Highly efficient DNA-free gene disruption in the agricultural pestCeratitis capitataby CRISPR-Cas9 RNPs

The Mediterranean fruitflyCeratitis capitata(medfly) is an invasive agricultural pest of high economical impact and has become an emerging model for developing new genetic control strategies as alternative to insecticides. Here, we report the successful adaptation of CRISPR-Cas9-based gene disruption in the medfly by injectingin vitropre-assembled, solubilized Cas9 ribonucleoprotein complexes (RNPs) loaded with gene-specific sgRNAs into early embryos. When targeting the eye pigmentation genewhite eye(we), we observed a high rate of somatic mosaicism in surviving G0 adults. Germline transmission of mutatedwealleles by G0 animals was on average above 70%, with individual cases achieving a transmission rate of nearly 100%. We further recovered large deletions in thewegene when two sites were simultaneously targeted by two sgRNAs. CRISPR-Cas9 targeting of theCeratitisortholog of theDrosophilasegmentationpairedgene (Ccprd) caused segmental malformations in late embryos and in hatched larvae. Mutant phenotypes correlate with repair by non-homologous end joining (NHEJ) lesions in the two targeted genes. This simple and highly effective Cas9 RNP-based gene editing to introduce mutations inCeratitis capitatawill significantly advance the design and development of new effective strategies for pest control management.