilia cuprina reveals dCas9-associated lethality

Kriete, A.·Basika, T.·Novas, R.·Belikoff, E. J.·Scott, M. J.

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Abstract

Conditional sex transformation systems are promising tools in the fight against insect pests. In this study, we developed and tested CRISPR-based, tetracycline-repressible sex transformation strains in the Australian sheep blowfly, Lucilia cuprina. Two CRISPR effector molecules, Cas9 and dCas9, were employed to target the sex-determining gene transformer with the goal of turning female blowflies into males. The Cas9 version of the system induced robust knockout of a visual marker gene but failed to trigger sex transformation without external provision of transformer-targeting sgRNAs. Furthermore, we found that dCas9 expression was linked to several deleterious phenotypes, including developmental delays, reduced body weight, and death. Our study provides the first proof-of-concept conditional CRISPR systems in L. cuprina, and suggests that while dCas9 is toxic at high levels in this species, Cas9 is well-tolerated and may be able to induce sex transformation with minor modifications to the system.

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Kriete, A., Basika, T., Novas, R., Belikoff, E. J., Scott, M. J.. 2025-08-13. Conditional expression of Cas9 and dCas9 in Lucilia cuprina reveals dCas9-associated lethality. https://doi.org/10.1101/2025.08.12.669956

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