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.

Gene drives hold promise for the genetic control of malaria vectors. The development of vector population modification strategies hinges on the availability of effector mechanisms impeding parasite development in transgenic mosquitoes. We augmented a midgut gene of the malaria mosquito Anopheles gambiae to secrete two exogenous antimicrobial peptides, magainin 2 and melittin. This small genetic modification, capable of efficient nonautonomous gene drive, hampers oocyst development in both Plasmodium falciparum and Plasmodium berghei . It delays the release of infectious sporozoites, while it simultaneously reduces the life span of homozygous female transgenic mosquitoes. Modeling the spread of this modification using a large-scale agent-based model of malaria epidemiology reveals that it can break the cycle of disease transmission across a range of transmission intensities.

Background Females of the Mediterranean fruit fly Ceratitis capitata (Medfly) are major agricultural pests, as they lay eggs into the fruit crops of hundreds of plant species. In Medfly, female sex determination is based on the activation of Cctransformer (Cctra). A maternal contribution of Cctra is required to activate Cctra itself in the XX embryos and to start and epigenetically maintain a Cctra positive feedback loop, by female-specific alternative splicing, leading to female development. In XY embryos, the male determining Maleness-on-the-Y gene (MoY) blocks this activation and Cctra produces male-specific transcripts encoding truncated CcTRA isoforms and male differentiation occurs. Results With the aim of inducing frameshift mutations in the first coding exon to disrupt both female-specific and shorter male-specific CcTRA open reading frames (ORF), we injected Cas9 ribonucleoproteins (Cas9 and single guide RNA, sgRNA) in embryos. As this approach leads to mostly monoallelic mutations, masculinization was expected only in G1 XX individuals carrying biallelic mutations, following crosses of G0 injected individuals. Surprisingly, these injections into XX-only embryos led to G0 adults that included not only XX females but also 50% of reverted fertile XX males. The G0 XX males expressed male-specific Cctra transcripts, suggesting full masculinization. Interestingly, out of six G0 XX males, four displayed the Cctra wild type sequence. This finding suggests that masculinization by Cas9-sgRNA injections was independent from its mutagenic activity. In line with this observation, embryonic targeting of Cctra in XX embryos by a dead Cas9 (enzymatically inactive, dCas9) also favoured a male-specific splicing of Cctra, in both embryos and adults. Conclusions Our data suggest that the establishment of Cctra female-specific autoregulation during the early embryogenesis has been repressed in XX embryos by the transient binding of the Cas9-sgRNA on the first exon of the Cctra gene. This hypothesis is supported by the observation that the shift of Cctra splicing from female to male mode is induced also by dCas9. Collectively, the present findings corroborate the idea that a transient embryonic inactivation of Cctra is sufficient for male sex determination.

Gene drives hold promise for the genetic control of malaria vectors. The development of vector population modification strategies hinges on the availability of effector mechanisms impeding parasite development in transgenic mosquitoes. We augmented a midgut gene of the malaria mosquito Anopheles gambiae to secrete two exogenous antimicrobial peptides, Magainin 2 and Melittin. This small genetic modification, capable of efficient non-autonomous gene drive, hampers oocyst development in both Plasmodium falciparum and Plasmodium berghei. It delays the release of infectious sporozoites while it simultaneously reduces the lifespan of homozygous female transgenic mosquitoes. Modeling the spread of this modification using a large-scale agent-based model of malaria epidemiology reveals that it can break the cycle of disease transmission across a range of endemic settings.

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...

Genetically modified (GM) mosquitoes expressing anti-plasmodial effectors propagating through wild mosquito populations by means of gene drive is a promising tool to support current malaria control strategies. The process of generating GM mosquitoes involves genetic transformation of mosquitoes from a laboratory colony and, often, interbreeding with other GM lines to cross in auxiliary traits. These mosquito colonies and GM lines thus often have different genetic backgrounds and GM lines are invariably highly inbred, which in conjunction with their independent rearing in the laboratory may translate to differences in their susceptibility to malaria parasite infection and life history traits. Here, we show that laboratory Anopheles gambiae colonies and GM lines expressing Cas9 and Cre recombinase vary greatly in their susceptibility to Plasmodium falciparum NF54 infection. Therefore, the choice of mosquitoes to be used as a reference when conducting infection or life history trait assays requires careful consideration. To address these issues, we established an experimental pipeline involving genetic crosses and genotyping of mosquitoes reared in shared containers throughout their lifecycle. We used this protocol to examine whether GM lines expressing the antimicrobial peptide (AMP) Scorpine in the mosquito midgut interfere with parasite infection and mosquito survival. We demonstrate that Scorpine expression in the Peritrophin 1 (Aper1) genomic locus reduces both P. falciparum sporozoite prevalence and mosquito lifespan; both these phenotypes are likely to be associated with the disturbance of the midgut microbiota homeostasis. These data lead us to conclude that the Aper1-Sco GM line could be used in proof-of-concept experiments aimed at mosquito population replacement, although the impact of its reduced fitness on the spread of the transgene through wild populations requires further investigation.