The Mediterranean fruitfly Ceratitis capitata (medfly) is an invasive agricultural pest of high economic impact and has become an emerging model for developing new genetic control strategies as an alternative to insecticides. Here, we report the successful adaptation of CRISPR-Cas9-based gene disruption in the medfly by injecting in vitro pre-assembled, solubilized Cas9 ribonucleoprotein complexes (RNPs) loaded with gene-specific single guide RNAs (sgRNA) into early embryos. When targeting the eye pigmentation gene white eye (we), a high rate of somatic mosaicism in surviving G0 adults was observed. Germline transmission rate of mutated we alleles by G0 animals was on average above 52%, with individual cases achieving nearly 100%. We further recovered large deletions in the we gene when two sites were simultaneously targeted by two sgRNAs. CRISPR-Cas9 targeting of the Ceratitis ortholog of the Drosophila segmentation paired gene (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 in C. capitata will significantly advance the design and development of new effective strategies for pest control management.

Background Genetic sex ratio distorters are systems aimed at effecting a bias in the reproductive sex ratio of a population and could be applied for the area-wide control of sexually reproducing insects that vector disease or disrupt agricultural production. One example of such a system leading to male bias is X-shredding, an approach that interferes with the transmission of the X-chromosome by inducing multiple DNA double-strand breaks during male meiosis. Endonucleases targeting the X-chromosome and whose activity is restricted to male gametogenesis have recently been pioneered as a means to engineer such traits. Results Here, we enabled endogenous CRISPR/Cas9 and CRISPR/Cas12a activity during spermatogenesis of the Mediterranean fruit fly Ceratitis capitata, a worldwide agricultural pest of extensive economic significance. In the absence of a chromosome-level assembly, we analysed long- and short-read genome sequencing data from males and females to identify two clusters of abundant and X-chromosome-specific sequence repeats. When targeted by gRNAs in conjunction with Cas9, cleavage of these repeats yielded a significant and consistent distortion of the sex ratio towards males in independent transgenic strains, while the combination of distinct distorters induced a strong bias (~ 80%). Conclusion We provide a first demonstration of CRISPR-based sex distortion towards male bias in a non-model organism, the global pest insect Ceratitis capitata. Although the sex ratio bias reached in our study would require improvement, possibly through the generation and combination of additional transgenic lines, to result in a system with realistic applicability in the field, our results suggest that strains with characteristics suitable for field application can now be developed for a range of medically or agriculturally relevant insect species.

Genetic sex ratio distorters have potential for the area-wide control of harmful insect populations. Endonucleases targeting the X-chromosome and whose activity is restricted to male gametogenesis have recently been pioneered as a means to engineer such traits. Here we enabled endogenous CRISPR/Cas9 and CRISPR/Cas12a activity during spermatogenesis of the Mediterranean fruit fly Ceratitis capitata, a worldwide agricultural pest of extensive economic significance. In the absence of a chromosome-level assembly, we analysed long and short-read genome sequencing data from males and females to identify two clusters of abundant and X-chromosome specific sequence repeats. When targeted by gRNAs in conjunction with Cas9 they yielded a significant and consistent distortion of the sex ratio in independent transgenic strains and a combination of distorters induced a strong bias towards males (~80%). Our results demonstrate the design of sex distorters in a non-model organism and suggest that strains with characteristics suitable for field application could be developed for a range of medically or agriculturally relevant insect species.

41The Mediterranean fruitfly Ceratitis capitata (medfly) is an invasive agricultural pest of 42 high economical impact and has become an emerging model for developing new genetic 43 control strategies as alternative to insecticides. Here, we report the successful adaptation 44 of CRISPR-Cas9-based gene disruption in the medfly by injecting in vitro pre-assembled, 45 solubilized Cas9 ribonucleoprotein complexes (RNPs) loaded with gene-specific sgRNAs 46 into early embryos. When targeting the eye pigmentation gene white eye (we), we 47 observed a high rate of somatic mosaicism in surviving G0 adults. Germline transmission 48 of mutated we alleles by G0 animals was on average above 70%, with individual cases 49 achieving a transmission rate of nearly 100%. We further recovered large deletions in the 50 we gene when two sites were simultaneously targeted by two sgRNAs. CRISPR-Cas9 51 targeting of the Ceratitis ortholog of the Drosophila segmentation paired gene (Ccprd) 52 caused segmental malformations in late embryos and in hatched larvae. Mutant 53 phenotypes correlate with repair by non-homologous end joining (NHEJ) lesions in the 54 two targeted genes. This simple and highly effective Cas9 RNP-based gene editing to 55 introduce mutations in Ceratitis capitata will significantly advance the design and 56 development of new effective strategies for pest control management. 57 58 59 60 61 62 63 64 65 The Mediterranean fruitfly Ceratitis capitata (medfly) is an economically relevant 66 agricultural pest infesting more than 260 crop species, including fruits, vegetables, and 67 nuts 1 . Wild populations can be contained by the Sterile Insect Technique (SIT), an 68 eradication strategy based on the repeated release of large numbers of factory-grown 69 sterile males into infested areas 2,3 . C. capitata was the first non-Drosophilidae insect 70 species in which transposon-mediated germline transformation was established 4,5 . 71Various Ceratitis transgenic strains have been developed, aiming to improve SIT and 72 other pest control strategies [8][9][10][11][12][13][14][15][16] . Also embryonic RNA interference was successfully 73 applied to study in vivo functions of key Ceratitis genes controlling sex determination 6,7 . 74Nonetheless, a more comprehensive study of gene functions in Ceratitis will be needed to 75 further improve existing control strategies. To generate long-lasting and hereditable 76 changes in gene function, the CRISPR-Cas9 system with its modular and simple 77 components provides a promising tool to implement scalable, reproducible pest control 78 strategies 17,18 . Furthermore, transgene-based CRISPR-Cas9 can be used to produce 79 homozygous loss-of-function mutations as well as a novel gene drive system for insect 80 population control 19,21 . 81 Various studies have reported the successful use of the Cas9 system to introduce 82 genome modifications in Drosophila melanogaster based on injecting different 83 combinations CRISPR-Cas9 reagents into embryos, such as DNA plasmids expressing 84 Cas9 prot...