Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 RNPs

The Mediterranean fruitfly Ceratitis 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 injecting in vitro pre-assembled Cas9 ribonucleoparticles (RNP) loaded with gene-specific sgRNAs into early embryos. When targeting the eye pigmentation white eye (we), a high rate of somatic mosaicism was observed in surviving G0 adults. Germline transmission of mutated we alleles was found in the progeny of more than 70% of G0 flies. Large deletions were recovered in the we gene when two sites were simultaneously targeted by duplex sgRNAs. CRISPR-Cas9 targeting of the Ceratitis ortholog of the Drosophila segmentation paired gene (Ccprd) caused segmental malformations in late embryos and irregular muscular contractions in hatched larvae. Mutant phenotypes correlate with presence of non-homologous end joining (NHEJ) lesions in the two targeted genes. This simple and highly effective method for gene editing together with the availability of genome and transcriptome sequences will significantly advance our knowledge on gene function and regulation in Ceratitis as well as improve design and development of new effective strategies for pest control management.