Conditional Expression of Cas9 and dCas9 in Lucilia cuprina Reveals dCas9-Associated Lethality

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.