Gene targeting of the female determining transformer gene in the mediterranean fruit fly: an unexpected CRISPR/Cas9 interference effect. b) More on the future of MoY study and how to molecularly screen for tsl genes.

The Mediterranean fruitfly Ceratitis capitata (medfly) is an agricultural pest of high economic impact. We targeted medfly transformer, the key female determining gene with the ability to autoregulate, in a way similar to Sex-lethal in Drosophila. We injected Cas9-sgRNA RNPs into Ceratitis female-only XX embryos which developed into G0 adult XX flies, with up to 50% presenting a completely masculinized phenotype and an exclusively male-specific Cctra splicing pattern. However, Cctra DNA sequence analysis revealed a lack of gene editing events in both G0 and G1 progenies. We reasoned that Cctra transcription was transiently suppressed by an unplanned CRISPR interference (CRISPRi), usually achieved by a defective Cas9 variant (dCas9) unable to cut DNA. This unexpected CRISPRi seems to have caused a masculinization of XX embryos by transient biallelic transcriptional silencing rather than biallelic DNA mutations. We propose that, similarly to dCas9, a wild type Cas9 protein can cause CRISPR interference in autoregulated genes that are activated very early during embryogenesis. b) We will discuss our projects to understand how MoY functions molecularly in Tephritidae. Furthermore, we will present first steps for a proof of principle of a novel method to quickly screen for putative tsl genes, using embryo injections of large genomic tsl+ fragments into an elav mutant strain of Drosophila.